Dynamics of fluorescence dequenching of ostrich-quenched fluorescein biotin: A multifunctional quantitative assay for biotin

We describe a simple and rapid quantitative assay for biotin and biotin conjugates. The assay is based on the kinetic analysis of the enhancement of fluorescence of streptavidin/fluorescein biotin complexes in the presence of biotin. The kinetic response of fluorescence enhancement is proportional to the concentration of biotin. Standard calibration curves based on the kinetic response are obtained and detection limits of ≈10−9 M are established. Because the assay is amenable for use in small volumes of 5-50 μL or bead-based assays, the detection limits can be extended to the femtomole range. Since the assay depends on kinetic analysis, routine quantitation can be achieved without reference to standard curves. The dynamic aspects allow the assay to be extended to a broader range of applications including its use as an indicator of reagent mixing in laminar-flow assays carried out in microfluidic devices.