Synthesis and application of FRET nanoparticles in the profiling of a protease**

A novel colloidal bar coding method utilizing nanoscale fluorescence resonance energy transfer (FRET) reporters decoded by confocal laser scanning microscopy combined with spectral unmixing, was reported. Succinimidyl ester dyes Atto-488, Atto-550, and Atto-590 were covalently linked to a silane coupling agent (3-aminopropyl)-trimethoxysilane, forming an APS-dye conjugate. The three dyes were incorporated into the reporters at five molar ratios. The reporters were loaded on 10μm amine functionalized polystyrene microspheres in eleven different combinations of between three and seven codes to evaluate the utility of the reporters to form a colloidal barcode. Following characterization, the reporters were used to encode a combinatorial peptide library that was applied to profiling the affinity of the West Nile flavivirus protease (WNV-pro). Varying the ratios of energy transfer dyes served as an innovative route to produce unique spectral signatures.