Time-resolved enzymatic determination of glucose using a fluorescent europium probe for hydrogen peroxide

An enzymatic assay for glucose based on the use of the fluorescent probe for hydrogen peroxide, europium(III) tetracycline (EuTc), is described. The weakly fluorescent EuTc and enzymatically generated H2O2 form a strongly fluorescent complex (EuTc-H2O2) whose fluorescence decay profile is significantly different. Since the decay time of EuTc-H2O2 is in the microseconds time domain, fluorescence can be detected in the time-resolved mode, thus enabling substantial reduction of background fluorescence. The scheme represents the first H2O2-based time-resolved fluorescence assay for glucose not requiring the presence of a peroxidase. The time-resolved assay (with a delay time of 60 mus and using endpoint detection) enables glucose to be determined at levels as low as 2.2 mumol L-1, with a dynamic range of 2.2-100 mumol L-1. The method also was adapted to a kinetic assay in order to cover higher glucose levels (mmol L-1 range). The latter was validated by analyzing spiked serum samples and gave a good linear relationship for glucose levels from 2.5 to 55.5 mmol L-1. Noteworthy features of the assay include easy accessibility of the probe, large Stokes' shift, a line-like fluorescence peaking at 616 nm, stability towards oxygen, a working pH of approximately 7, and its suitability for both kinetic and endpoint determination.