| Popis: |
The aim of this study is to advance the technique of fluorescent assays by using metallic nanostructures, which can enhance the fluorescence of molecules placed at nanometric distances. Enhancing fluorescence signals while keeping a good signal-to-noise ratio is very important for detection of low amounts of analytes in diagnosis of infectious diseases and cancer cells; and in monitoring of healthy, food and environment. For reproducibility reasons, a choice was made to work with structures constructed by electron-beam lithography in conjunction with atomic layer deposition and thermal evaporation. The structures consisted of two-dimensional periodic arrays of silver nanocylinders and dimers with and without an underlying thin silver layer. The reported investigations comprised three parts: fluorescence bead assays for RNA detection, a study of the optical/plasmonic properties of the nanostructures, and fluorescence experiments on these nanostructures. Before entering in the plasmonics field, we explored a fluorescence-based technique for detection of labelled RNA of a specific pathogen using microspheres ow cytometry. We used a 2100 Bioanalyzer (Agilent Technologies), a commercially available desktop lab-on-a-chip ow cytometer. We demonstrated the detection down to 125 ng of RNA, 16 times less than previously reported. Subsequently, we studied the plasmonic properties of specific electron-beam fabricated nanostructures, and to this aim we examined the dispersion relations of nanoscale planar multilayer metallic-dielectric films. For the first time, to our knowledge, it was obtained a solution for an IIMI (insulator-insulator-metal-insulator) configuration, using metal permittivity given by the lossless Drude's model as well as tabulated in the literature. This IIMI geometry is related to the fabricated nanostructures with an underlying silver layer. We showed that the studied structures and excitation can match the wave vectors required for excitation of propagating surface plasmons on the planar metal layer. We also showed that an extraordinary transmission achieved for the nanoparticles over that metal layer is due to the periodic array, but it cannot be attributed to propagating surface plasmons. Further, a thorough study of the optical/plasmonic properties on the nanostructures was performed by finite element method (FEM) using the software COMSOL Multiphysics 3.5a with the RF module. We found that clear dipolar and quadrupolar resonant modes of localised surface plasmons were excited on the nanoparticles. These modes can be tuned by controlling some parameters, such as nanoparticles-metal layer thickness, refractive index of dielectric layer, the thickness of a cap dielectric layer and the cylinder diameter. The structure can also be applied in SPR sensing based on wavelength interrogation. The near-_eld of the metals enhances the second power of the electric _eld averaged over the top surface of the structure, where a fluorescence assay can be performed. The presence of the underlying silver layer red shifts the resonances and provides further enhancement to the squared electric _eld. The highest enhancement was achieved by a dimer in longitudinal polarisation. The factor was about 21.8 times higher than the one obtained by a simple dielectric substrate. The fluorescence experiments were carried out on 55 to 60nm layers of polyvinyl alcohol (PVA) embedded with fluorochromes over the nanostructures. Fluorescence enhancement of up to 30.8 times, compared to bare dielectric substrate, was achieved on experiments with a homogeneous silver layer without nanoparticles. Most experiments with the nanocylinders over a planar silver layer showed reduced enhancement compared to structure with just the silver layer. This can be explained by modifications in the non-radiative routes, quenching the fluorescence. In conclusion, we investigated the properties of a periodic array of silver cylinders and dimers, and the effects of an underlying thin silver layer. We showed how to tune the surface plasmon resonances by varying material and geometric parameters. The enhanced electric near-field provided by these structures can be applied in surface-enhanced fluorescence. Experiments showed fluorescence enhancement factors up to 20 times. With further numerical studies of electric field and modifications of radiative and non-radiative decay routes, it is possible to offer a complete description of fluorescence enhancement and to optimise it. |
