Direct determination of glucose in serum by fluorimetry using a labeled enzyme

Abstract

In this paper we propose the use of derivatized glucose oxidase (GOx) with fluorescein-5(6)-carboxamido-caproic acid ester (FS) for the direct fluorometric determination of glucose in blood serum. This method is based on the modification of the fluorescence of GOx–FS ( λ excitation=489 nm and λ emission=520 nm) with the analyte concentration during its enzymatic reaction with the glucose. This makes a second enzymatic reaction unnecessary. The mechanism by which the change in the fluorescence of GOx–FS takes place is discussed and a mathematical equation which models the process is given. The derivatization and measurement conditions were optimized: in the optimum conditions GOx–FS is stable for at least 3 weeks, the detection limit obtained is 85 mg l −1 and the precision 2.5% ( n=9). The results were compared with those obtained by a commercial clinical analyzer and no systematic errors were observed.