In this application we propose the development of technology with potential of at least 100-fold improved sensitivity over current fluorometric methods. Specifically we integrate a metal enhanced fluorescence (MEF) with time-resolved detection technique to obtain high sensitivity and ability for real time monitoring of biomolecular interactions. We propose to specifically integrate Metal-Enhanced Fluorescence (MEF) phenomena and time-resolved phase-modulation (PM) detection technique with surface-based assays to obtain high sensitivity and large analyte concentration range as well as to simplify the biochemical procedure. The new approach represents a significant advance on fluorometric analyses of biomolecule interactions, with sensitivity comparable to ELISA and a simplified sample procedure. Specifically, we will demonstrate the potential of MEF-PM technology using a panel of cytokines such as IFN?, TNFa, IL-5, IL-8, IL-16, VEGF, and RANTES. Currently, detection of multiple cytokines requires the use of the most sensitive detection technologies such as enzyme linked immunosorbent assay (ELISA), radioimmunoassay and chemiluminescence because of their low concentration in human blood. Within projected work we will perform feasibility studies on reproducible fabrication of fluorescence enhancing substrates, functionalization and optimization of surface chemistry, performing feasibility on clinical assays that require high sensitivity and reduction in cost, and optimization of time-resolved detection modality, and integration of components into practical systems for clinical diagnostics and for research. This will be accomplished by (1) Developing a procedure for reproducible fabrication of substrates with metallic nanostructures. (2) Optimization of biomolecules immobilization on the surface of the MEF substrates. (3) Validation of MEF-PM method using a panel of cytokines with comparison with ELISA method. The proposed technology will meet requirements for high sensitivity (1-10 pg/ml), broad analytical range (4 - 5 orders of magnitude), ease of use, and versatility. MEF-PM will be of broad use in basic and cancer research applications, and will provide a tool for proteomics, bioassay developments and clinical diagnostics.
