A homogeneous assay principle for universal substrate quantification via hydrogen peroxide producing enzymes

Abstract

H2O2 is a widely occurring molecule which is also a byproduct of a number of enzymatic reactions. It can therefore be used to quantify the corresponding enzymatic substrates. In this study, the time-resolved fluorescence emission of a previously described complex consisting of phthalic acid and terbium (III) ions (PATb) is used for H2O2 detection. In detail, glucose oxidase and choline oxidase convert glucose and choline, respectively, to generate H2O2 which acts as a quencher for the PATb complex. The response time of the PATb complex toward H2O2 is immediate and the assay time only depends on the conversion rate of the enzymes involved. The PATb assay quantifies glucose in a linear range of 0.02–10mmolL−1, and choline from 1.56 to 100μmolL−1 with a detection limit of 20μmolL−1 for glucose and 1.56μmolL−1 for choline. Both biomolecules glucose and choline could be detected without pretreatment with good precision and reproducibility in human serum samples and infant formula, respectively. Furthermore, it is shown that the detected glucose concentrations by the PATb system agree with the results of a commercially available assay. In principle, the PATb system is a universal and versatile tool for the quantification of any substrate and enzyme reaction where H2O2 is involved.