Studies of Surface-Enhanced Fluorescence of Dyes Using LSPR Phenomenon in Au and Ag Nanostructures

Fluorescence techniques are widely used in biomedical research due to their high sensitivity, multiplex sensing possibility, compatibility with living organisms, and high response speed [184-186]. It is possible to study living cells and even whole organisms by using fluorescence spectroscopy [185, 187, 188]. Fluorescence is a rapid and sensitive method for studying the structure, kinetics, and functions of biological macromolecules such as nucleic acids and proteins [189-192].

Recently, nanoscale fluorescent emitters have attracted special attention for application in biological studies [193, 194]. In a short time, several applications have been found for these objects in clinical diagnostics [195], environmental monitoring [196], food quality control [197], biological weapons detection [198], and in various related fields. This diversity of applications is provided by bionanointeractions that allow delivery of nanoparticles to the biologically important systems to obtain information about them and even to change their properties [199-201]. Significant progress has been achieved in the application of high-conductive plasmonic nanomaterials for biochemical and diagnostic applications [202].

This subsection describes existing approaches for the development of sensors and biodiagnostic instruments based on surface-enhanced fluorescence [203-205].

Fluorescence enhancement by using high-conductive nanostructures

Studying the possibilities of using high-conductive metal nanoparticles (MNPs) has attracted great interest due to the prospects of MNP applications in biophysics and biochemistry, materials science, and fluorescent spectroscopy based on the surface enhancement methods [206]. The optical properties of MNPs are mostly determined by the surface plasmons, which are inherent collective electron oscillations in the metal. LSPR that occurs in metal nanostructures is widely used in highly sensitive sensors and optical devices [170, 207]. To enhance the fluorescence, silver and gold nanoparticles that have unique optical, electronic, and catalytic properties are usually exploited. This is due to their attractive properties, which include not only the simple fabrication, but also such features as the intense plasmon resonance band in the visible spectral region and significant extinction at the LSPR wavelength [154].

Enhancement of fluorescence of the dyes by using silver and gold nanostructures significantly depends on the resonance energy transfer from MNPs generating the plasmon field to the dye molecule, which is located near MNP [208-210], shape and size of MNP [211], distance between the dye molecule and the MNP [170, 207, 208], as well as the characteristics of dye molecule such as intrinsic quantum yield and excited state lifetime [208, 209, 212].

In general, the fluorescence emission enhancement Ф is caused by the resonance energy transfer from plasmon-generating nanostructures to the dye molecule and subsequent change in the quantum yield of the "plasmonic nanostructure-dye molecule” system and can be described by the following equation [213]:

where Ep is the plasmon electric field strength in the observation point that is generated by the incident light E0; q is the quantum yield of the dye molecule induced by the plasmon field Ep; and is the intrinsic quantum yield of the dye molecule.

Factor of optimal distance between fluorophore molecule and plasmonic nanoparticle

The optimal distance between the fluorophore and the metal surface is a very important factor in the mechanism of surface-enhanced fluorescence. This optimal distance depends on the immanent properties of the molecule relevant to energy exchange between the molecule and the surface plasmon. There are three cases for the location of dye molecule near the surface of nanoparticles. First of them is very close to the metal surface when the quenching of fluorescence is observed; second is an optimal distance resulting in the most significant enhancement of fluorescence; and the third is much greater than the distance at which the enhancement occurs

[214] .

The optimal distance can be determined both theoretically and experimentally [212]. For example, the emission simulation for a layer of fluorescent dye molecules with the thickness of 5 nm placed on the 80 nm spherical gold nanoparticle has shown that the optimal distance between the fluorophores and the metal surface to observe fluorescence enhancement was about 20 nm; in other cases there were weak enhancement or quenching [212]. In Ref.

