We propose to develop a new generic approach to fluorescence sensing based on metal-enhance fluorescence (MEF). """"""""The spectral properties of fluorophores can be favorably modified by proximity to conducting metallic surfaces (metals). The useful changes include increased rates of excitation, increased quantum yields, increased photostability resulting from decreased lifetimes, increased multi-photon excitation and increased distances for resonance energy transfer (RET). We propose to use these spectral changes in fluorescence sensing. ? Specific Aim 1. Evaluate the optimal configuration for high sensitivity detection. a. Develop the use of excitation by the surface plasmon resonance (SPR). b. Evaluate the use of directional emission using semi-transparent silver coated surfaces. c. Extend the use of SPR excitation and/or directional emission to metal particle coated surfaces. ? Specific Aim 2. Optimize the metallic surface chemistry for high sensitivity detection. a. Determine the signal levels with labeled proteins on the silver surfaces. b. Determine the optimal distances from the surface for highest intensity and directional emission. ? Specific Aim 3. Evaluation of long range resonance energy transfer near metallic surfaces. a. Examine RET near silver particles in a readily accessible antibody-antigen interaction. b. Examine RET for protein linked to silver island films or colloid coated surfaces. c. Examine the effect of distance from the surface on RET. ? Specific Aim 4. In collaboration with Dr. Robert Christenson (Director of Rapid Resonance, Clinical Chemistry and Toxicology Laboratory) apply the knowledge gained in Specific Aims 1 to 3 to develop assays for PSA and ACT-PSA. a. Develop sandwich (capture) immunoassays for PSA using anti-PSA (or anti-ACT-PSA) labeled with low and high quantum yield fluorophores. b. Develop RET immunoassays for PSA (or ACT-PSA) with the capture and detection antibodies. ? ?
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