NanoWiki - Brukerbidrag [nb]

Transmission Electron Microscopy

2009-05-25T11:39:57Z

Bjornoy: /* High Angle Annular Dark Field */

Dette er et av to typer elektronmikroskop. Med en TEM får man god oppløsning, omtrent ned på atomært nivå. Denne saken sender elektroner ned gjennom et rør som innehar en del elektromagnetiske linser som får alt til å bli bra. Det er flere forskjellige ting man kan få ut av og gjøre med en TEM og noen forskjellige saker står derfor under. Den viktikste egenskapen til en TEM er at den kombinerer informasjon fra både det virkelige rom (real space) og det resiproke rom (reciprocal space).

== Oppsett ==
Kilden er en elektronkanon med en svært høy spenning (100-400kV). Elektronkanonen kan være av forskjellige typer (filamenter eller "tip"er), men det mest vanlige materialet er Wolfram (Tungsten). Elektronene blir så akselrert av en anode holdt ved jord. Strålen er karakteristert av den effektive størrelsen, vinkelen, elektronenergien og spredningen av elektronenergier. En liten effektiv størrelse forbedrer strålens koherens.

"Condenser" aperturen er elektromagnetiske linser som fokuserer elektronstrålen. De har samme funksjon som condenser aperturen i [[optiske mikropskop]]. Forskjellen er at man her kan variere fokal lengden, ved å variere strømmen som går i linsene. Elektronene vil følge en heliks bane pga det elektromagnetsike feltet.

Prøvematerialet kan både roteres og vippes. Diameteren på prøven må være under 3mm, og tykkelsen må være under 0.1 <math>\mu m</math>, for å få elastisk spredning (scattering).

Bildet blir laget av en "flourescent" skjerm som konverterer høy-energi-elektronbildet til et bildet vi kan se, eller ved et CCD-kamera.

== Oppløsning ==
Ved 100kV er oppløsningen ca. 0.2nm.

De "vanlige" ligningen for depth of field and focus fra optisk mikroskopi, kan ikke lenger brukes med sikkerhet, nå som linsene ikke lenger kan defineres som "tynne" linser. Man kan likevel finne approksimerte verdier. Depht of field blir dermed 20-200nm (noe som avgrenser tykkelsen til en prøve enda mer), mens depth of focus blir flere meter!

Begrensninger på oppløsningen er gitt ved Rayleighs kriterium som for [[optisk mikroskopi]]. Man kan altså få høyere oppløsning ved høyere spenning, men en spenning opp mot 1MV vil som regel skade prøven. Man får analogt med optisk mikroskopi også sfæriske og kromatiske avik (spherical and cromatic aberrations). Sfæriske avik kommer fra at stråler i forskjellig avstand fra den optiske aksen (forskjellig vinkel) vil fokuseres ved forskjellige fokal punkt. Der en stråle lengre unna aksen vil fokuseres på et punkt nærmere linsen. Kromatiske avik kommer fra at stråler med forskjellig energi vil fokuserers ved forskjellige fokalpunkt. En stråle med høyere energi vil fokuseres på et punkt nærmere linsen.

Astigmatisme kommer fra manglende aksial symmetri ved objektiv linsene, noe som også fører til en usikkerhet i fokalpunktet. Dette kan som regel korrigeres ved magnetiske felt.

== Kontrast ==
*Mass-thickness contrast: Sannsynligheten for at et elektron blir spredd elastisk avhenger av spredningsfaktoren (atomic scattering factor), som øker med atom nummeret og det totale antall atomer. Spredningen er altså avhengig av prøvens tykkelse og tetthet, noe som gir kontrast i bildet. Amorfe og biologiske prøver vil dominerers at mass-thickness contrast.

*Diffraction contrast: Denne typen kontrast kommer fra avik i enten den eksakte betingelsen fra Braggs lov eller avik i krystallstrukturen. Det er altså både avik i det virkelige rom og i det resiproke rom som fører til kontrast. I krystallinske materialer er dette den dominate kontrasten.

*Phase contrast: I TEM kan man velge hvilke ståler man vil se på (en spredd stråle, eller den direkte strålen). For å få phase contrast må man sette aperturen slik at man velger flere stråler samtidig. Det vil dermed oppstå et interferensmønster ut i fra disse strålene pga en veiforskjell. Dette interferensmønsteret gir kontrast. Ved phase-contrast vil oppløsningen kunne bli mye bedre enn ved mass-thickness eller diffraction, helt ned til 0.07nm.

== Teknikker ==
I TEM bruker man objektivaperturen til å bestemme hvilke(n) stråle(r) man vil se på. Dette fører til mange forskjellige teknikker og kontraster.

=== Bright field ===
Her brukes den direkte elektronstrålen som har gått rett gjennom prøven. Bakgrunnen blir dermed lys, sånn som i optisk mikroskopi. Denne teknikken bruker mass-thickness contrast.

=== Dark field ===
Her brukes en spredd stråle, og analogt med optisk mikroskopi får man svart bakgrunn og bedre kontrast enn ved bright field. Denne teknikken bruker diffraction contrast. Dersom "incident beam" er rett og man velger en spredd stråle får man det man kaller dirty DF, eller lav-oppløsnings DF. Dersom man heller tilter strålen og velger strålen langs den opprinnelige optiske aksen får man høy-oppløsnings DF.

=== High Resolution TEM ===
Bruker en kombinasjon av DF og BF, altså den velger flere stråler, og bruker phase contrast. Bildet man får er i meget høy oppløsning, men kan være vanskelig å tolke pga mange interferensfenomener.

Høy oppløsning. HØY!

=== High Angle Annular Dark Field ===
Kommer av elektroner som blir avbøyd kraftig av tunge atom-kjerner (Z-kontrast). HAADF er altså avhengig av størrelsen på atomene og ikke strukturen til latticen, som vanlig DF er. Brukes kun i sammenheng med STEM (har ikke funnet noen eksempler på HAADF-TEM, kun HAADF-STEM).

=== Electron Energy-Loss Spectroscopy ===
Hvis man fjerner den vanlige detektoren i en TEM, og i stedet lar elektronene passere igjennom et magnetisk prisme slik at de blir avbøyd på grunn av [[Lorentz-kraften]], vil man kunne filtrere elektronene etter energi. Dette fordi elektroner med høy energi, og dermed høy hastighet, vil kreve en lengre distanse til å bli avbøyd 90 grader enn elektroner som går saktere. Konsekvensen blir dermed at man får skilt ut elektronene i rommet basert på deres energi, på samme måte som man kan spre lys til et spekter vha. et optisk prisme.

Slik energiatskillelse er interessant siden elektroner som blir sendt gjennom en prøve i mange tilfeller vil tape litt energi. Energien elektronene taper skyldes vekselvirkninger med materialet i prøven, og energien elektronene taper vil dermed være karakteristisk for materialet det passerer igjennom.

=== Energy Filtered TEM ===
Gitt et oppsett som ved [[EELS]], vil man etter det magnetiske prismet ha signalet spredd i rommet etter elektronenergi, og signalet vil være i [[resiprokt rom]]. Dette vil si at i et hvert "energibånd" (høydeutsnitt), vil ha et komplett romlig bilde av prøven. Hvis man da bruker et apertur til å kun velge seg ut elektroner med en viss energi, og konverterer signalet tilbake til reelt rom, vil man få et bildet av ''hvor i prøven elektroner med en viss energi kommer i fra''. Da forskjellige materialer gjerne gir større energitap for elektronene som passerer, vil man kunne bruke EFTEM til å lokalisere spesifikke materialer i prøven.

=== Scanning TEM ===
Fokuserer strålen slik at man kan scanne bildet, i stedet for å få informasjonen simultant. STEM har alle de samme funksjonene som vanlig TEM, og man kan dermed få bright field, dark field, HAADF osv. I HAADF får man nå noe som kalles z-contrast, altså en direkte sammenheng mellom den lokale kontrasten og mass-thickness kontrasten som avhenger av atom nummeret (Z).

Transmission Electron Microscopy

2009-05-25T11:32:45Z

Bjornoy: /* Dark field */

Dette er et av to typer elektronmikroskop. Med en TEM får man god oppløsning, omtrent ned på atomært nivå. Denne saken sender elektroner ned gjennom et rør som innehar en del elektromagnetiske linser som får alt til å bli bra. Det er flere forskjellige ting man kan få ut av og gjøre med en TEM og noen forskjellige saker står derfor under. Den viktikste egenskapen til en TEM er at den kombinerer informasjon fra både det virkelige rom (real space) og det resiproke rom (reciprocal space).

== Oppsett ==
Kilden er en elektronkanon med en svært høy spenning (100-400kV). Elektronkanonen kan være av forskjellige typer (filamenter eller "tip"er), men det mest vanlige materialet er Wolfram (Tungsten). Elektronene blir så akselrert av en anode holdt ved jord. Strålen er karakteristert av den effektive størrelsen, vinkelen, elektronenergien og spredningen av elektronenergier. En liten effektiv størrelse forbedrer strålens koherens.

"Condenser" aperturen er elektromagnetiske linser som fokuserer elektronstrålen. De har samme funksjon som condenser aperturen i [[optiske mikropskop]]. Forskjellen er at man her kan variere fokal lengden, ved å variere strømmen som går i linsene. Elektronene vil følge en heliks bane pga det elektromagnetsike feltet.

Prøvematerialet kan både roteres og vippes. Diameteren på prøven må være under 3mm, og tykkelsen må være under 0.1 <math>\mu m</math>, for å få elastisk spredning (scattering).

Bildet blir laget av en "flourescent" skjerm som konverterer høy-energi-elektronbildet til et bildet vi kan se, eller ved et CCD-kamera.

== Oppløsning ==
Ved 100kV er oppløsningen ca. 0.2nm.

De "vanlige" ligningen for depth of field and focus fra optisk mikroskopi, kan ikke lenger brukes med sikkerhet, nå som linsene ikke lenger kan defineres som "tynne" linser. Man kan likevel finne approksimerte verdier. Depht of field blir dermed 20-200nm (noe som avgrenser tykkelsen til en prøve enda mer), mens depth of focus blir flere meter!

Begrensninger på oppløsningen er gitt ved Rayleighs kriterium som for [[optisk mikroskopi]]. Man kan altså få høyere oppløsning ved høyere spenning, men en spenning opp mot 1MV vil som regel skade prøven. Man får analogt med optisk mikroskopi også sfæriske og kromatiske avik (spherical and cromatic aberrations). Sfæriske avik kommer fra at stråler i forskjellig avstand fra den optiske aksen (forskjellig vinkel) vil fokuseres ved forskjellige fokal punkt. Der en stråle lengre unna aksen vil fokuseres på et punkt nærmere linsen. Kromatiske avik kommer fra at stråler med forskjellig energi vil fokuserers ved forskjellige fokalpunkt. En stråle med høyere energi vil fokuseres på et punkt nærmere linsen.

