Functional Biomolecule-Nanoparticle Structures on Surfaces for Application as Sensors
Intro
- How we interpreted the research question
- The sensors studied in this discussion
- Why Nanoparticles and biomolecules?
Theory
Nanoparticle
Optical properties
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 whishes 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. 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"/>".
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.
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 with an exact chosen sequence over and over again 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 environment 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
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.
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 fibroblasts <ref name="marcel"/>".
The report argues that "the development of nanocrystals for biological labeling opens up new possibilities for many multicolor experiments and diagnostics. Further, it establishes a class of fluorescent probe for which no small organic molecule equivalent exists <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;
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"/>".
Nanowiring of redox enzymes
«Plugging into Eznymes»: Nanowiring of Redox Enzynes by a Gold Nanoparticle Experimental: 1.4nm gold nanocrystals are functionalized with the cofactor flavin adenine dinucleotide (FAD). FAD is a redox cofactor; a non-protein chemical compound that binds to an enzyme and is required for catalysis. FAD is involved in several important reactions in metabolism. In functionalizing gold nanocrystals with FAD, one creates «a bioelectrocatalytic system with exceptional electrical contact with the electrode support».
Experiment: Gold nanocrystal functionalized NAD is incorporated into the redox enzyme Apo-Glucose oxidase, and connected to a conducting Au film (figure 1b in report). Glucose oxidase (GOx) is an enzyme which binds to beta-D-glucopyranose (a hemiacetal form of the six-carbon sugar glucose) and aids in breaking the sugar down into its metabolites4. Remember the reducing site of the hemiacetal binding in 6-ring sugar. Therefore, the arrangement described above may be used to route the electrons from the redox protein to an electrode. Thus the current induced by the redox reactions with glucose may be measured. What is especially interesting is the fact that the «reconstituted GOx exceeds the electron transfer features of the native enzyme». There are two possible routes of making the arrangement: 1.Reconstitution of apo-glucose oxidase with the nanoparticle-functionalized-NAD is then assembled on a thilated monolayer by using different dithiols as linkers. 2.The nanoparticle functionalised NAD is assembled on a thiolated monolayer associated with an electrode, and apo-GOx could be subsequently reconstituted on the functional nanoparticles. It turns out that these two procedures reveal similar surface coverages of about 1*10^-12 molcm^-2 of the GOx protein. «From the anodic current density plateau (..) and from the surface coverage of the enzyme molecules, we calculate a unimolecular electron transfer rate constant5 of 5*10^3 s^-1. This value is seven times as high as the electron-transfer rate constant of native GOx with 02 as electron acceptor (~700s^-1), which leads us to the unexpected conclusion that the incorporation of Au-NPs in the enzyme greatly increases the maximum turnover rate». Due to this exceeding rate, neither the concentration of 02 or an oxidizable interferant (such as ascorbic acid) affect the measurements. See figures showing current vs potential and current vs concentration.
An overpotential is required. This might originate from the tunneling barrier introduced by the dithiolate bridge.
DNA connected to CNT
Advantages and disadvantages
Importance to Nanotechnology
References
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