[215] , the experimental studies of the dye fluorescence emission near the silver and gold nanostructures were carried out. It was found that the enhancement of fluorescence was observed when the distance between dye molecules and metal surface was 24-25 nm and the quenching was observed at 15 nm. If we consider the model "fluorescent sphere-silver nanoparticle” system [216], it turns out that when the distance between them is equal to 90 nm, the emission quenching by 30% is observed. When the distance is decreased to some optimal value, 1.5-fold enhancement occurs. And if the second silver particle with the same properties is added to the system, 2.7-fold enhancement of fluorescence emission of the fluorescent sphere will be produced.

Consideration of the influence of the distance between the fluorophore and the surface of the metal nanostructures has led to the development of the methods enabling the fluorescence enhancement observation. The most effective technique for distance- controlled surface enhancement is to use the artificially created spacer layer between the fluorescent molecule and the silver or gold nanostructure, which prevents the emission quenching effect [170,

210,215]. The function of spacers could be provided by organic (e.g., lipid or protein) or inorganic (e.g., silicon dioxide) separation layers [210,217].

For fluorescence enhancement studies, organic "sandwich" structures are often used [210], which are dye-labeled protein molecules located directly on the surface of the high-conductive nanostructures. Here, protein molecules themselves act as the separation layers between the nanostructure and fluorophore. Such structures allow obtaining up to 18 times enhanced emission of dye molecules. In some experiments, the distance between the nanoparticle and the dye molecule was adjusted by a double- stranded DNA molecule [209] or its replicating form [218]. Another interesting option is to exploit spacer layers fabricated from silicon dioxide. This coating deposited on nanostructures provides strength, chemical inertness, and versatility for binding to biomolecules or any hydrophobic fluorophores, and also allows adjusting the distance between the fluorophores and the nanostructures [205, 217]. For precise spacing of fluorophore molecules from nanostructured surface, the layer-by-layer (LbL) technology is often used [219] to produce oppositely charged polymer "sandwich" that enables fluorescence signal enhancement.

Influence of the size, position, and shape of nanostructures on the enhancement effect

As mentioned above, the fluorescence enhancement using high- conductive nanostructures depends on their size and shape. In Ref. [220], a comparative analysis of the fluorescent enhancement of the dye using colloidal gold nanoparticles of different diameters with protein-modified surface was carried out. It was found that nanoparticles with a 40 nm diameter allow obtaining 1.5-fold enhancement of fluorescence signal and nanoparticles of 200 nm diameters provide 2-fold enhancement, respectively. Forty nm cubic and spherical silver nanoparticles were used as the plasmon- generating nanostructures for fluorescence enhancement [221]. The authors experimentally found that the cubic silver nanoparticles provide higher fluorescence enhancement in comparison with the spherical silver nanoparticles of the same size. The effect of the size and the shape of nanoparticles on the enhancement value can be considered through the spectral overlap of the absorption spectra of nanostructure and the emission spectra of the fluorophore [209], because there is a direct relationship of the wavelength position and the half-width of the light extinction spectra with the geometrical parameters of the nanoparticles.

Another important fact is that the emission intensity of the dye placed near the nanostructure exhibits nonlinear dependence on their relative position [173, 218, 222]. In Refs. [170, 173, 223, 224], it was found that the largest enhancement of fluorescence was observed in the gap between closely spaced nanoparticles, where the intensity of the electric field near their surface is greater than at the surface of individual nanostructures. The study in Ref. [173] showed that the enhancement of fluorescence of the dye placed near a plasmon-generating separate silver nanoparticle is about two times smaller than in the case of placing the dye molecule between two silver nanoparticles.

It should be noted that the fluorescence emission enhancement of the dye molecule located between two silver or gold nanoparticles depends on the geometry of this "sandwich” structure. Namely, when the fluorophore is sandwiched in the hot spot region right between the two gold nanoparticles on the axis connecting their centers, it leads to a strong fluorescence enhancement; in the other case, the fluorescence enhancement is less pronounced [216].

