OPTICAL SENSING OF GLUCOSE USING PHASE-MODULATION FLUOROMETRY

We describe a fluorescence assay of glucose based on fluorescence resonance energy transfer and phase-modulation measurements of the donor decay times. The assay is based on the decreased decay time of a donor fluorophore linked to Concanavalin A (ConA) upon binding of acceptor-labeled sugars. Displacement of the labeled sugars by glucose results in a decrease in energy transfer and an increase in the donor decay time. The assay was demonstrated with several donor-acceptor pairs, demonstrating the robustness and generality of this approach. A competitive glucose assay was demonstrated with both low-molecular-weight acceptors, and with acceptor-labeled dextran. Use of a polymeric acceptor would allow the glucose sensor to be placed behind a glucose-permeable barrier, as may be needed in clinical applications. The use of energy transfer allows the selection of excitation and emission wavelengths compatible with the desired light sources and optical properties of the samples. The use of fluorescence decay times rather than intensities, makes the measurements mostly independent of probe photobleaching, light losses in the optics, instrumental drifts, and mostly independent of scattering and/or absorption of the sample.