Astigmatisme kommer fra manglende aksial symmetri ved objektiv linsene, noe som også fører til en usikkerhet i fokalpunktet. Dette kan som regel korrigeres ved magnetiske felt.

== Kontrast ==
*Mass-thickness contrast: Sannsynligheten for at et elektron blir spredd elastisk avhenger av spredningsfaktoren (atomic scattering factor), som øker med atom nummeret og det totale antall atomer. Spredningen er altså avhengig av prøvens tykkelse og tetthet, noe som gir kontrast i bildet. Amorfe og biologiske prøver vil dominerers at mass-thickness contrast.

*Diffraction contrast: Denne typen kontrast kommer fra avik i enten den eksakte betingelsen fra Braggs lov eller avik i krystallstrukturen. Det er altså både avik i det virkelige rom og i det resiproke rom som fører til kontrast. I krystallinske materialer er dette den dominate kontrasten.

*Phase contrast: I TEM kan man velge hvilke ståler man vil se på (en spredd stråle, eller den direkte strålen). For å få phase contrast må man sette aperturen slik at man velger flere stråler samtidig. Det vil dermed oppstå et interferensmønster ut i fra disse strålene pga en veiforskjell. Dette interferensmønsteret gir kontrast. Ved phase-contrast vil oppløsningen kunne bli mye bedre enn ved mass-thickness eller diffraction, helt ned til 0.07nm.

== Teknikker ==
I TEM bruker man objektivaperturen til å bestemme hvilke(n) stråle(r) man vil se på. Dette fører til mange forskjellige teknikker og kontraster.

=== Bright field ===
Her brukes den direkte elektronstrålen som har gått rett gjennom prøven. Bakgrunnen blir dermed lys, sånn som i optisk mikroskopi. Denne teknikken bruker mass-thickness contrast.

=== Dark field ===
Her brukes en spredd stråle, og analogt med optisk mikroskopi får man svart bakgrunn og bedre kontrast enn ved bright field. Denne teknikken bruker diffraction contrast. Dersom "incident beam" er rett og man velger en spredd stråle får man det man kaller dirty DF, eller lav-oppløsnings DF. Dersom man heller tilter strålen og velger strålen langs den opprinnelige optiske aksen får man høy-oppløsnings DF.

=== High Resolution TEM ===
Bruker en kombinasjon av DF og BF, altså den velger flere stråler, og bruker phase contrast. Bildet man får er i meget høy oppløsning, men kan være vanskelig å tolke pga mange interferensfenomener.

Høy oppløsning. HØY!

=== High Angle Annular Dark Field ===
Kommer av elektroner som blir avbøyd kraftig av tunge atom-kjerner (Z-kontrast). HAADF er altså avhengig av størrelsen på atomene og ikke strukturen til latticen, som vanlig DF er.

=== Electron Energy-Loss Spectroscopy ===
Hvis man fjerner den vanlige detektoren i en TEM, og i stedet lar elektronene passere igjennom et magnetisk prisme slik at de blir avbøyd på grunn av [[Lorentz-kraften]], vil man kunne filtrere elektronene etter energi. Dette fordi elektroner med høy energi, og dermed høy hastighet, vil kreve en lengre distanse til å bli avbøyd 90 grader enn elektroner som går saktere. Konsekvensen blir dermed at man får skilt ut elektronene i rommet basert på deres energi, på samme måte som man kan spre lys til et spekter vha. et optisk prisme.

Slik energiatskillelse er interessant siden elektroner som blir sendt gjennom en prøve i mange tilfeller vil tape litt energi. Energien elektronene taper skyldes vekselvirkninger med materialet i prøven, og energien elektronene taper vil dermed være karakteristisk for materialet det passerer igjennom.

=== Energy Filtered TEM ===
Gitt et oppsett som ved [[EELS]], vil man etter det magnetiske prismet ha signalet spredd i rommet etter elektronenergi, og signalet vil være i [[resiprokt rom]]. Dette vil si at i et hvert "energibånd" (høydeutsnitt), vil ha et komplett romlig bilde av prøven. Hvis man da bruker et apertur til å kun velge seg ut elektroner med en viss energi, og konverterer signalet tilbake til reelt rom, vil man få et bildet av ''hvor i prøven elektroner med en viss energi kommer i fra''. Da forskjellige materialer gjerne gir større energitap for elektronene som passerer, vil man kunne bruke EFTEM til å lokalisere spesifikke materialer i prøven.

=== Scanning TEM ===
Fokuserer strålen slik at man kan scanne bildet, i stedet for å få informasjonen simultant. STEM har alle de samme funksjonene som vanlig TEM, og man kan dermed få bright field, dark field, HAADF osv. I HAADF får man nå noe som kalles z-contrast, altså en direkte sammenheng mellom den lokale kontrasten og mass-thickness kontrasten som avhenger av atom nummeret (Z).

Transmission Electron Microscopy

2009-05-25T08:15:10Z

Bjornoy: /* High Angle Annular Dark Field */

Transmission Electron Microscopy

2009-05-25T08:13:21Z

Bjornoy: /* High Resolution TEM */

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-04-05T11:16:02Z

Bjornoy: /* DNA */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short summary of our findings and a discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">[http://www.alibris.co.uk/search/books/isbn/9781429201247?cid=null Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition]''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, analog to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information. The number of combinations between different nano-particles and biomolecules is vast. Therefore the main focus of this article is DNA-NP and enzyme-NP structures.
====DNA====
[[Bilde:Antibody.gif|right|thumb|350px| '''Schematic of an antibody.''' The variable structure of both the light and heavy chains determines which antigen it will bind to. ]]
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antibody-antigen====
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref name="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor where a solution changes color because of LSPR when antigenes are attached to the antibodies on the particles surface.

====Hormone-receptor====
Similar to antibody-antigen, the reactions between hormones and receptors are highly specific. Hormones are chemicals released by cells that affects other celltypes. The cells respond to a hormone if it has the given receptor. This can be used with functionalized nano-particles to produce cell-specific reactions.

====Enzymes====
Similar to the examples mentioned above, enzymes have highly selective recognition properties <ref name="katz"/>. In addition, enzymes are very interesting due to their specific catalytic properties. As will be discussed later in this article, the enzyme glucose oxidase may be functionalised unto a gold nanoparticle to create a glucose sensor.

==Applications==
===Desired properties===
A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution was still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets was introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a concentration value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.45V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Summary===
In this article the possibilities of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors'' have been discussed. It has been shown that the optical and electrical properties of NPs together with the specific and catalytic properties of biomaterials may be combined to create novel biosensors.

Though the applications of the NP-biomaterials have shown to be useful in many areas, the technology is still young and in need of further development. Although there are some examples of commercial NP-biomolecule sensors available<ref>[http://www.biacore.com/lifesciences/index.html Swedish based BIAcore]</ref>, some examples of the challenges this new technology faces will be:

*Functionalization of nano-particles. How do we attach biomolecules on nano-particles?
*Stability over time. How long can the probes/sensors be stored?
*Re-use.
*Costs.
*Nano-particles inside our bodies.
*Durability. The ability to stay intact in the crowded environment of our bodies.

Many of these problems are yet to be solved, but the future looks brigth. For instance in the case of molecular beacons:
''Molecular beacons are expected to be capable of functioning as highly specific, highly sensitive recognition and signaling elements in state-of-the art biological detection strategies for biomolecules''<ref>[http://www3.interscience.wiley.com/cgi-bin/fulltext/71005735/PDFSTART Weihong Tan, Xiaohong Fang, Jeffery Li, and Xiaojing Liu ''Molecular Beacons: A Novel DNA Probe for Nucleic Acid and Protein Studies''].</ref>.

If these challenges are met, it could [http://en.wikipedia.org/wiki/Utopia#Scientific_and_technological_utopia one day] be possible to have not only sensors that can simultaneously detect several diseases at once, but even bring drugs to the exact location of damaged cells or bacterial colonies.

For further reading you are referred to our references below which will provide ease of access to the sources used in this article.

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-23T11:12:43Z

Bjornoy: /* Challenges */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, analog to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information. The number of combinations between different nano-particles and biomolecules is vast. Therefore the main focus of this article is DNA-NP and enzyme-NP structures.
====DNA====
[[Bilde:Antibody.gif|right|thumb|350px| '''An antibody.''' The variable structure of both the light and heavy chains determines which antigen it will bind to. ]]
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antibody-antigen====
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref name="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor where a solution changes color because of LSPR when antigenes are attached to the antibodies on the particles surface.

====Hormone-receptor====
Similar to antibody-antigen, the reactions between hormones and receptors are highly specific. Hormones are chemicals released by cells that affects other celltypes. The cells respond to a hormone if it has the given receptor. This can be used with functionalized nano-particles to produce cell-specific reactions.

====Enzymes====

==Applications==
===Desired properties===
A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution was still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets was introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a concentration value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.45V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Challenges===

It may sound like these new sensor have changed the entire field of biosensors, but that is not the case. The techniques and sensors presented here are still in development. Although there are some biomolecule sensors using the techniques disscussed here,<ref>[http://www.biacore.com/lifesciences/index.html Swedish based BIAcore]</ref> the challenges with these techniques are considerable:
*Functionalization of nano-particles. How do we attach biomolecules on nano-particles?
*Stability over time. How long can the probes/sensors be stored?
*Re-use.
*Costs.
*Nano-particles inside our bodies.
*Durability. The ability to stay intact in the crowded environment of our bodies.

Some of these questions remains unanswered, but the future looks brigth. For instance:
''Molecular beacons are expected to be capable of functioning as highly specific, highly sensitive recognition and signaling elements in state-of-the art biological detection strategies for biomolecules''<ref>[http://www3.interscience.wiley.com/cgi-bin/fulltext/71005735/PDFSTART Weihong Tan, Xiaohong Fang, Jeffery Li, and Xiaojing Liu ''Molecular Beacons: A Novel DNA Probe for Nucleic Acid and Protein Studies''].</ref>

If these challenges are met, it could [http://en.wikipedia.org/wiki/Utopia#Scientific_and_technological_utopia one day] be possible to have not only sensors that can simultaneously detect several diseases at once, but even bring drugs to the exact location of damaged cells or bacterial colonies.

