title: 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 0.02-10 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.
authors.affiliation: Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.
authors.forename: Kristin
authors.initials: K
authors.lastname: Zscharnack
authors.affiliation: Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Kreisig
authors.affiliation: Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.
authors.forename: Agneta A
authors.initials: AA
authors.lastname: Prasse
authors.affiliation: Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany; Center for Biotechnology and Biomedicine, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany. Electronic address: Thole.Zuechner@octapharma.com.
authors.forename: Thole
authors.initials: T
authors.lastname: Zuchner
mesh_terms: D001786:Blood Glucose / Q000032:analysis; D002794:Choline / Q000032:analysis; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007225:Infant Food / Q000032:analysis; D013379:Substrate Specificity
chemical_list: D001786:Blood Glucose; D006861:Hydrogen Peroxide; D002794:Choline
keywords: Choline; Glucose; Kinetics; Matrix compatibility; Oxidase; Time-resolved fluorescence