Measurements of fluorescence resonance energy transfer in the concanavalin A-dextran affinity system using frequency-domain lifetime spectroscopy

Abstract

A glucose affinity sensor based on a homogeneous fluorescence resonance energy transfer (FRET) assay system was developed to monitor the competitive binding between concanavalin A (con A), a sugar-binding protein labeled with acceptor fluorophore, Alexa Fluor 647 (AF647) and polysaccharides conjugated with donor fluorophore, Alexa Fluor 568 (AF568). Confounding factors such as: (i) the impact of scattering due to tissue optical properties; (ii) the reabsorption of propagated donor fluorescence by the acceptor fluorophore; (iii) photobleaching; and (iv) fluorophore loading must be accounted for before quantitative glucose measurements can be made from fluorescence intensity measurements. Fluorescence lifetime spectroscopy made in the frequency domain circumvents most of these artifacts by measuring phase-shift and modulation ratio related with the fluorophore lifetime change. Experiments were performed to assess the FRET effects of this affinity sensing system, using dextran (MW 2000K) labeled with donor molecule, AF568 (donor-dextran), and con A labeled with acceptor molecule, AF647 (acceptor- con A). Herein we demonstrate that the FRET decay kinetics can indicate changes in the competitive binding of 0.09 μM dextran as con A concentration (from 0 to 10.67 μM) and glucose concentration (from 0 to 224 mg/dL) are changed. Preliminary work also presented here shows that quantitative frequency-domain lifetime measurement of FRET changes could be achieved in tissue-like scattering media using the photon diffusion equation.