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-23T10:22:51Z

Bjornoy: /* Importance to Nanotechnology */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, analog to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information. The number of combinations between different nano-particles and biomolecules is vast. Therefore the main focus of this article is DNA-NP and enzyme-NP structures.
====DNA====
[[Bilde:Antibody.gif|right|thumb|350px| '''An antibody.''' The variable structure of both the light and heavy chains determines which antigen it will bind to. ]]
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antibody-antigen====
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref name="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor where a solution changes color because of LSPR when antigenes are attached to the antibodies on the particles surface.

====Hormone-receptor====
Similar to antibody-antigen, the reactions between hormones and receptors are highly specific. Hormones are chemicals released by cells that affects other celltypes. The cells respond to a hormone if it has the given receptor. This can be used with functionalized nano-particles to produce cell-specific reactions.

====Enzymes====

==Applications==
===Desired properties===
A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution was still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets was introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.45V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Challenges===

It may sound like these new sensor have changed the entire field of biosensors, but that is not the case. The techniques and sensors presented here are still in development. Although there are some biomolecule sensors using the techniques disscussed here,<ref>[http://www.biacore.com/lifesciences/index.html Swedish based BIAcore]</ref> the challenges with these techniques are considerable:
*Functionalization of nano-particles. How do we attach biomolecules on nano-particles?
*Stability over time. How long can the probes/sensors be stored?
*Re-use.
*Costs.
*Nano-particles inside our bodies.
*Durability. The ability to stay intact in the crowded environment of our bodies.

Some of these questions remains unanswered, but the future looks brigth. For instance:
''Molecular beacons are expected to be capable of functioning as highly specific, highly sensitive recognition and signaling elements in state-of-the art biological detection strategies for biomolecules''<ref>[http://www3.interscience.wiley.com/cgi-bin/fulltext/71005735/PDFSTART Molecular Beacons: A Novel DNA Probe for Nucleic Acid and Protein Studies].</ref>

If these challenges are met, it could [http://en.wikipedia.org/wiki/Utopia#Scientific_and_technological_utopia one day] be possible to have not only sensors that can simultaneously detect several diseases at once, but even bring drugs to the exact location of damaged cells or bacterial colonies.

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-23T10:22:27Z

Bjornoy: /* Challenges */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, analog to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information. The number of combinations between different nano-particles and biomolecules is vast. Therefore the main focus of this article is DNA-NP and enzyme-NP structures.
====DNA====
[[Bilde:Antibody.gif|right|thumb|350px| '''An antibody.''' The variable structure of both the light and heavy chains determines which antigen it will bind to. ]]
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antibody-antigen====
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref name="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor where a solution changes color because of LSPR when antigenes are attached to the antibodies on the particles surface.

====Hormone-receptor====
Similar to antibody-antigen, the reactions between hormones and receptors are highly specific. Hormones are chemicals released by cells that affects other celltypes. The cells respond to a hormone if it has the given receptor. This can be used with functionalized nano-particles to produce cell-specific reactions.

====Enzymes====

==Applications==
===Desired properties===
A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution was still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets was introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.45V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Challenges===

It may sound like these new sensor have changed the entire field of biosensors, but that is not the case. The techniques and sensors presented here are still in development. Although there are some biomolecule sensors using the techniques disscussed here,<ref>[http://www.biacore.com/lifesciences/index.html Swedish based BIAcore]</ref> the challenges with these techniques are considerable:
*Functionalization of nano-particles. How do we attach biomolecules on nano-particles?
*Stability over time. How long can the probes/sensors be stored?
*Re-use.
*Costs.
*Nano-particles inside our bodies.
*Durability. The ability to stay intact in the crowded environment of our bodies.

Some of these questions remains unanswered, but the future looks brigth. For instance:
''Molecular beacons are expected to be capable of functioning as highly specific, highly sensitive recognition and signaling elements in state-of-the art biological detection strategies for biomolecules''<ref>[http://www3.interscience.wiley.com/cgi-bin/fulltext/71005735/PDFSTART Molecular Beacons: A Novel DNA Probe for Nucleic Acid and Protein Studies].</ref>

If these challenges are met, it could [http://en.wikipedia.org/wiki/Utopia#Scientific_and_technological_utopia one day] be possible to have not only sensors that can simultaneously detect several diseases at once, but even bring drugs to the exact location of damaged cells or bacterial colonies.

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-23T09:59:39Z

Bjornoy: /* SPR */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, analog to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information. The number of combinations between different nano-particles and biomolecules is vast. Therefore the main focus of this article is DNA-NP and enzyme-NP structures.
====DNA====
[[Bilde:Antibody.gif|right|thumb|350px| '''An antibody.''' The variable structure of both the light and heavy chains determines which antigen it will bind to. ]]
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antibody-antigen====
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref name="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor where a solution changes color because of LSPR when antigenes are attached to the antibodies on the particles surface.

====Hormone-receptor====
Similar to antibody-antigen, the reactions between hormones and receptors are highly specific. Hormones are chemicals released by cells that affects other celltypes. The cells respond to a hormone if it has the given receptor. This can be used with functionalized nano-particles to produce cell-specific reactions.

====Enzymes====

==Applications==
===Desired properties===
A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution was still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets was introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Challenges===

It may sound like these new sensor have changed the entire field of biosensors, but that is not the case. The techniques and sensors presented here are still in development. Although there are some biomolecule sensors out there<ref>[http://www.biacore.com/lifesciences/index.html Swedish based BIAcore]</ref> the challenges with these techniques are massive:
*Functionalization of nano-particles. How do we attach biomolecules on nano-particles?
*Stability over time. How long can the probes/sensors be stored?
*Re-use.
*Costs.
*Nano-particles inside our bodies.
*Durability. The ability to stay intact in the crowded environment of our bodies.

Some of these questions remains unanswered, but the future looks brigth:
''Molecular beacons are expected to be capable of functioning as highly specific, highly sensitive recognition and signaling elements in state-of-the art biological detection strategies for biomolecules''<ref>[http://www3.interscience.wiley.com/cgi-bin/fulltext/71005735/PDFSTART Molecular Beacons: A Novel DNA Probe for Nucleic Acid and Protein Studies].</ref>

If these challenges are met, it could [http://en.wikipedia.org/wiki/Utopia#Scientific_and_technological_utopia one day] be possible to have not only sensors that can simultaneously detect several diseases at once, but even bring drugs to the exact location of damaged cells or bacterial colonies.

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-23T09:55:09Z

Bjornoy: /* Challenges */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information. The number of combinations between different nano-particles and biomolecules is vast. Therefore the main focus of this article is DNA-NP and enzyme-NP structures.
====DNA====
[[Bilde:Antibody.gif|right|thumb|350px| '''An antibody.''' The variable structure of both the light and heavy chains determines which antigen it will bind to. ]]
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antibody-antigen====
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref name="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor where a solution changes color because of LSPR when antigenes are attached to the antibodies on the particles surface.

====Hormone-receptor====
Similar to antibody-antigen, the reactions between hormones and receptors are highly specific. Hormones are chemicals released by cells that affects other celltypes. The cells respond to a hormone if it has the given receptor. This can be used with functionalized nano-particles to produce cell-specific reactions.

====Enzymes====

==Applications==
===Desired properties===
A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution was still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets was introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Challenges===

It may sound like these new sensor have changed the entire field of biosensors, but that is not the case. The techniques and sensors presented here are still in development. Although there are some biomolecule sensors out there<ref>[http://www.biacore.com/lifesciences/index.html Swedish based BIAcore]</ref> the challenges with these techniques are massive:
*Functionalization of nano-particles. How do we attach biomolecules on nano-particles?
*Stability over time. How long can the probes/sensors be stored?
*Re-use.
*Costs.
*Nano-particles inside our bodies.
*Durability. The ability to stay intact in the crowded environment of our bodies.

Some of these questions remains unanswered, but the future looks brigth:
''Molecular beacons are expected to be capable of functioning as highly specific, highly sensitive recognition and signaling elements in state-of-the art biological detection strategies for biomolecules''<ref>[http://www3.interscience.wiley.com/cgi-bin/fulltext/71005735/PDFSTART Molecular Beacons: A Novel DNA Probe for Nucleic Acid and Protein Studies].</ref>

If these challenges are met, it could [http://en.wikipedia.org/wiki/Utopia#Scientific_and_technological_utopia one day] be possible to have not only sensors that can simultaneously detect several diseases at once, but even bring drugs to the exact location of damaged cells or bacterial colonies.

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-23T09:51:15Z

Bjornoy: /* Challenges */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information. The number of combinations between different nano-particles and biomolecules is vast. Therefore the main focus of this article is DNA-NP and enzyme-NP structures.
====DNA====
[[Bilde:Antibody.gif|right|thumb|350px| '''An antibody.''' The variable structure of both the light and heavy chains determines which antigen it will bind to. ]]
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antibody-antigen====
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref name="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor where a solution changes color because of LSPR when antigenes are attached to the antibodies on the particles surface.

====Hormone-receptor====
Similar to antibody-antigen, the reactions between hormones and receptors are highly specific. Hormones are chemicals released by cells that affects other celltypes. The cells respond to a hormone if it has the given receptor. This can be used with functionalized nano-particles to produce cell-specific reactions.

====Enzymes====

==Applications==
===Desired properties===
A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution was still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets was introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Challenges===

It may sound like these new sensor have changed the entire field of biosensors, but that is not the case. The techniques and sensors presented here are still in development. Although there are some biomolecule sensors out there<ref>[http://www.biacore.com/lifesciences/index.html Swedish based BIAcore]</ref> the challenges with these techniques are massive:
*Functionalization of nano-particles. How do we attach biomolecules on nano-particles?
*Stability over time. How long can the probes/sensors be stored?
*Re-use.
*Costs.
*Nano-particles inside our bodies.
*Durability. The ability to stay intact in the crowded environment of our bodies.

Some of these questions remains unanswered, but the future looks brigth:
''Molecular beacons are expected to be capable of functioning as highly specific, highly sensitive recognition and signaling elements in state-of-the art biological detection strategies for biomolecules''<ref>[http://www3.interscience.wiley.com/cgi-bin/fulltext/71005735/PDFSTART Molecular Beacons: A Novel DNA Probe for Nucleic Acid and Protein Studies].</ref>

If these challenges are met, it could one day be possible to have not only sensors that can simultaneously detect several diseases at once, but even bring drugs to the exact location of damaged cells or bacterial colonies.

