Recently we have explained and developed a new fluorescence phenomenon that relies on the interactions of fluorophores with metallic particles and surfaces. These interactions can increase the quantum yield of weakly fluorescing species, provide spatially localized excitation and even improve probe photostability. Our new technology clearly demonstrates that these novel effects can result in up to a million-fold more photons per fluorophore, which when applied to assay sensing platforms will provide the equivalent of PCR or ELISA sensitivity, without any amplification steps. We subsequently envisage developing a new metallic-surface micro-assay based detection system, to serve downstream as a field deployable bio-terrorism sensor or a rapid gene profiling platform for clinical diagnostic applications. This R21 proposal is designed to demonstrate that we can indeed detect RNA targets with extremely high sensitivity and selectivity. Furthermore we believe that the likely successful outcome of high sensitivity detection in this proposal will lead to a generic platform for the development of high-sensitivity micro-assays for both RNA and DNA targets. The Metal-Enhanced Fluorescence RNA sensing assay functions as follows. Two DNA oligonucleotide probes complimentary to different regions of the target mRNA will be synthesized. One probe will be 5'-labeled with biotin, and the other with fluorescein. The DNA probes will be annealed in solution to RNA targets in a bulk RNA population under reaction conditions optimized for each probe pair. The annealing mixture will then be applied to a streptavidin-coated silver surface at the same temperature, permitting capture of specific RNA:DNA hybrids through binding of the biotin moiety. After extensive washing, the amount of bound RNA will be quantified by Metal-Enhanced Fluorescence of the retained fluorescein-coupled DNA probe. We intend to determine the sensitivity of the assay by using series dilution of the RNA and subsequently determine and compare the detection levels using both 1 and 2-photon excitation of fluorescein. In addition we aim to demonstrate the enhanced photostability of fluorescein on the silver assay.
Showing the most recent 10 out of 12 publications
