Spectrophotometric or fluorometric methods with glucose oxidase (GOD)–peroxidase (POD)–chromogen(s) systems

oxidase (GOD)–peroxidase (POD)–chromogen(s) systems are well known as useful tools for determination of glucose. Indicator reactions in these methods are based on oxidative formation of a colored or fluorescent substance from a chromogen(s) with H2O2, generated through GODcatalyzed oxidation of glucose, in the presence of POD. These color reactions are well-characterized chemical processes. However, the POD-dependent indicator reactions are known to be inevitably disturbed by electron donors present in biological samples such as ascorbic acid, uric acid, and bilirubin. Major processes of interference by these compounds are as follows: 1) reduction of oxidatively formed colored or fluorescent substance to its original chromogen(s); and 2) competition with a chromogen in reduction of H2O2. Several methodologies have been developed for elimination of such interference in glucose determination with GOD–POD–chromogen(s) systems. However, the most straightforward approach to glucose determination free from such interference is design of a novel indicator reaction without recourse to redox reactions coupled with H2O2 and POD. To our knowledge, only a few methods with non-redox color reactions for glucose determination using only GOD have been reported. These methods utilize transformation of a vanadium(V), titanium(IV) or dinuclear iron(III) complex to its adduct with H2O2 accompanied by a bathochromic shift as a spectrophotometric indicator reaction. As expected, it was demonstrated that glucose determination with vanadium(V) and titanium(IV) complexes was not affected by various substances usually present in serum or added to test solution. However, spectrophotometric or fluorometric determination of glucose with high accuracy should use formation of a colored or fluorescent substance from a chromogen rather than a color change of a dye as an indicator reaction. Recently, resorufin (2) was shown to be able to reoxidize a reduced form of GOD at 37 °C, being transformed to a colorless dihydro derivative, although the reductive bleaching of the dye is of no use for GOD-based determination of glucose as an indicator reaction. This finding prompted us to examine how resorufin derivatives such as acetyl resorufin (1) would behave in GOD-catalyzed oxidation of glucose, and it was found that 1 behaves in a manner different from 2 in the enzymatic reaction. Here, we report that deacetylation of non-fluorescent 1 to fluorescent 2 is induced by H2O2 generated in GOD-catalyzed oxidation of glucose, and the transformation is promising as an indicator reaction for fluorometric determination of glucose without significant effects of ascorbic acid, uric acid, or bilirubin.