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-23T09:26:24Z

Bjornoy: /* Challenges */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information. The number of combinations between different nano-particles and biomolecules is vast. Therefore the main focus of this article is DNA-NP and enzyme-NP structures.
====DNA====
[[Bilde:Antibody.gif|right|thumb|350px| '''An antibody.''' The variable structure of both the light and heavy chains determines which antigen it will bind to. ]]
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antibody-antigen====
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref name="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor where a solution changes color because of LSPR when antigenes are attached to the antibodies on the particles surface.

====Hormone-receptor====
Similar to antibody-antigen, the reactions between hormones and receptors are highly specific. Hormones are chemicals released by cells that affects other celltypes. The cells respond to a hormone if it has the given receptor. This can be used with functionalized nano-particles to produce cell-specific reactions.

====Enzymes====

==Applications==
===Desired properties===
A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution was still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets was introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Challenges===

It may sound like these new sensor have changed the entire field of biosensors, but that is not the case. The techniques and sensors presented here are still in development. Although there are some biomolecule sensors out there<ref>[http://www.biacore.com/lifesciences/index.html Swedish based BIAcore]</ref> the challenges with these techniques are massive:
*Functionalization of nano-particles. How do we attach biomolecules on nano-particles?
*Stability over time. How long can the probes/sensors be stored?
*Re-use.
*Costs.

Some of these questions remains unanswered, but the future looks brigth:
''Molecular beacons are expected to be capable of functioning as highly specific, highly sensitive recognition and signaling elements in state-of-the art biological detection strategies for biomolecules''<ref>[http://www3.interscience.wiley.com/cgi-bin/fulltext/71005735/PDFSTART Molecular Beacons: A Novel DNA Probe for Nucleic Acid and Protein Studies].</ref>

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-23T09:25:49Z

Bjornoy: /* Advantages and disadvantages */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information. The number of combinations between different nano-particles and biomolecules is vast. Therefore the main focus of this article is DNA-NP and enzyme-NP structures.
====DNA====
[[Bilde:Antibody.gif|right|thumb|350px| '''An antibody.''' The variable structure of both the light and heavy chains determines which antigen it will bind to. ]]
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antibody-antigen====
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref name="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor where a solution changes color because of LSPR when antigenes are attached to the antibodies on the particles surface.

====Hormone-receptor====
Similar to antibody-antigen, the reactions between hormones and receptors are highly specific. Hormones are chemicals released by cells that affects other celltypes. The cells respond to a hormone if it has the given receptor. This can be used with functionalized nano-particles to produce cell-specific reactions.

====Enzymes====

==Applications==
===Desired properties===
A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution was still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets was introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Challenges===

It may sound like these new sensor have changed the entire field of biosensors, but that is not the case. The techniques and sensors presented here are still in development. Although there are some biomolecule sensors out there[http://www.biacore.com/lifesciences/index.html Swedish based BIAcore] the challenges with these techniques are massive:
*Functionalization of nano-particles. How do we attach biomolecules on nano-particles?
*Stability over time. How long can the probes/sensors be stored?
*Re-use.
*Costs.

Some of these questions remains unanswered, but the future looks brigth:
''Molecular beacons are expected to be capable of functioning as highly specific, highly sensitive recognition and signaling elements in state-of-the art biological detection strategies for biomolecules''<ref>[http://www3.interscience.wiley.com/cgi-bin/fulltext/71005735/PDFSTART Molecular Beacons: A Novel DNA Probe for Nucleic Acid and Protein Studies].</ref>

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-23T08:48:46Z

Bjornoy: /* Advantages and disadvantages */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information. The number of combinations between different nano-particles and biomolecules is vast. Therefore the main focus of this article is DNA-NP and enzyme-NP structures.
====DNA====
[[Bilde:Antibody.gif|right|thumb|350px| '''An antibody.''' The variable structure of both the light and heavy chains determines which antigen it will bind to. ]]
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antibody-antigen====
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref name="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor where a solution changes color because of LSPR when antigenes are attached to the antibodies on the particles surface.

====Hormone-receptor====
Similar to antibody-antigen, the reactions between hormones and receptors are highly specific. Hormones are chemicals released by cells that affects other celltypes. The cells respond to a hormone if it has the given receptor. This can be used with functionalized nano-particles to produce cell-specific reactions.

====Enzymes====

==Applications==
===Desired properties===
A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution was still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets was introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Advantages and disadvantages===
*Combining molecular beacon with tuned fluorescent NP -> lab-on-a-chip
*Difficult to functionalize nano-particle surfaces
*stability
''Molecular beacons are expected to be capable of functioning as highly specific, highly sensitive recognition and signaling elements in state-of-the art biological detection strategies for biomolecules''<ref>[http://www3.interscience.wiley.com/cgi-bin/fulltext/71005735/PDFSTART Molecular Beacons: A Novel DNA Probe for Nucleic Acid and Protein Studies].</ref>

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-23T08:48:21Z

Bjornoy: /* Advantages and disadvantages */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information. The number of combinations between different nano-particles and biomolecules is vast. Therefore the main focus of this article is DNA-NP and enzyme-NP structures.
====DNA====
[[Bilde:Antibody.gif|right|thumb|350px| '''An antibody.''' The variable structure of both the light and heavy chains determines which antigen it will bind to. ]]
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antibody-antigen====
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref name="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor where a solution changes color because of LSPR when antigenes are attached to the antibodies on the particles surface.

====Hormone-receptor====
Similar to antibody-antigen, the reactions between hormones and receptors are highly specific. Hormones are chemicals released by cells that affects other celltypes. The cells respond to a hormone if it has the given receptor. This can be used with functionalized nano-particles to produce cell-specific reactions.

====Enzymes====

==Applications==
===Desired properties===
A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution was still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets was introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Advantages and disadvantages===
*Combining molecular beacon with tuned fluorescent NP -> lab-on-a-chip
*Difficult to functionalize nano-particle surfaces
*stability
''Molecular beacons are expected to be capable of functioning as highly specific, highly sensitive recognition and signaling elements in state-of-the art biological detection strategies for biomolecules''<ref>[http://www3.interscience.wiley.com/cgi-bin/fulltext/71005735/PDFSTART Molecular Beacons: A Novel DNA Probe for Nucleic Acid and Protein Studies].

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-23T08:47:52Z

Bjornoy: /* Advantages and disadvantages */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information. The number of combinations between different nano-particles and biomolecules is vast. Therefore the main focus of this article is DNA-NP and enzyme-NP structures.
====DNA====
[[Bilde:Antibody.gif|right|thumb|350px| '''An antibody.''' The variable structure of both the light and heavy chains determines which antigen it will bind to. ]]
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antibody-antigen====
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref name="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor where a solution changes color because of LSPR when antigenes are attached to the antibodies on the particles surface.

====Hormone-receptor====
Similar to antibody-antigen, the reactions between hormones and receptors are highly specific. Hormones are chemicals released by cells that affects other celltypes. The cells respond to a hormone if it has the given receptor. This can be used with functionalized nano-particles to produce cell-specific reactions.

====Enzymes====

==Applications==
===Desired properties===
A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution was still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets was introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Advantages and disadvantages===
*Combining molecular beacon with tuned fluorescent NP -> lab-on-a-chip
*Difficult to functionalize nano-particle surfaces
*stability
''Molecular beacons are expected to be capable of functioning
as highly specific, highly sensitive recognition and signaling
elements in state-of-the-art biological detection strategies for
biomolecules''<ref>[http://www3.interscience.wiley.com/cgi-bin/fulltext/71005735/PDFSTART Molecular Beacons: A Novel DNA Probe for Nucleic Acid and Protein Studies].

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-23T08:27:35Z

Bjornoy: /* LSPR Sensor */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information. The number of combinations between different nano-particles and biomolecules is vast. Therefore the main focus of this article is DNA-NP and enzyme-NP structures.
====DNA====
[[Bilde:Antibody.gif|right|thumb|350px| '''An antibody.''' The variable structure of both the light and heavy chains determines which antigen it will bind to. ]]
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antibody-antigen====
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref name="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor where a solution changes color because of LSPR when antigenes are attached to the antibodies on the particles surface.

====Hormone-receptor====
Similar to antibody-antigen, the reactions between hormones and receptors are highly specific. Hormones are chemicals released by cells that affects other celltypes. The cells respond to a hormone if it has the given receptor. This can be used with functionalized nano-particles to produce cell-specific reactions.

====Enzymes====

==Applications==
===Desired properties===
A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution was still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets was introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Advantages and disadvantages===
*Combining molecular beacon with tuned fluorescent NP -> lab-on-a-chip
*Difficult to functionalize nano-particle surfaces
*stability

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-22T16:47:30Z

Bjornoy: /* LSPR Sensor */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information. The number of combinations between different nano-particles and biomolecules is vast. Therefore the main focus of this article is DNA-NP and enzyme-NP structures.
====DNA====
[[Bilde:Antibody.gif|right|thumb|350px| '''An antibody.''' The variable structure of both the light and heavy chains determines which antigen it will bind to. ]]
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antibody-antigen====
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref name="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor where a solution changes color because of LSPR when antigenes are attached to the antibodies on the particles surface.

====Hormone-receptor====
Similar to antibody-antigen, the reactions between hormones and receptors are highly specific. Hormones are chemicals released by cells that affects other celltypes. The cells respond to a hormone if it has the given receptor. This can be used with functionalized nano-particles to produce cell-specific reactions.

====Enzymes====

==Applications==
===Desired properties===
A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution was still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Advantages and disadvantages===
*Combining molecular beacon with tuned fluorescent NP -> lab-on-a-chip
*Difficult to functionalize nano-particle surfaces
*stability

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-22T16:42:26Z

Bjornoy: /* Advantages and disadvantages */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information. The number of combinations between different nano-particles and biomolecules is vast. Therefore the main focus of this article is DNA-NP and enzyme-NP structures.
====DNA====
[[Bilde:Antibody.gif|right|thumb|350px| '''An antibody.''' The variable structure of both the light and heavy chains determines which antigen it will bind to. ]]
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antibody-antigen====
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref name="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor where a solution changes color because of LSPR when antigenes are attached to the antibodies on the particles surface.

====Hormone-receptor====
Similar to antibody-antigen, the reactions between hormones and receptors are highly specific. Hormones are chemicals released by cells that affects other celltypes. The cells respond to a hormone if it has the given receptor. This can be used with functionalized nano-particles to produce cell-specific reactions.

