Homogeneous non-competitive bioaffinity assay based on fluorescence resonance energy transfer

A homogeneous non-competitive assay principle for measurement of small analytes based on quenching of fluorescence is described. Fluorescence resonance energy transfer (FRET) occurs between the donor, intrinsically fluorescent europium(III)-chelate conjugated to streptavidin, and the acceptor, quencher dye conjugated to biotin derivative when the biotin-quencher is bound to Eu-streptavidin. Fluorescence can be measured only from those streptavidins that are bound to biotin of the sample, while the fluorescence of the streptavidins that are not occupied by biotin are quenched by quencher-biotin conjugates. The quenching efficiencies of the non-fluorescent quencher dyes were over 95% and one dye molecule was able to quench the fluorescence of more than one europium(III)-chelate. This, however, together with the quadrovalent nature of streptavidin limited the measurable range of the assay to 0.2-2 nmol L⁻¹. In this study we demonstrated that FRET could be used to design a non-competitive homogeneous assay for a small analyte resulting in equal performance with competitive heterogeneous assay.