Metal-Enhanced Fluorescent Probes in DNA Microarrays

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Theoretical Background

This chapter describes the theory upon which our research was based. It points out the interaction of light with metallic NPs and the effect of metal NPs on fluorescent dye molecules placed in their vicinity.

Surface Plasmons or Surface Plasmon Polaritons

The term “surface plasmon” is often used in metal thin films in order to describe the electromagnetic waves that propagate along the interface between a metal film and a dielectric material such as organic films [42a]. The light absorption by metallic thin films results in a coherent motion of electrons at their surface, causing a wave. Therefore, this wave is called a surface plasmon wave.
Three conditions are necessary for the existence of a surface plasmon:
a) the materials meeting at the interface must be of different dielectric constants, εd and εm, where εd is the dielectric constant of the environment, and εm, the metal dielectric constant, is equal to the sum of its real and imaginary components (εm = ε1+ iε2);
b) ε1 < 0; and
c) ε1 = -2 εd
These conditions can be satisfied simply by putting a metallic film in contact with vacuum, air, or glass. Once these conditions are met, surface plasmons can be appropriately excited.
The excitation of a surface plasmon requires a match between the wave vector of the incident light in the direction of surface plasmon propagation and the wave vector of the surface plasmon expected to be produced, where ksp is the momentum of the surface plasmon, k0 is the momentum of light in vacuum, εm is the complex dielectric constant of the metal, and εd is the complex dielectric constant of the environment in contact with the metal layer.
Electromagnetic waves propagate in a metal film in the frequency and wavelength for which light propagation is forbidden in either of the two media. In other words, light cannot couple directly as a surface plasmon at a flat metal surface. This is because the wavevector k sp r of the surface plasmon wave is much larger than that of light propagating in air or vacuum [42b]. Hence, no direct excitation of the surface plasmon is possible, and special geometries are required in order to generate a surface plasmon. The Kretschmann configuration of attenuated total reflection (ATR) is widely used for that purpose (see Figure 2.1) [42a]. A microscopic glass slide coated with a metallic thin film (usually a 40-to-50-nm thick gold or silver film) is coupled to a prism through an index matching fluid or a polymer layer. Then a beam of light, whose reflection is controlled, is impinged
on the prism. At a particular angle, θsp the electromagnetic wave couples to the metalglass interface as a surface plasmon, resulting in a drop in the intensity of the reflected light (the ATR signal). The angle is given by the relation: 0 sin sp p sp k = k n θ (2.3)
where ksp is the momentum of the surface plasmon, k0 is the momentum of light in vacuum, np is the refractive index of the prism, and θsp is the angle of incidence of light needed to generate a surface plasmon.
The concept of surface plasmon can also be applied to metallic nanostructures or nanoparticles (NPs), in which case it is often called “localized surface plasmon” since the wave vector of the surface plasmon cannot characterize the movement of the electrons.
Optical excitation in NPs produces a collective oscillation of electrons across the NPs, creating a dipole. To excite these localized surface plasmons, no special geometry (such as the Kretschmann configuration) is needed. The specific wavelengths of light absorption necessary to produce plasmon oscillations are called surface plasmon bands or plasmon bands [42c].
A dipole approximation is often used to describe the optical properties of small metallic particles (for which the radius is much smaller than the wavelength of light).
This approximation gives rise to an extinction coefficient kex (measure of both absorption and scattering strengths) of the metal NP by the following relation (adapted from ref. [42g])
where λ is the wavelength of light, εd is the dielectric constant of the surrounding medium, ε1 and ε2 represent the real and imaginary parts of the dielectric constant εm of the metal ( m 1 2 ε =ε + iε ), and r is the radius of the NP. The highest transfer of energy from a photon to the metal NP,  causing the oscillation of the electrons, occurs for the resonance condition: 2ε d = −ε1 . Surface plasmon resonance (SPR) is then achieved, implying maximum absorption of light. From equation 2.4, we notice that a change in the dielectric constant around the NPs causes a variation in the amplitude and the wavelength of the SPR peak. We also observe that the size of the NPs has a direct impact on the amplitude and the wavelength of the localized surface plasmon peak as both the real and imaginary components of εm are a function of the radius of the nanoparticle.
The shape of metal NPs has also an effect on their resonance properties. When particles (like nanorods or ellipsoidal NPs) are nonsymmetrical in shape, they display a SPR peak for each of their dimensions. This is the result of different orientations and magnitudes of the dipoles inside these nonsymmetrical particles. For instance, for a nanorod-shaped metallic NP, the plasmon band shifts into two bands corresponding to the oscillation of free electrons in the longitudinal and transverse directions with respect to the long axis of the nanorod [43]. A spherical NP may have a single plasmon band associated with its singular dimension, i.e. its radius.
It is informative to consider that, in a nanoscale material, scattering processes can be observed since light can be scattered outside the NP (in which the number of atoms is very limited compared to a bulk material). This leads us to talk about Rayleigh scattering over which the absorption spectrum of a metal NP is usually superposed. Rayleigh scattering theory addresses the scattering of light by particles whose sizes are much smaller than the wavelength of the incident light (wavelength > π * particle size). When a photon is scattered by a NP, its angle of propagation changes while its frequency stays the same. The intensity of the scattered light Is is inversely proportional to the fourth power of the wavelength.
Now that we have introduced the theory describing the effect of an incident light impinging on metallic NPs, we may study the interaction of the NPs with fluorescent dye molecules.