====Enzymes====

==Applications==
===Desired properties===
A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Advantages and disadvantages===
*Combining molecular beacon with tuned fluorescent NP -> lab-on-a-chip
*Difficult to functionalize nano-particle surfaces
*stability

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-22T15:37:44Z

Bjornoy: /* Antigen-antibody */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information. The number of combinations between different nano-particles and biomolecules is vast. Therefore the main focus of this article is DNA-NP and enzyme-NP structures.
====DNA====
[[Bilde:Antibody.gif|right|thumb|350px| '''An antibody.''' The variable structure of both the light and heavy chains determines which antigen it will bind to. ]]
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antibody-antigen====
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref name="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor where a solution changes color because of LSPR when antigenes are attached to the antibodies on the particles surface.

====Hormone-receptor====
Similar to antibody-antigen, the reactions between hormones and receptors are highly specific. Hormones are chemicals released by cells that affects other celltypes. The cells respond to a hormone if it has the given receptor. This can be used with functionalized nano-particles to produce cell-specific reactions.

====Enzymes====

==Applications==
===Desired properties===
A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Advantages and disadvantages===
Combining molecular beacon with tuned fluorescent NP -> lab-on-a-chip

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-22T15:08:49Z

Bjornoy: /* Advantages and disadvantages */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information. The number of combinations between different nano-particles and biomolecules is vast. Therefore the main focus of this article is DNA-NP and enzyme-NP structures.
====DNA====
[[Bilde:Antibody.gif|right|thumb|350px| '''An antibody.''' The variable structure of both the light and heavy chains determines which antigen it will bind to. ]]
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antigen-antibody====
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref name="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor where a solution changes color because of LSPR when antigenes are attached to the antibodies on the particles surface.
====Hormone-receptor====
Similar to antibody-antigen, the reactions between hormones and receptors are highly specific. Hormones are chemicals released by cells that affects other celltypes. The cells respond to a hormone if it has the given receptor. This can be used with functionalized nano-particles to produce cell-specific reactions.

====Enzymes====

==Applications==
===Desired properties===
A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Advantages and disadvantages===
Combining molecular beacon with tuned fluorescent NP -> lab-on-a-chip

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-22T14:04:31Z

Bjornoy: /* Desired properties */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information. The number of combinations between different nano-particles and biomolecules is vast. Therefore the main focus of this article is DNA-NP and enzyme-NP structures.
====DNA====
[[Bilde:Antibody.gif|right|thumb|350px| '''An antibody.''' The variable structure of both the light and heavy chains determines which antigen it will bind to. ]]
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antigen-antibody====
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref name="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor where a solution changes color because of LSPR when antigenes are attached to the antibodies on the particles surface.
====Hormone-receptor====
Similar to antibody-antigen, the reactions between hormones and receptors are highly specific. Hormones are chemicals released by cells that affects other celltypes. The cells respond to a hormone if it has the given receptor. This can be used with functionalized nano-particles to produce cell-specific reactions.

====Enzymes====

==Applications==
===Desired properties===
A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-22T14:03:48Z

Bjornoy: /* Hormone-receptor */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information. The number of combinations between different nano-particles and biomolecules is vast. Therefore the main focus of this article is DNA-NP and enzyme-NP structures.
====DNA====
[[Bilde:Antibody.gif|right|thumb|350px| '''An antibody.''' The variable structure of both the light and heavy chains determines which antigen it will bind to. ]]
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antigen-antibody====
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref name="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor where a solution changes color because of LSPR when antigenes are attached to the antibodies on the particles surface.
====Hormone-receptor====
Similar to antibody-antigen, the reactions between hormones and receptors are highly specific. Hormones are chemicals released by cells that affects other celltypes. The cells respond to a hormone if it has the given receptor. This can be used with functionalized nano-particles to produce cell-specific reactions.

====Enzymes====

==Applications==
===Desired properties===
*What would we like to measure?
*What properties would we like our sensors to have?
*etc

A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-22T13:58:13Z

Bjornoy: /* Bio-molecules */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information. The number of combinations between different nano-particles and biomolecules is vast. Therefore the main focus of this article is DNA-NP and enzyme-NP structures.
====DNA====
[[Bilde:Antibody.gif|right|thumb|350px| '''An antibody.''' The variable structure of both the light and heavy chains determines which antigen it will bind to. ]]
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antigen-antibody====
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref name="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor where a solution changes color because of LSPR when antigenes are attached to the antibodies on the particles surface.
====Hormone-receptor====
====Enzymes====

==Applications==
===Desired properties===
*What would we like to measure?
*What properties would we like our sensors to have?
*etc

A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-22T13:50:01Z

Bjornoy: /* Bio-molecules */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information.
====DNA====
[[Bilde:Antibody.gif|right|thumb|350px| '''An antibody.''' The variable structure of both the light and heavy chains determines which antigen it will bind to. ]]
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antigen-antibody====
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref name="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor where a solution changes color because of LSPR when antigenes are attached to the antibodies on the particles surface.
====Hormone-receptor====
====Enzymes====

==Applications==
===Desired properties===
*What would we like to measure?
*What properties would we like our sensors to have?
*etc

A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-22T13:48:29Z

Bjornoy: /* DNA */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information.
====DNA====
[[Bilde:Antibody.gif|right|thumb|350px| '''An antibody.''' The variable structure of both the light and heavy chains determines which antigen it will bind to. ]]
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antigen-antibody====
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref name="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor where a solution changes color because of LSPR when antigenes are attached to the antibodies on the particles surface.

==Applications==
===Desired properties===
*What would we like to measure?
*What properties would we like our sensors to have?
*etc

A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-22T13:48:17Z

Bjornoy: /* Antigen-antibody */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information.
====DNA====
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antigen-antibody====
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref name="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor where a solution changes color because of LSPR when antigenes are attached to the antibodies on the particles surface.

==Applications==
===Desired properties===
*What would we like to measure?
*What properties would we like our sensors to have?
*etc

A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-22T13:47:48Z

Bjornoy: /* Antigen-antibody */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information.
====DNA====
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antigen-antibody====
[[Bilde:Antibody.gif|right|thumb|350px| '''An antibody.''' The variable structure of both the light and heavy chains determines which antigen it will bind to. ]]
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref name="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor where a solution changes color because of LSPR when antigenes are attached to the antibodies on the particles surface.

==Applications==
===Desired properties===
*What would we like to measure?
*What properties would we like our sensors to have?
*etc

A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-22T13:47:21Z

Bjornoy: /* Antigen-antibody */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information.
====DNA====
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antigen-antibody====
[[Bilde:Antigen.gif|right|thumb|350px| '''An antibody.''' The variable structure of both the light and heavy chains determines which antigen it will bind to. ]]
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref name="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor where a solution changes color because of LSPR when antigenes are attached to the antibodies on the particles surface.

==Applications==
===Desired properties===
*What would we like to measure?
*What properties would we like our sensors to have?
*etc

A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />

Fil:Antibody.gif

2009-03-22T13:44:45Z

Bjornoy:

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-22T13:44:00Z

Bjornoy: /* Antigen-antibody */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information.
====DNA====
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antigen-antibody====
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref name="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor that changes color because of LSPR when antigenes are attached to the antibodies.

==Applications==
===Desired properties===
*What would we like to measure?
*What properties would we like our sensors to have?
*etc

A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-22T13:43:36Z

Bjornoy: /* Antigen-antibody */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Introduction==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information.
====DNA====
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antigen-antibody====
Antibodies are proteinstructures that makes up part of our immune system<ref>[http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html Antibodies explaines by the University of Arizona]</ref>. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific<ref="katz" />. Covering a ~15 nm nano-particle with antibodies could make a sensor that changes color because of LSPR when antigenes are attached to the antibodies.

==Applications==
===Desired properties===
*What would we like to measure?
*What properties would we like our sensors to have?
*etc

A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-22T13:40:09Z

Bjornoy: /* DNA */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Intro==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information.
====DNA====
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antigen-antibody====
Antibodies are proteinstructures that makes up part of our immune system. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific. Covering a ~15 nm nano-particle with antibodies could make a sensor that changes color because of LSPR when antigenes are attached to the antibodies.

==Applications==
===Desired properties===
*What would we like to measure?
*What properties would we like our sensors to have?
*etc

A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-22T13:39:31Z

Bjornoy: /* Bio-molecules */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Intro==
This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, in this article we choose to examine sensors that ''transduce biological phenomena into optical and electronic information''. Therefore, in order to properly evaluate the topic at hand, an introduction to the specific properties of nanoparticles and biomaterials will be given in the following section, before evaluating examples where these properties have been implemented in practice. Finally a short discussion of the opportunities these technologies pose will be presented.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
The use of bio-molecules in sensors is interesting because of the specific recognition interactions many of these molecules have. For example the strong complementary hybridization of DNA, the interactions between antigen-antibody or hormone-receptor and the specific catalytic properties of enzymes. Functionalization of nano-particles i.e. attaching DNA- or antigen-probes on the surface, leads to self-assembly of the nano-particles and changes in their properties. In other words, a way of reading the information.
====DNA====
=====Structure and Favourable properties=====
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

====Antigen-antibody====
Antibodies are proteinstructures that makes up part of our immune system. The Y-structure contains two heavy chains and to light chains where the variable parts on top of each chain determines which antigen it hybridizes with. These binding are very specific. Covering a ~15 nm nano-particle with antibodies could make a sensor that changes color because of LSPR when antigenes are attached to the antibodies.

==Applications==
===Desired properties===
*What would we like to measure?
*What properties would we like our sensors to have?
*etc

A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-22T12:39:27Z

Bjornoy: /* Desired properties */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Intro==
*How we interpreted the research question: applications of biomolecule-nanoparticle structures on surfaces for use as sensors. Therefore a very wide field.
*The sensors studied in this discussion
*Why Nanoparticles and biomolecules?

This article discusses the topic of ''Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors''. The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, sensors that transduce biological phenomena into optical and electronic information has been an area of much research.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
====DNA====
=====Structure and Favourable properties=====
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

==Applications==
===Desired properties===
*What would we like to measure?
*What properties would we like our sensors to have?
*etc

A sensor may be defined as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''<ref>[http://www.websters-online-dictionary.org/definition/sensor Websters dictionary] </ref>". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

====DNA connected to CNT====

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-22T12:37:24Z

Bjornoy: /* Desired properties */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Intro==
*How we interpreted the research question: applications of biomolecule-nanoparticle structures on surfaces for use as sensors. Therefore a very wide field.
*The sensors studied in this discussion
*Why Nanoparticles and biomolecules?

