Fluorescent molecules that are positioned in close proximity to silver nanoparticles produce increased fluorescence intensity along with increased photostability and decreased lifetimes. These silver structures have been studied in great detail for their ability to increase fluorescent intensities when deposited onto solid surfaces, such as glass or quartz. Little information exists, however, on the ability of silver nanostructures to enhance fluorophores deposited onto porous membrane surfaces. The overall objective of this proposal is to use metal enhanced fluorescence to increase near- infrared fluorescence emissions on membrane substrates. Membrane based assays, such as western blots are routinely used in clinical settings to confirm serious diagnoses suggested by other screening tests, and in research settings to characterize and quantitate proteins of interest. Fluorescence based assays in the near-infrared offer the additional advantage of significantly lowering the background compared to that generated in the visible spectrum, helping to lower the limit of detection. In the long term, this work should be applicable to most systems that utilize fluorescence in a manner that traps the fluorescent dyes on a membrane substrate. Near-infrared metal enhanced fluorescence on membrane surfaces should provide a reliable and efficient procedure to increase fluorescent emissions and decrease the limit of detection, providing a tool that is compatible with current proteomic and genomic applications. Future endeavors could use metal enhanced fluorescence to increase the detectability of membrane based DNA, protein, and antibody arrays on membranes. This project focuses on developing an enhancement reagent for Western and dot blot analyses, which integrates the use of low background near-infrared LI-COR dyes with Metal Enhanced Fluorescence (MEF), to produce more sensitive membrane based assays capable of reducing the limit of detection by an order of magnitude. Advances in sensitivity for these Western and dot blot assays will aid in the analysis of molecular, cellular, and inter-cellular processes, and the relationship of these processes with respect to one another. ? ? ?
