Rapid assay for avidin and biotin based on fluorescence quenching

Biotin was covalently tagged with a BODIPY dye which can undergo an efficient distance-dependent fluorescence self-quenching. Multivalent binding of avidin with the BODIPY-labeled biotin (B581/591-biotin, either in aqueous buffer, or anchored on the surfaces of lipid vesicles or lipid bilayers coated on glass beads) induces aggregation of the BODIPY dye (up to four dyes for each avidin) to result in a decrease in fluorescence intensity due to fluorescence self-quenching. The system can be used to perform a rapid, direct assay for avidin and competitive assay for biotin with high sensitivity (<50 pM for avidin and <0.2 nM for biotin) and selectivity. The assay method is generally applicable for detection of all the species involved in a multivalent binding interaction.