This article discusses the topic of "Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors". The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, sensors that transduce biological phenomena into optical and electronic information has been an area of much research.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
====DNA====
=====Structure and Favourable properties=====
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

==Applications==
===Desired properties===
*What would we like to measure?
*What properties would we like our sensors to have?
*etc

Websters dictionary [http://www.websters-online-dictionary.org/definition/sensor defines a sensor] as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

====DNA connected to CNT====

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-22T12:35:49Z

Bjornoy: /* Desired properties */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Intro==
*How we interpreted the research question: applications of biomolecule-nanoparticle structures on surfaces for use as sensors. Therefore a very wide field.
*The sensors studied in this discussion
*Why Nanoparticles and biomolecules?

This article discusses the topic of "Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors". The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, sensors that transduce biological phenomena into optical and electronic information has been an area of much research.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
====DNA====
=====Structure and Favourable properties=====
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

==Applications==
===Desired properties===
*What would we like to measure?
*What properties would we like our sensors to have?
*etc

Websters dictionary [http://www.websters-online-dictionary.org/definition/sensor defines a sensor] as "''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it''". Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In this article we looked at sensors that utilize biomolecules for recognition combined with nano-particles for signalling. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

====DNA connected to CNT====

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-22T12:35:25Z

Bjornoy: /* Desired properties */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Intro==
*How we interpreted the research question: applications of biomolecule-nanoparticle structures on surfaces for use as sensors. Therefore a very wide field.
*The sensors studied in this discussion
*Why Nanoparticles and biomolecules?

This article discusses the topic of "Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors". The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, sensors that transduce biological phenomena into optical and electronic information has been an area of much research.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
====DNA====
=====Structure and Favourable properties=====
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

==Applications==
===Desired properties===
*What would we like to measure?
*What properties would we like our sensors to have?
*etc

Websters dictionary [http://www.websters-online-dictionary.org/definition/sensor defines a sensor] as ''any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it.'' Herein we look at sensors that either detect or measure a biological phenomena and tranduce this into a readable signal. In this article we looked at sensors that utilize biomolecules for recognition combined with nano-particles for signalling. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

====DNA connected to CNT====

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-22T12:26:44Z

Bjornoy: /* Desired properties */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Intro==
*How we interpreted the research question: applications of biomolecule-nanoparticle structures on surfaces for use as sensors. Therefore a very wide field.
*The sensors studied in this discussion
*Why Nanoparticles and biomolecules?

This article discusses the topic of "Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors". The convergence of biotechnology and nanotechnology have over recent years allowed for the incorporation of the unique optical and electronic properties of nanoparticles with the selective catalytic and recognititon properties of biomaterials <ref name="katz"> [http://www3.interscience.wiley.com/cgi-bin/fulltext/109785984/PDFSTART Eugenii Katz and Itamar Willner ''Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications''] </ref>. Hence, sensors that transduce biological phenomena into optical and electronic information has been an area of much research.

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
====DNA====
=====Structure and Favourable properties=====
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

==Applications==
===Desired properties===
*What would we like to measure?
*What properties would we like our sensors to have?
*etc

A sensor is a device that either detects or measures something. In this article we looked at sensors that utilize biomolecules for recognition combined with nano-particles for signalling. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

====DNA connected to CNT====

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-22T12:21:54Z

Bjornoy: /* Theory */

<Big> Under construction. Please do not edit until after we have been assessed for our literature project.</Big>
-Sindre and Christopher
==Intro==
*How we interpreted the research question: applications of biomolecule-nanoparticle structures on surfaces for use as sensors. Therefore a very wide field.
*The sensors studied in this discussion
*Why Nanoparticles and biomolecules?

==Theory==
===Nanoparticle===

====Favourable characteristics: Tuneability====
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of these properties and some applications will be discussed below.

====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. As is visualised for a semiconductor material in the figure to the right, nanoparticles lie in between the atomic and molecular region of discrete and continuous density of state regions respectively. Moving from right to left in the figure (this may be thought of as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center of the band first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>. It should be noted that this effect occurs most readily for semiconductor nanoparticles (that is, at greater size dimensions for semiconductor nanoparticles) as the fermienergi of these are placed between the HOMO-LUMO bands. For details on optical properties of non-semiconductor nanoparticles you are referred to the sources used in this section <ref name="pers"/>.

Nanoparticles electrical transport properties also depend strongly on size. In a nanocrystal, the presence of one charge acts to prevent the addition of the other <ref name="pers"/>. Due to this "Coulumb blockade" the current-voltage curves of individual crystallites resemble a staircase <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Thermodynamic properties of nanoparticles====
Many of the special properties found in nanoparticles is a result of the large fraction of the atoms located at the surface compared to that of a large bulk material. This is because there is a large energy associated with surface atoms arising from the fact that surface atoms tend to "..be coordinately unsaturated" <ref name="pers"/>. This may help to explain why melting points for nanoparticles decrease with the size. There is less surface energy in the liquid phase than in the solid state. As the surface area of a constant mass of nanoparticles increase as their size decreases, the energy difference between the solid state and liquid phase increases.

===Bio-molecules===
====DNA====
=====Structure and Favourable properties=====
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

==Applications==
===Desired properties===
*What would we like to measure?
*What properties would we like our sensors to have?
*etc

A sensor is a device that either detects or measures something. In this article we looked at sensors that utilize biomolecules for recognition combined with nano-particles for signaling. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles as fluorescent labels====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] the nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the [http://en.wikipedia.org/wiki/F-actin F-actin] filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

====DNA connected to CNT====

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-21T19:46:38Z

Bjornoy: /* Electrical properties */

Under construction. Please do not edit
==Intro==
*How we interpreted the research question: applications of biomolecule-nanoparticle structures on surfaces for use as sensors. Therefore a very wide field.
*The sensors studied in this discussion
*Why Nanoparticles and biomolecules?

==Theory==
===Nanoparticle===
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of the properties that may be altered will be discussed below.
====Optical and Electrical properties of Semiconductor Nanoparticles====
[[Bilde:Bandgaps2.jpg|right|thumb|300px|''' Density of states in a semiconductor material''' The fermi level is situated between the bands in the semiconductor materials. As the dimensions of the semiconductor decrease the band gap increases, therefore corresponding to higher excitation energy.
(Source: A. P. Alivisatos ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals'' <ref name="pers"/>)]]

The size dependance of optical and electrical properties of nanoparticles may be explained qualitatively by simple band gap theory. Nanoparticles lie in between the atomic and molecular limit of discrete and continuous density of state regions respectively, as is visualised for a semiconductor material in the figure to the right. Moving from right to left in the figure (this may be visualised as moving single atoms together to a large bulk material) one may see that a band of density of states develops at the center first and the edges develop last as. Therefore, in the case of the semiconductor nanoparticle, the bandgap between the unfilled and filled band (HOMO-LUMO) increases as the size of the particles decrease. This is because less states are available at the edges as the particle size decreases. The excitation energy therefore increases as the size of the semiconductor nanoparticle decreases. Optical excitations across the gap therefore depend strongly on the size, even for crystallites as large as 10000 atoms <ref name="pers"/>.

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Chemical properties====

====Advantages====

===DNA===
====Properties====
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

====Advantages====
The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

==Applications==
===Desired properties===
*What would we like to measure?
*What properties would we like our sensors to have?
*etc

A sensor is a device that either detects or measures something. In this article we looked at sensors that utilize biomolecules for recognition combined with nano-particles for signaling. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] The nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the F-actin filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

====DNA connected to CNT====

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-21T18:53:33Z

Bjornoy: /* Desired properties */

Under construction. Please do not edit
==Intro==
*How we interpreted the research question: applications of biomolecule-nanoparticle structures on surfaces for use as sensors. Therefore a very wide field.
*The sensors studied in this discussion
*Why Nanoparticles and biomolecules?

==Theory==
===Nanoparticle===
The properties of solids change when their size dimensions fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>. Over the past years tailoring of material characteristics by size control has been demonstrated. For instance, the semiconductor material CdS has an enourmous range of fundamental properties all realized by altering the size; band gap sizes, conductivity, melting temperature, and so on <ref name="pers"/>. It is the tuneability of these properties that render nanoparticles as interesting components for bio-sensor technology. Some of the properties that may be altered will be discussed below.
====Optical and Electrical properties of Semiconductor Nanoparticles====

=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Chemical properties====
====Electrical properties====
====Advantages====

===DNA===
====Properties====
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

====Advantages====
The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

==Applications==
===Desired properties===
*What would we like to measure?
*What properties would we like our sensors to have?
*etc

A sensor is a device that either detects or measures something. In this article we looked at sensors that utilize biomolecules for recognition combined with nano-particles for signaling. In the future this type of sensors may be able to perform diagnostics and/or localization of damaged cells. Combined with some kind of medicine carrier vessel they may even be able to diagnose and repair damaged cells in one step. The use of nucleotide sensors could give us the ability to search for specific genes in our DNA. The possibilities are only limited to our imagination. Such sensors are still part of science fiction, but the fiction part is decreasing. The method of using natures own molecules incorporated in sensors, grants us the ability to study reactions and processes at molecular level. Evolution has refined different types of molecules to match each other perfectly. This specificity is ideal for sensors and location-specific medicine.

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] The nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the F-actin filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

====DNA connected to CNT====

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-21T18:16:05Z

Bjornoy: /* Semiconductor Nanoparticles */

Under construction. Please do not edit
==Intro==
*How we interpreted the research question: applications of biomolecule-nanoparticle structures on surfaces for use as sensors. Therefore a very wide field.
*The sensors studied in this discussion
*Why Nanoparticles and biomolecules?

==Theory==
===Nanoparticle===
The properties of solids change when their sizes fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>.
====Optical properties====
=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Chemical properties====
====Electrical properties====
====Advantages====

===DNA===
====Properties====
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

====Advantages====
The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

==Applications==
===Desired properties===
*What would we like to measure?
*What properties would we like our sensors to have?
*etc

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] The nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the F-actin filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|350px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

====DNA connected to CNT====

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-21T18:15:48Z

Bjornoy: /* Semiconductor Nanoparticles */

Under construction. Please do not edit
==Intro==
*How we interpreted the research question: applications of biomolecule-nanoparticle structures on surfaces for use as sensors. Therefore a very wide field.
*The sensors studied in this discussion
*Why Nanoparticles and biomolecules?

==Theory==
===Nanoparticle===
The properties of solids change when their sizes fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>.
====Optical properties====
=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Chemical properties====
====Electrical properties====
====Advantages====

===DNA===
====Properties====
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

====Advantages====
The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

==Applications==
===Desired properties===
*What would we like to measure?
*What properties would we like our sensors to have?
*etc

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] The nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the F-actin filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|300px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

====DNA connected to CNT====

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-21T18:15:31Z

Bjornoy: /* Semiconductor Nanoparticles */

Under construction. Please do not edit
==Intro==
*How we interpreted the research question: applications of biomolecule-nanoparticle structures on surfaces for use as sensors. Therefore a very wide field.
*The sensors studied in this discussion
*Why Nanoparticles and biomolecules?

