Amperometric Detection of the Urinary Disease Biomarker p-HPA by Allosteric Modulation of a Redox Polymer-Embedded Bacterial Reductase

Teanphonkrang, S. and Ernst, A. and Janke, S. and Chaiyen, P. and Sucharitakul, J. and Suginta, W. and Khunkaewla, P. and Schuhmann, W. and Schulte, A. and Ruff, A.

Volume: 4 Pages: 1270-1278
DOI: 10.1021/acssensors.9b00144
Published: 2019

We report an amperometric biosensor for the urinary disease biomarker para-hydroxyphenylacetate (p-HPA) in which the allosteric reductase component of a bacterial hydroxylase, C1-hpah, is electrically wired to glassy carbon electrodes through incorporation into a low-potential Os-complex modified redox polymer. The proposed biosensing strategy depends on allosteric modulation of C1-hpah by the binding of the enzyme activator and analyte p-HPA, stimulating oxidation of the cofactor NADH. The pronounced concentration-dependence of allosteric C1-hpah modulation in the presence of a constant concentration of NADH allowed sensitive quantification of the target, p-HPA. The specific design of the immobilizing redox polymer with suitably low working potential allowed biosensor operation without the risk of co-oxidation of potentially interfering substances, such as uric acid or ascorbic acid. Optimized sensors were successfully applied for p-HPA determination in artificial urine, with good recovery rates and reproducibility and sub-micromolar detection limits. The proposed application of the allosteric enzyme C1-hpah for p-HPA trace electroanalysis is the first successful example of simple amperometric redox enzyme/redox polymer biosensing in which the analyte acts as an effector, modulating the activity of an immobilized biocatalyst. A general adVantage of the concept of allosterically modulated biosensing is its ability to broaden the range of approachable analytes, through the move from substrate to effector detection. © 2019 American Chemical Society.

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