Mechanisms of surface enhancement

The interaction between the metal surface and fluorescent molecule provides such effects as increasing the radiative or nonradiative decay rates of fluorescent molecule and, accordingly, increasing or decreasing the quantum yield [212]. Enhancement of fluorescence depends on the plasmon resonance energy transfer from the nanostructured metal surface to the dye molecules located near the surface [177, 208, 210, 216, 225]. This transfer is determined by two mechanisms: first of them is the plasmon field generated around the nanoparticle by the incident light that, depending on wavelength, can enhance the excitation of the fluorophore, which, in turn, determines the level of fluorescence emission. The second one is the nanoparticle-fluorophore interaction that reduces the ratio of radiative to nonradiative decay rate and, depending on the presence of the dielectric layer, influences the quantum yield of the fluorophore, resulting in fluorescence quenching [213]. It should be noted that the enhancement of fluorescent emission depends not only on the properties of metal nanoparticle, but also on the properties of the fluorescent molecule. These properties are the quantum yield and the excitation relaxation time, which depend on the probability of quantum transitions involved in the radiative processes. Accordingly, the probability of quantum transitions under the certain conditions increases when fluorescent molecule is located near the metal surface. Quantum yield, which is defined as the ratio of the number of emitted to absorbed photons of the molecule, determines the efficiency of the emission, which, in turn, depends on the distance between the fluorophore and the surface of nanostructures [212, 224]. In Ref. [212], it was theoretically and experimentally shown that the quantum yield of a molecule decreases when the distance between the fluorophore and the surface of metal nanostructure is too small, even in the case of the sample excitation increase, and results in fluorescence quenching.

Studies on fluorescence of the dye solutions containing the high- conductive nanoparticles established that the quantum yield also depends on the pH. In Ref. [206], scientists found that the high pH of the dye solution has no effect on the enhancement; but when pH reduces, the fluorescence enhancement occurs, which is caused by the increasing overlap of the emission spectrum of the dye with the plasmon resonance band of the metal nanoparticles.

The presence of the nanostructured metal surface near the dye molecule affects not only the quantum yield and fluorescence enhancement, but also the radiative lifetime of the excited molecule [172, 226, 227]. For example, a 13-fold emission enhancement of the fluorophore is accompanied by a decrease in the fluorescence lifetime by a factor of 22 [172].

The orientation of the dipole moment of the dye molecules near gold and silver nanostructures and the polarization of excitation light have a considerable effect on the fluorescence enhancement [226, 228]. In the study in Ref. [216], the fluorescence intensity of the "Au nanoparticle-fluorophore-Au nanoparticle” sandwich structure is enhanced when the laser light is polarized parallel to the axis of the sandwich, whereas the fluorescence is decreased when the laser is polarized perpendicular to it. Theoretical calculations showed [177] that the maximum enhancement was observed in the case of perpendicular orientation of the dipole moment of the molecules to the nanostructured surface and when the distance between the molecule and the metal surface was optimal. A singlemolecule fluorescence rate as a function of the particle-sample distance for an 80 nm silver particle was studied in the case of perpendicular orientation of the dipole moment of the molecule [225]. Experimentally, it is difficult to identify molecules with such orientation of the dipole moment, but the results of the performed studies indicate that even at the angle of the dipole moment to the normal of the plane in which the nanostructures are placed equal to 15 degrees, 19 times enhancement of fluorescence can be observed [172].

Modeling and comparative analysis of gold and silver nanostructures as fluorescent signal amplifiers

Random silver and gold nanostructure arrays were obtained by thermal annealing of silver (mass thickness 8 nm, 250°C, 1 h) and gold (mass thickness 10 nm, 450°C, 2 h) island films [158]. The light extinction spectra of random silver and gold NSA were measured using the LSPR biosensor [136]. Silver and gold NSA exhibited the excitation of localized surface plasmon at 480 nm (Fig. 3.35b) and at 555 nm (Fig. 3.35d), respectively. The average sizes of nanostructures forming the NSA were calculated by matching the wavelength peak positions in the spectra obtained experimentally and theoretically.

Thus, for silver nanostructures, the equivalent base diameter was found to be equal to 20 nm and height equal to 10 nm, and for gold nanostructures, the equivalent base diameter is 10 nm and height is 20 nm.