==Theory==
===Nanoparticle===
The properties of solids change when their sizes fall below 10nm <ref name="pers"> [http://pubs.acs.org/doi/abs/10.1021/jp9535506 A. P. Alivisatos Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ''Perspectives on the Physical Chemistry of Semiconductor Nanocrystals''] </ref>.
====Optical properties====
=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Chemical properties====
====Electrical properties====
====Advantages====

===DNA===
====Properties====
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

====Advantages====
The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

==Applications==
===Desired properties===
*What would we like to measure?
*What properties would we like our sensors to have?
*etc

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] The nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the F-actin filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |right|thumb|300px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

[[Bilde:spacer.JPG]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

====DNA connected to CNT====

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-21T16:19:25Z

Bjornoy: /* LSPR Sensor */

Under construction. Please do not edit
==Intro==
*How we interpreted the research question
*The sensors studied in this discussion
*Why Nanoparticles and biomolecules?

==Theory==
===Nanoparticle===
====Optical properties====
=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Chemical properties====
====Electrical properties====
====Advantages====
===DNA===
====Properties====
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

====Advantages====
The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

==Applications==
===Desired properties===
*What would we like to measure?
*What properties would we like our sensors to have?
*etc

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.

[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] The nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the F-actin filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |center|thumb|300px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

====DNA connected to CNT====

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-21T16:19:13Z

Bjornoy: /* LSPR Sensor */

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-21T16:10:00Z

Bjornoy: /* LSPR Sensor */

Under construction. Please do not edit
==Intro==
*How we interpreted the research question
*The sensors studied in this discussion
*Why Nanoparticles and biomolecules?

==Theory==
===Nanoparticle===
====Optical properties====
=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Chemical properties====
====Electrical properties====
====Advantages====
===DNA===
====Properties====
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

====Advantages====
The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

==Applications==
===Desired properties===
*What would we like to measure?
*What properties would we like our sensors to have?
*etc

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted.

When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.
[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]

A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] The nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the F-actin filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |center|thumb|300px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

====DNA connected to CNT====

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-21T15:25:01Z

Bjornoy: /* Semiconductor Nanoparticles */

Under construction. Please do not edit
==Intro==
*How we interpreted the research question
*The sensors studied in this discussion
*Why Nanoparticles and biomolecules?

==Theory==
===Nanoparticle===
====Optical properties====
=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Chemical properties====
====Electrical properties====
====Advantages====
===DNA===
====Properties====
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

====Advantages====
The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

==Applications==
===Desired properties===
*What would we like to measure?
*What properties would we like our sensors to have?
*etc

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted. When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion
band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.
[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|350px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] The nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the F-actin filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |center|thumb|300px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

====DNA connected to CNT====

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />

Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors

2009-03-21T15:23:42Z

Bjornoy: /* LSPR Sensor */

Under construction. Please do not edit
==Intro==
*How we interpreted the research question
*The sensors studied in this discussion
*Why Nanoparticles and biomolecules?

==Theory==
===Nanoparticle===
====Optical properties====
=====Fluorescence=====
[[Bilde:Emission.jpg|right|thumb|350px|'''Excitation (dashed) and fluorescence (solid) spectra of:''' '''a)'''fluorescein (a common fluorophore) '''b)'''a typical water-soluble nanocrystal (NC) sample in PBS.
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

Fluorescence is a widely used tool in biology. An example of fluorescence is as follows: by exciting an atom with ultraviolet light, it will then undergo a series of transitions to one or more intermediate states as it returns to its ground state. By doing this it emits visible light, which is of less energy <ref name="tipler_fluoro">Paul A. Tipler and Gene Mosca ''Physics for Scientists and Engineers sixth edition''</ref>. Hence one may light a sample with UV light, and the sample will light up in visible light (for an example follow the link: [http://www.youtube.com/watch?v=16PAgK2mBz4 Fluorescent confocal microscopy]).

In fluorescent labeling of biological material, one would like to measure several fluorescence probes simultaneously. Ideal probes for this purpose should therefore emit at spectrally resolvable energies and have a narrow, symmetric [http://en.wikipedia.org/wiki/Emission_spectrum emission spectrum], and the whole group of probes should be exciteable at a single wavelength. However, conventional dye molecules fail to meet these requirements; they have broad emission spectrums and narrow excitation spectrums (as may be seen in figure showing the fluorescence characteristics of fluorescein). Broad emission spectrums make it difficult to identify the different probes since their emission spectrums will overlap. The narrow excitation spectrums makes simultaneous excitation difficult as it is difficult to excite several dye molecules with a single wavelength.

Semiconductor nanocrystals, on the other hand, meet the requirements well as may be seen in the figure showing the fluorescence characteristics of a typical water-soluble nanocrystal to the right. For semiconductor nanoparticles the emission maximum and excitation onset shifts to higher energy with decreasing size of the particle; hence "..resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength <ref name="marcel"/>". It has been observed that by varying the materials used to create the nanoparticle and by varying its size, peak emissions in the spectral range of 400nm to 2 <math>\mu m</math> with typical emission widths of 20 to 30 nm in the visible region of the spectrum are obtained <ref name="marcel"/>. "Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously <ref name="marcel"/>".

Semiconductor nanoparticles have other notable features. They may posess high [http://en.wikipedia.org/wiki/Quantum_yield quantum yields], which is a measure of efficiency, and photochemical stability compared with conventional dye molecules.

=====SPR=====
Surface plasmons are electron-density waves at the surface of metals along the metal and medium interface. When shining light onto the surface, some of these electrones are excited by the photons and some of the lights energy is absorbed<ref>[http://www.astbury.leeds.ac.uk/facil/SPR/spr_intro2004.htm Surface plasmon resonance explained by the Astbury Centre for Structural Molecular Biology]</ref>. A dielectric constant dependent on the surface and environment of the metal determines the resonance frequency of the electron waves, similar to the spring-constant of a physical spring. By altering the surface or the environment of the metal, the resonance frequency is altered and another spectrum of the light is absorbed. This effect is called localized surface plasmon resonance (LSPR) when we talk about particles in the nanometer scale.

====Chemical properties====
====Electrical properties====
====Advantages====
===DNA===
====Properties====
The structure of DNA is typically a double-helix with complementary base-pairs on each side of the helix. It can also be found as more flexible single-stranded DNA (ssDNA) which tends to hybridize with a complementary ssDNA sequence to form a double helix.

====Advantages====
The use of ssDNA is ideal for sensors as the hybridization with targets are both higly tuneable and selective. The ssDNA-probes can be synthesized in great numbers with an exact chosen sequence and due to the chemical properties of the DNA-molecule, hybridization with even a single base-pair difference is detectable (for 15-30'polynucleotides)<ref name="plasmon" /><ref name="singlemismatch"/>. The DNA-NP hybrids are also stable over a long time and in different types of environments such as strong electrolyte concentrations or in solutions containing random sequence DNA<ref name="plasmon" />.

==Applications==
===Desired properties===
*What would we like to measure?
*What properties would we like our sensors to have?
*etc

====Molecular Beacon====
[[Bilde:Hairpin1.JPG|right|thumb|350px| Schematic of the unfolding of the hairpin upon hybridizing with target nucleotide (The gold-particle and the dye are not to scale with the DNA-molecule).]]
A molecular beacon is the use of a single stranded oligonucleotide with a fluorescent chemical at one end, and a quencher at the other, to create a sensor for nucleic acid detection<ref>[http://www.molecular-beacons.org Molecular beacons explained by the University of Medicine of New Jersey.]</ref>. The nucleotide has complementary bases at each end so it forms a hairpin-loop bringing the fluorescent and the quencher in close proximity. When in contact with a target the two single strand nucleotides hybridizes to a double-helix molecule. The double-helix is not as flexible as the single stranded version and thus the loop is straightened, bringing the fluorescent away from the quencher enabling detection. Scientists have developed a hybrid material consisting of a 1.4 nm gold particle, an organic fluorescent dye and a 25-base-nucleotide single-strand DNA molecule<ref name="singlemismatch">[http://www.nature.com/nbt/journal/v19/n4/pdf/nbt0401_365.pdf B.Dubertret, M.Calame, A.J.Libchaber ''Single-mismatch detection using gold-quenched fluorescent oligonucleotides''].</ref> The dye is connected to the 3'end with a primary amine and the gold is connected to the 5'end with a disulfide. The loop is stable at room-temperature, but will easily open when reacted with the target. When in loop-structure, the fluorescent chemical is quenched by 99 % due to proximity to the gold-particle. When the target, a complementary ssDNA, is introduced in a solution containing the sensor, the DNA hybridizes to the loop. As the double-helix structure of DNA is less flexible than the single strand, the sensor straightens and the fluorescent is moved away from the quencher. This enables detection of the target through a spectrofluorometer. To be able to apply this as a sensor we must be able to separate detection of the target from detection of mismatched targets. This is achieved by manipulating the stem-length of the loop. Optimization causes a mismatch to shift towards non-hybridization. Then the ratio between the intensity of the fluorescence between a solution containing targets and mismatched targets and a solution containing mismatched targets only is measured.

<math>R=\frac{(I_p(c_0)+I_m(c_m))}{I_m(c_m)}</math>

<math>\alpha=\frac{c_0}{c_m}</math>

R defines the resolution of the sensor. When <math>R>3</math> the target is detected. This particular sensor has in experiments showed an <math>\alpha<50</math> when <math>R>3</math> meaning the target is detectable if there is one target present per 50 nucleotides. One of the reasons this type of sensor is of interest is that the gold cluster attached at the 5' end is highly tunable. This means we can use different colored dyes for simultaneous targeting of multiple DNA-sequences. Optimists think this type of sensor can be linked to some kind of medicine-carrier that triggers when the sensor hybridizes with the target, enabling diagnostic and treatment in one step.

