Large Fluorescence Enhancements of Fluorophore Ensembles with Multilayer Plasmonic Substrates: Comparison of Theory and Experimental Results.

Multilayer substrates consisting of a glass slide, silver mirror, silica layer, and silver nanoparticles were fabricated using magnetron sputtering. This new geometry of substrates with backplane mirror and dielectric photonic cavity produced large average fluorescence enhancements up to 190-fold. Fluorescence enhancements of five fluorescent probes were measured over the broad spectral range from 470 to 800 nm. Fluorescent probes were streptavidin conjugates attached to the substrate surface through a layer of biotinylated bovine serum albumin. The protein layers represent a common surface modification for surface-based bioassays such as immunoassays or molecular diagnostic assays. We found that optimal enhancement is dependent on the thickness of the dielectric layer separating the silver mirror and the silver nanoparticles and on the spectral range. We performed numerical calculations for enhancement in both the excitation and emission using finite element method (FEM) the results of which were in qualitative agreement with the experimental results. The described method for fabrication multilayered substrates and the results obtained with protein layers demonstrate great potential for the design of simple and ultrasensitive fluorometric bioassays with large optical amplifications compared to the standard approaches of enzyme-based bioassays with dielectric surfaces.