Phase-resolved fluorimetric determination of two albumin-bound fluorescein species

Abstract

A new approach is described for phase-resolved fluorescence spectroscopy, for use in resolving mixtures of two components with very similar fluorescence spectra and life-times. Results are given for application of the technique to solutions containing fluorescein physically bound to albumin and fluorescein isothiocyanate covalently bound to albumin. Because the two fluorescein species have essentially identical fluorescence spectra and a phase-angle difference of only 2°, the conventional phase-resolved method in which measurements are made at the two phase angles at which the fluorescence contribution from one or the other of the components is zero will not resolve the components. Solutions containing 25–50 nM of each component were successfully resolved by making measurements at two other phase angles and solving the pair of simultaneous equations that is generated. Accuracy is best (average relative error, 3%) using detector phase angles corresponding to a 45° shift from the phase angles of the components. Relative standard deviations of ±16% are obtained at these phase angles. Solutions containing 5–50 nM fluorescein and 50–500 nM fluorescein isothiocyanate conjugated to albumin could also be resolved, with an average relative error of 16% and ±2.4 r.s.d. The method could be used for simultaneous determination of a fluorophore in two different microenvironments, as in protein-ligand binding studies and in homogeneous immunoassay.