Fluorescence anisotropy: application in quantitative enzymatic determinations

In this work a method is presented for the enzymatic determination of glucose using fluorescence anisotropy. During the enzymatic reaction a change in the fluorescence anisotropy of the glucose oxidase (GOx) is produced; the reaction time at which this change appears (tm) depends on the glucose concentration. A theoretical study has been developed which enables: (a) the correlation of this change in anisotropy with changes in the intensity and the lifetime of the enzyme fluorescence; from this a model which could be generalized to other flavo-enzymes is proposed; (b) the linking of tm with glucose concentration. optimisation, the proposed method allows the determination of glucose over the range 100–1000 mg l−1. The detection limit is 90 mg l−1and the reproducibility is better than 4% (n = 6, [glucose] = 250 mg l−1). Anisotropy is more selective than conventional fluorescence intensity, and this method has therefore been applied to direct glucose determination in fruit juices without the interference caused by the inner filter effect.