Plasmon-Dye Interactions

Metal NPs and other metallic nanostructures can increase the fluorescence intensity of fluorescent dye molecules located in their proximity. Two distinct mechanisms have been proposed in order to explain the enhancement of the optical properties of these fluorophores placed in close proximity to metallic NPs. The first is energy transfer between the dye molecules and the metal NPs, resulting either in surface enhanced fluorescence or surface enhanced absorption of the dye molecules. In order to obtain an effective energy transfer, the SPR peak of the NPs can be tuned by monitoring
the physical dimensions and composition of the particles. The second mechanism that can result in surface enhancement of the fluorescence of the dye molecules is the decrease of their radiative and non radiative lifetimes due to the coupling of the dye dipole moment with the electric field induced by the surface plasmon polaritons [44]. Indeed, when light is coupled as a surface plasmon in a metal NP, it induces an evanescent electromagnetic field coming out of the surface of the NP and decaying into the surrounding dielectric field [42d]. The distance between the dye molecule and the particle is crucial in both mechanisms.

Energy Transfer

When a dye molecule is located in the vicinity of a metal surface, the energy levels of the dye and the surface plasmon may couple, resulting in energy transfer between them. Depending on the nature of the coupling, the effect of this energy transfer may be observed as an increase in the dye’s absorption coefficient or fluorescence efficiency (see Figure 2.2).
The interaction between light, dyes and NPs can result in two different scenarios (see Figures 2.2a and 2.2b, respectively):
a) The metal NP acts as a reservoir, which can receive the energy from the dye molecule in the excited state. Therefore, the fluorescence of the excited dye molecule is quenched when the energy of the excited dye molecule is transferred to the NP to excite a surface plasmon polariton. As a result, fewer dye molecules are available to return to their ground state through the radiative path (dashed line). Moreover, the dye molecule rapidly drops back to its ground state. Hence, the population of ground state dye molecules that may absorb additional photons is increased, resulting in the enhancement of the absorption of the dye.
b) The dye molecule is excited by a photon or through the transfer of energy from the optically excited surface plasmon polariton. When the metal transfers its energy to the fluorophore, an enhancement of fluorescence is expected due to the higher absorption cross-section of the metal compared to that of the dye molecule.
Two resonance conditions exist: one in which the energy level of the surface plasmon is equal to that of the upper energy level of the excited dye molecule (see Figure 2.3a) and the other in which the energy level of the surface plasmon is equal to that of the radiative transition of the dye molecule (see Figure 2.3b).