For determined sizes of the gold and silver nanostructures, the profiles of electric field intensity enhancement around the nanoparticles were calculated (Figs. 3.35a,c). These enhancement values correlate with the increase in the fluorescence excitation rate [209]. The calculation was performed at 532 nm, which is an inherent excitation wavelength for the organic dye Rhodamine 6G (R6G). The maximum electric field intensity enhancement value calculated on the 30x30 nm2 area outside the nanostructure in the plane in which the propagation vector and polarization vector of incident light wave lie was about 100 times for silver nanostructure and about 30 times for gold nanostructure. The broadening in the extinction spectra of silver NSA, observed in the experiment, compared with the simulation results (Fig. 3.35b), can be explained by the growing mismatch between the model semi-ellipsoid and experimental nanostructure shapes and variety of sizes of nanostructures in the NSA. Thus, silver nanostructures produced by thermal annealing of island films are potentially more promising in creating the nanochip. However, the gold nanostructures are mostly used in most scientific researches at the present time due to their higher chemical stability.

Simulated profiles of electric field intensity distributions for

Figure 3.35 Simulated profiles of electric field intensity distributions for (a) Ag and (c) Au semi-ellipsoids on glass substrate; light extinction spectra of unordered (b) Ag and (d) Au nanostructure arrays. Reprinted from Ref. [229] under a Creative Commons Attribution 4.0 International License. Figure and caption were adapted.

Studies of Rhodamine 6G dye fluorescence enhancement by using random gold nanostructure arrays

Using the developed method of surface-enhanced fluorometry [205], based on the phenomenon of LSPR in unordered gold nanostructure arrays (GNAs), the fluorescence measurements of R6G were carried out. The dye was placed on the plasmon-generating GNA with various thicknesses of the dielectric Si02 coating. The Si02 layer deposited on the GNA provides strength, chemical inertness, versatility required to compound the biomolecule or any hydrophobic fluorophore, and also allows adjusting the distance between the fluorophore and the nanoparticle.

The samples of GNA with the thicknesses of dielectric spacer equal to 10, 15, 20, and 25 nm coated with a polymer composite, consisting of an aqueous solution of R6G and polyacrylic acid (PAA), were studied. The R6G concentration in the polymer composite was equal to 10~5 mol/L. The fluorescence measurements of R6G on GNA were carried out with the 532 nm green laser used as an excitation light source. For all samples, the enhancement of R6G fluorescence near the GNA was observed compared with the signal on the sample without GNA (Fig. 3.36).

Fluorescence spectra of R6G dye for various thicknesses of the dielectric Si0 coating placed on the gold nanostructure arrays

Figure 3.36 Fluorescence spectra of R6G dye for various thicknesses of the dielectric Si02 coating placed on the gold nanostructure arrays. Reprinted from Ref. [229] under a Creative Commons Attribution 4.0 International License. Figure was adapted.

The intensity of the fluorescent signal dependence on the Si02 coating thickness was nonlinear with an expressed peak. The maximum enhancement of R6G fluorescence was obtained for the sample with 20 nm thickness of Si02. Figure 3.37 shows the enhancement factor dependence on the thickness of dielectric

Si02 coating. The enhancement factor is defined as the ratio of R6G fluorescence intensities for GNA with Si02 shell and bare glass substrate at the maximum emission wavelength. The experimental results of maximum R6G fluorescence enhancement (about 23 times) obtained by using nanochips agree with the calculated value of the electric field enhancement on the gold nanostructures. The disagreement between the calculated and experimental enhancement values is due to the influence of the dielectric substrate and the spread of the size and shape of nanoparticles on the real nanochip.

Enhancement factor of R6G fluorescence for different thicknesses of Si0 coating. Reprinted from Ref. [229] under a Creative Commons Attribution 4.0 International License. Figure was adapted

Figure 3.37 Enhancement factor of R6G fluorescence for different thicknesses of Si02 coating. Reprinted from Ref. [229] under a Creative Commons Attribution 4.0 International License. Figure was adapted.

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