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

• Application of the TRF-based PATb system for universal oxidase substrate detection.
• H2O2 generated by choline or glucose oxidase quenches the TRF signal of PATb.
• The assay time is only limited by the oxidase catalysis rate.
• Glucose is precisely detected in human serum consistent to a commercial assay.
• A reliable quantification of choline in infant formula is shown.

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 0.02–10 mmol L−1, and choline from 1.56 to 100 μmol L−1 with a detection limit of 20 μmol L−1 for glucose and 1.56 μmol L−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.

Figure optionsDownload as PowerPoint slide