In the first resonance condition (Figure 2.3a), the energy transfer can either go from the excited surface plasmon polaritons to the dye molecule in the ground state, causing surface enhanced fluorescence, or can originate from the excited dye molecule in order to excite a surface plasmon polariton, causing surface enhanced absorption and quenching of fluorescence. The question as to which process dominates depends on the lifetimes of the surface plasmon polariton and excited dye molecule. The lifetime of the excited dye molecule decreases in the presence of a metallic NP. The magnitude of the reduction is based on the dimensions and composition of the NP and the spatial separation between the dye molecule and the NP [45]. The lifetime of the surface plasmon polariton in gold and silver thin films has been measured and falls between 25 and 800 fs, depending on the morphology of the film [44]. As far as we know, data about the lifetime of surface plasmon polaritons in either gold or silver NPs is not available.
The second resonance condition (Figure 2.3b) usually leads to a quenching of the fluorescence of the dye since the energy transfer generally originates from the excited dye molecules located in the intermediate energy level. The energy transfer process is much localized as the maximum distance separating the dye molecule from the metal NP needs to be between 10 and 30 nm. Quenching decreases with the cube of the distance (d) between the metal surface and the fluorophore molecule [46].

Electric Field Effects

As mentioned earlier, light can couple into a metal NP, giving rise to a localized surface plasmon. This localized surface plasmon induces an electric field which has a very high intensity near the surface of the NP, and decays as the distance is increased away from the surface of the particle. Hence, there is a confinement of electromagnetic radiation around the metallic nanostructure. This electric field enhancement is wavelength-dependent. Large particles give smaller enhancement, with a shift of the plasmon resonance to longer wavelengths [42c].
When a dye molecule is within the extended electric field of a surface plasmon, the radiative decay rate of the dye can increase. The manipulation of the radiative decay properties of a fluorescent molecule is sometimes referred to as radiative decay engineering [12]. To understand the importance of controlling the radiative decay rate, it is necessary to study how this rate influences the quantum yield Q0 and lifetime τ0 of a dye molecule in the absence of a metal surface. The quantum yield and lifetime in the absence of other quenching interaction are given respectively.

1 Introduction
1.1 Theoretical Overview
1.2 Applications of Metal Enhanced Fluorescence to Biosensing
1.2.1 MEF in ultra Bright Labeled Proteins: New Class of Probes for Immunoassays and Immunostaining
1.2.2 MEF in Deoxyribonucleic Acid (DNA) Hybridization for Biotechnological and Diagnostic Applications
1.2.3 Metal-Enhanced Fluorescent Probes in DNA Microarrays
1.2.4 MEF for Medical Imaging
1.3 Conclusions and scope of the project
2 Theoretical Background 
2.1 Surface Plasmons or Surface Plasmon Polaritons
2.2 Plasmon-Dye Interactions
2.2.1 Energy Transfer
2.2.2 Electric Field Effects
3 Materials and Experimental Method 
3.1 Generalities
3.2 Synthesis of Aux-Ag1-x Nanoalloys
3.3 Preparation of Dye Stock Solutions
3.4 Preparation of Dye Control Solutions and Dye-NPs Solutions
4 Results and Discussion 
4.1 Characterization of Aux-Ag1-x Nanoalloys
4.2 Fluorescence Analysis of Fluorophore-NPs Solutions
4.2.1 Rose Bengal / NPs Interactions
4.2.2 Rhodamine B / NPs Interactions
4.2.3 Fluorescein Sodium / NPs Interactions
4.3 Parameters for Optimum Surface Enhanced Fluorescence
5 Conclusion and Future Work 
6 References
Effects of Metallic Nanoalloys on Dye Fluorescence

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