Forskjell mellom versjoner av «Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors»

Revisjonen fra 22. mar. 2009 kl. 13:54 (vis kilde) Chrisdi (diskusjon | bidrag) (→‎Favourable characteristics) ← Forrige endring		Revisjonen fra 22. mar. 2009 kl. 14:21 (vis kilde) Bjornoy (diskusjon | bidrag) (→‎Theory) Neste endring →
Linje 39:		Linje 39:
	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.		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.
−	===DNA===	+	===Bio-molecules===
		+	====DNA====
−	====Structure and Favourable properties====	+	=====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 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.

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Revisjonen fra 22. mar. 2009 kl. 14:21

Under construction. Please do not edit until after we have been assessed for our literature project. -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"> 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

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

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: 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 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 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>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

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>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">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

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.

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>M.S.Urdea et al., Nucleic Acids 16,4937(1988)</ref>

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 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; 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">wikipedia article on the avidin protein </ref>). By use of a wide field microscope (a type of fluorescence microscope <ref> 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% 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.

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 co-enzyme flavin adenine dinucleotide (FAD), and then connected to both the enzyme 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">Yi Xiao, Fernando Patolsky, Eugenii Katz, James F. Hainfeld, Itamar Willner "Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle </ref> ". 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 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 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.

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

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