Tuned Amperometric Detection of Reduced β-Nicotinamide Adenine Dinucleotide by Allosteric Modulation of the Reductase Component of the p-Hydroxyphenylacetate Hydroxylase Immobilized within a Redox PolymerClick to copy article linkArticle link copied!
- Somjai TeanphonkrangSomjai TeanphonkrangSchool of Chemistry, Institute of Science, Biochemistry−Electrochemistry Research Unit (BECRU), Suranaree University of Technology, 30000 Nakhon Ratchasima, ThailandMore by Somjai Teanphonkrang
- Salome JankeSalome JankeAnalytical Chemistry, Center for Electrochemical Sciences (CES), Ruhr-University Bochum, 44780 Bochum, GermanyMore by Salome Janke
- Pimchai ChaiyenPimchai ChaiyenSchool of Biomolecular Science and Engineering (BSE), Vidyasirimedhi Institute of Science and Technology (VISTEC), 21210 Rayong, ThailandMore by Pimchai Chaiyen
- Jeerus SucharitakulJeerus SucharitakulDepartment of Biochemistry, Faculty of Dentistry, Chulalongkorn University, 10330 Bangkok, ThailandMore by Jeerus Sucharitakul
- Wipa SugintaWipa SugintaSchool of Chemistry, Institute of Science, Biochemistry−Electrochemistry Research Unit (BECRU), Suranaree University of Technology, 30000 Nakhon Ratchasima, ThailandCenter of Excellence (CoE) in Advanced Functional Materials, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, ThailandMore by Wipa Suginta
- Panida KhunkaewlaPanida KhunkaewlaSchool of Chemistry, Institute of Science, Biochemistry−Electrochemistry Research Unit (BECRU), Suranaree University of Technology, 30000 Nakhon Ratchasima, ThailandMore by Panida Khunkaewla
- Wolfgang SchuhmannWolfgang SchuhmannAnalytical Chemistry, Center for Electrochemical Sciences (CES), Ruhr-University Bochum, 44780 Bochum, GermanyMore by Wolfgang Schuhmann
- Adrian Ruff*Adrian Ruff*(A.R.) Tel.: +49-234-3225586. E-mail: [email protected]Analytical Chemistry, Center for Electrochemical Sciences (CES), Ruhr-University Bochum, 44780 Bochum, GermanyMore by Adrian Ruff
- Albert Schulte*Albert Schulte*(A.S.) Tel.: +66-8737-58518. E-mail: [email protected]School of Biomolecular Science and Engineering (BSE), Vidyasirimedhi Institute of Science and Technology (VISTEC), 21210 Rayong, ThailandMore by Albert Schulte
Abstract
We report the fabrication of an amperometric NADH biosensor system that employs an allosterically modulated bacterial reductase in an adapted osmium(III)-complex-modified redox polymer film for analyte quantification. Chains of complexed Os(III) centers along matrix polymer strings make electrical connection between the immobilized redox protein and a graphite electrode disc, transducing enzymatic oxidation of NADH into a biosensor current. Sustainable anodic signaling required (1) a redox polymer with a formal potential that matched the redox switch of the embedded reductase and avoided interfering redox interactions and (2) formation of a cross-linked enzyme/polymer film for stable biocatalyst entrapment. The activity of the chosen reductase is enhanced upon binding of an effector, i.e. p-hydroxy-phenylacetic acid (p-HPA), allowing the acceleration of the substrate conversion rate on the sensor surface by in situ addition or preincubation with p-HPA. Acceleration of NADH oxidation amplified the response of the biosensor, with a 1.5-fold increase in the sensitivity of analyte detection, compared to operation without the allosteric modulator. Repetitive quantitative testing of solutions of known NADH concentration verified the performance in terms of reliability and analyte recovery. We herewith established the use of allosteric enzyme modulation and redox polymer-based enzyme electrode wiring for substrate biosensing, a concept that may be applicable to other allosteric enzymes.
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