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 Polymer

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.

Drug Release from Polymer Thin Films and Gel Pellets: Insights from Programmed Microplate Electroanalysis

“… Molecular structures of the target analyte paracetamol (PCT), the commercially available polymers chitosan and agarose and the artificial polymers (PS(SS‐GMA‐BA)‐S1 and P(SS‐GMA‐BA)‐S2). P(SS‐GMA‐BA)‐S1 and P(SS‐GMA‐BA)‐S2 were prepared following previously described procedures with a nominal composition of x=50 mol%, y=30 mol%, z=20 mol% and x=80 mol%, y=10 mol%, z=10 mol%, respectively. Sulfonate groups (blue) in the backbone confer pH sensitivity, while epoxide groups (red) readily react with nucleophiles (e. g. amines in chitosan, green) for chain crosslinking.…”

“…Following previously published protocols for polymer synthesis, poly(4‐styrene sulfonate‐ co ‐glycidyl methacrylate‐ co ‐butyl acrylate), abbreviated P(SS‐GMA‐BA), was prepared in two forms that varied only in the relative ratios of the incorporated co‐monomers (SS=4‐styrene sulfonate, GMA=glycidyl methacrylate, and BA=butyl acrylate) entities. Polymer P(SS‐GMA‐BA)‐S1 had nominal SS, GMA and BA contents of 50, 30 and 20 mol‐% while polymer P(SS‐GMA‐BA)‐S2 had an 80 : 10 : 10 mol‐% ratio.…”

A fully protected hydrogenase/polymer-based bioanode for high-performance hydrogen/glucose biofuel cells

“…The architecture of the proposed O 2 -protected hydrogenase bioanode is based on a polymer multilayer architecture comprising an active H 2 oxidation layer in which a hydrogenase, i.e., [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F ( Dv MF-[NiFe]) 47 or [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough ( Dv H-[NiFeSe]) 48 , is wired via a low-potential viologen-modified polymer, i.e., the polymer matrix P(N 3 MA-BA-GMA)-vio (poly(3-azido-propyl methacrylate- co -butyl acrylate- co -glycidyl methacrylate)-viologen, Fig. 1 , right) 17 , and an O 2 removing top layer composed of an oxidase and catalase entrapped in the redox-silent hydrophilic polymer matrix P(SS-GMA-BA) (poly(4-styrenesulfonate- co -glycidyl methacrylate- co -butyl acrylate) 49 , Fig. 1 , right).…”

“…1 ) was described in ref. 49 , the nominal concentration of the individual co-monomers was k = 50 mol%, l = 30 mol%, and m = 20 mol%. Note that due to overlapping signals in the NMR spectrum of P(SS-GMA-BA) the actual composition cannot be determined via the integral ratios.…”

Molecularly imprinted polymeric shell coated monodisperse‐porous silica microspheres as a stationary phase for microfluidic boronate affinity chromatography

“…Hence, the main function of β‐NAD is to perform the electron transfer reactions in various chemical and biological systems. The isolation/purification of β‐NAD is of crucial importance since it is widely used in many applications including biosensors, biofuel cells and hydrogen production . For testing of boronate affinity isolation performance of imprinted sorbent, β‐nicotinamide adenine dinucleotide, β‐NAD was selected as a model target carrying two cis‐diol groups.…”