====LSPR Sensor====
[[Bilde:gullaggregatmindre.jpeg|right|thumb|350px|'''Schematic of two probes and one target.'''The actual sensor will have a lot of these probes connected to the gold-particle and a far greater number than two gold particles will be connected. The dashed lines are the 13' nucleotide spacers. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
Another type of sensor uses a similar set-up as the molecular beacon, but relies on a totally different method of detection. Single-strand DNA with two different sequences is hybridized with gold nano-particles. The two different sequences are complementary to the whole section of target DNA. When the target is present the gold particles will aggregate due to hybridization of the DNA and they will undergo a color-shift caused by surface plasmon resonance. The resonance is dependent on the surface environment and so by changing the environment of the gold-clusters, their color is shifted. When target nucleotide is present, the gold particles are brought in close proximity and the surface plasmon resonance frequency is altered. Advantages with this technique are that the color-shift is visible to the un-aided eye, the sensor has a longer shelf-storage and does not use radioactive components like other sensors for similar purposes<ref name="plasmon" />. A sensor of this type utilizes two 28-base nucleotides as probes to detect a 30-base target. 15 bases on each probe are complementary to the target and the other 13 acts as flexible spacers to separate the gold particles. The gold-particles are 13nm in diameter, chosen because they are fairly easy to produce without deviation in size and because they have a sharp absorbtion
band. This means that small changes in the environment or surface will give a clearly visible change of color. Using a "developing" technique involving freezing and thawing to speed up the process, the solution changes from red to purple/blue.
[[Bilde:color1.JPG|right|thumb|350px| '''Thermal stability of different nucleotides.''' '''A:''' Perfect target, '''B:''' no target, '''C:''' complementary to one probe, '''D:''' 6-bp deletion, '''E:''' 1-bp mismatch, '''F:''' 2-bp mismatch. Temperatures in celsius. (Source: R Elghanian, J Storhoff, R Mucic, R Letsinger, and C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles''.<ref name="plasmon">[http://www.sciencemag.org/cgi/reprint/277/5329/1078.pdf R Elghanian, J Storhoff, R Mucic, R Letsinger, C Mirkin ''Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles'']</ref>)]]
A remarkable feature of this probe is the stability of the hybridized gold aggregates. When heated to 58°C the solution were still blue, but when the temperature reached 59°C it turned pink indicating the gold-particles had been dispersed. The sharp transition from aggregates to dispersion is great for detecting mismatchs. When a solution of single mismatched targets were introduces in the probe solution, the gold-particles were re-dispersed at 53°C. The reason for this is that for shorter polynucleotides, just a single mismatched base-pair is enough to reduce the stability of the molecule noticeably. The probe's sensitivity is not yet confirmed, but the unoptimized probe used in this particular experiment detected 10 fmol of a target in a 2 µl solution. In comparison a former oligonucleotide detection technique using fluorescein detected 500 fmol<ref>[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=336708&blobtype=pdf M.S.Urdea ''et al., Nucleic Acids '''16''',4937(1988)]</ref>

[[Bilde:Spacer.JPG]]

====Semiconductor Nanoparticles====
In 1998 Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alivisatos published the article "Semiconductor Nanocrystals as Fluorescent Biological Labels <ref name="marcel"/>". Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. "The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse [http://en.wikipedia.org/wiki/Fibroblast fibroblasts] <ref name="marcel"/>".

Mouse fibroblasts were stained with two nanoparticles of different sizes, corresponding to red and green. The constituents of these particles are discussed in the next paragraph. The particles were engineered with affinities for different parts of the mouse fibroblast, which resulted in the corresponding image shown to the right; [[Bilde:Fibroblasts.jpg|right|thumb|300px|'''Mouse fibroblasts labelled by CdSe-CdS core-shell nanocrystals enclosed in a silica shell of different sizes.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]] The nucleus was stained by green-colored nanocrystals through electrostatic and hydrogen-bonding interactions between the nucleus and the engineered surface of the nanoparticle. Likewise, the F-actin filaments were labeled with red-colored nanocrystals by a avidin-biotin interaction (a strong and specific interaction between the avidin protein and the biotin vitamin <ref name="avidin">[http://en.wikipedia.org/wiki/Avidin wikipedia article on the avidin protein] </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> [http://support.svi.nl/wiki/WideFieldMicroscope Wide Field Microscope] </ref>) the green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera <ref name="marcel"/>". This therefore demonstrates how semiconductor nanoparticles allows for multicolor labeling taylored for specific colors and specific sites on the sample. However, one may notice that in this case the cell wall appears yellow due to nonspecific labeling by both red and green nanoparticles.

Although semiconductor nanoparticles have many favourable optical properties for fluorescence labelling, as discussed in a previous section, the use of these particles in a biological context can be more problematic. The article argues that "the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation <ref name="marcel"/>". In order to prevent photochemical degradation, the particles were coated with a shell of material with a higher bandgap than that of the core. This confines light excitation to the core of the nanoparticle, thus shielding the excited core from photochemical reactions with the surroundings. The particles were made water soluble by the addition of a layer of silica <ref name="marcel"/>.

For the labeling of the mouse fibroblast, two sizes of CdSe-CdS core-shell nanocrystals enclosed in a silica shell were used. "The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% [http://en.wikipedia.org/wiki/Quantum_yield quantum yield]), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield)<ref name="marcel"/>". The nanocyrstals employed in this article have long fluorescence lifetimes compared to that of conventional dye of fluorescein phalloidin. As may be seen in the figure below, the nanocrystal-labeled samples showed very little photo bleaching.

[[Bilde:Photostability.jpg |center|thumb|300px|'''Sequential scan photostability comparison of fluorescein-phalloidin-labeled actin fibers compared with nanocrystal-labeled actin fibers.'''
(Source: Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' <ref name="marcel">[http://www.sciencemag.org/cgi/reprint/281/5385/2013.pdf Marcel Bruchez Jr, Mario Moronne, Peter Gin, Shimon Weiss, A.Paul Alivisatos ''Semiconductor Nanocrystals as Fluorescent Biological Labels'' ]</ref>)]]

====Nanowiring of redox enzymes====
In 2003 Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner published ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''. A 1.4nm gold nanoparticle was functionalized with the [http://en.wikipedia.org/wiki/Co-enzyme co-enzyme] [http://en.wikipedia.org/wiki/FAD flavin adenine dinucleotide (FAD)], and then connected to both the enzyme [http://en.wikipedia.org/wiki/GOx apo-glucose oxidase (apo-GOx)] and to a gold conducting film (see the figure below to the right). This yields "a bioelectrocatalytic system with exceptional electrical contact with the electrode support<ref name="yixi">[http://www.sciencemag.org/cgi/reprint/299/5614/1877.pdf Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle''] </ref> ". [[Bilde:Plugging in.jpeg|right|thumb|400px|'''(A) Displaying the two paths in making the bioelectrocatalytic system: (a) the adsorption of Au-NP-reconstituted GOx to a dithiol monolayer associated with a Au electrode and (b) the adsorption of Au-NPs functionalized with FAD on the dithiol-modified Au electrode followed by the reconstitution of apo-GOx on the functional NPs, (B) A STEM image of GOx reconstituted with the Au-FAD hybrid NP. Arrows show Au clusters.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] As glucose oxidase is an enzyme that aids in breaking a sugar ring into its constituents; this arrangement may be used as an electrical sensor for the presence of glucose. When glucose is present, an oxidzing current may be collected through the nanoparticle arrangement from the apo-GOx redox enzyme. Therefore the amount of current collected is dependent on the glucose concentration.

As is shown in the figure to the right, the bioelectrocatalytics system was constructed by two different routes. It was found that both routes yield almost identical bioelectrocatalytic properties toward oxidation of glucose; therefore only route (a) will be discussed in detail here. A gold nanoparticle (Au-NP) is connected to the redox co-enzyme FAD by a peptide linkage. For interested readers: ''this petpide linkage is formed between "...a single N-hydroxysuccinimide functionality", which has been functionalized onto tha Au-NP, and a "..N6-(2-aminoethyl)-flavin adenine dinucleotide (1)"'' <ref name="yixi"/>. The obtained Au-NP-FAD units are then inserted into the apo-GOx enzyme; the result of which may be seen in the STEM image displayed in the figure to the right (the Au-NPs are marked with arrows). Finally the Au-NP-reconstituted GOx were then assembled on a Au electrode covered with a [http://en.wikipedia.org/wiki/Thiol thiolated] monolayer (1,4-dimercaptoxylene).

The article mentions that the "routing of electrons from redox proteins, particularly redox enzymes, to electrodes has been a subject of extensive research <ref name="yixi"/>". However, previously the electron transfer rate constants between the redox enzymes (f.ex. apo-GOx) and the gold film have either been far lower, or approximately the same as to those between the enzymes and their natural electron acceptor <math>O_2</math> (approximately <math>700_{\frac{1}{s}}</math> <ref name="yixi"/>). For this reason nanoparticles are incorporated into the redox enzymes: In doing so, the electron transfer rate constant is found to be <math>5000_{\frac{1}{s}}</math> <ref name="yixi"/>, which exceeds the electron transfer features of the native enzyme. Therefore the gold nanoparticle acts as an electron relay or "electrical nanoplug" <ref name="yixi"/> from the redox enzyme to the gold film. The article concludes that the "...incorporation of Au-NPs in the enzyme greatly increases its maximum [http://en.wikipedia.org/wiki/Turnover_number turnover rate] <ref name="yixi"/>". Since the turnover rate is so high, the arrangement is not affected by the changes in the dissolved <math>O_2</math> concentration or oxidizable interferants such as ascorbic acid. This is a good result if one whishes to use this arrangement as a means to measure glucose concentration.

[[Bilde:Plugging in current.jpeg|right|thumb|350px|'''CVs corresponding to the bioelectrocatalyzed oxidation of glucose by the Au-NP-GOx electrode prepared according to route (a) (Fig. 1A), in the presence of different glucose concentrations: (a) 0 mM, (b) 1 mM, (c) 10 mM, (d) 20 mM, and (e) 50 mM. Results were recorded in 0.1 M phosphate buffer (pH 7.0), under Ar, potential scan rate 5 mV·s1. (B) Calibration plot derived from the CVs at E = 0.6 V versus SCE.'''
(Source: Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner ''"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle'' <ref name="yixi"/>]] The arrangement described in this article gives rise to the cyclic voltammograms shown to the right. As expected the glucose electrooxidation current increases with glucose concentration. The current increases up to a value of about <math>1.2 \cdot 10^-2 </math> M glucose. One may observe that the bioelectrocatalysed oxidation of glucose proceeds at an applied potential of <math>E_{applied}> 0.4 V </math>, where E is the potential measured against SCE (saturated calomelectrode). Since the redox potential of FAD units are E°= -0.045V, there requires a considerable potential for the bioelectrocatalysed oxidation to occur. The article writes: "This overpotential (η) might originate from the tunneling barrier introduced by the dithiolate spacer that bridges the Au-NP to the bulk electrode" <ref name="yixi"/>. By testing with different dithiol bridges, it turns out that the tunneling barrier between the NPs and the electrode is lower for more conjugated bridging units.

====DNA connected to CNT====

===Advantages and disadvantages===

==Importance to Nanotechnology==

==References==
<references />