Tuned Amperometric Detection of Reduced β‑Nicotinamide Adenine Dinucleotide by Allosteric Modulation of the Reductase Component of the <i>p</i>‑Hydroxyphenylacetate Hydroxylase Immobilized within a Redox Polymer

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. <i>p</i>-hydroxy-phenylacetic acid (<i>p</i>-HPA), allowing the acceleration of the substrate conversion rate on the sensor surface by in situ addition or preincubation with <i>p</i>-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