Integration of Polyaniline/Poly(acrylic acid) Films and Redox Enzymes on Electrode Supports: An in Situ Electrochemical/Surface Plasmon Resonance Study of the Bioelectrocatalyzed Oxidation of Glucose or Lactate in the Integrated Bioelectrocatalytic Systems

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Abstract
Electropolymerization of aniline in the presence of poly(acrylic acid) on Au electrodes yields a polyaniline/poly(acrylic acid) composite film, exhibiting reversible redox functions in aqueous solutions at pH = 7.0. In situ electrochemical-SPR measurements are used to identify the dynamics of swelling and shrinking of the polymer film upon the oxidation of the polyaniline (PAn) to its oxidized state (PAn2+) and the reduction of the oxidized polymer (PAn2+) back to its reduced state (PAn), respectively. Covalent attachment of N6-(2-aminoethyl)-flavin adenin dinucleotide (amino-FAD, 1) to the carboxylic groups of the composite polyaniline/poly(acrylic acid) film followed by the reconstitution of apoglucose oxidase on the functional polymer yields an electrically contacted glucose oxidase of unprecedented electrical communication efficiency with the electrode: electron-transfer turnover rate ∼1000 s-1 at 30 °C. In situ electrochemical-SPR analyses are used to characterize the bioelectrocatalytic functions of the biomaterial−polymer interface. The current responses of the bioelectrocatalytic system increase as the glucose concentrations are elevated. Similarly, the SPR spectra of the system are controlled by the concentration of glucose. The glucose concentration controls the steady-state concentration ratio of PAn/PAn2+ in the film composition. Therefore, the SPR spectrum of the film measured upon its electrochemical oxidation is shifted from the spectrum typical for the oxidized PAn2+ at low glucose concentration to the spectrum characteristic of the reduced PAn at high glucose concentration. Similarly, the polyaniline/poly(acrylic acid) film acts as an electrocatalyst for the oxidation of NADH. Accordingly, an integrated bioelectrocatalytic assembly was constructed on the electrode by the covalent attachment of N6-(2-aminoethyl)-β-nicotinamide adenine dinucleotide (amino-NAD+, 2) to the polymer film, and the two-dimensional cross-linking of an affinity complex formed between lactate dehydrogenase and the NAD+-cofactor units associated with the polymer using glutaric dialdehyde as a cross-linker. In situ electrochemical-SPR measurements are used to characterize the bioelectrocatalytic functions of the system. The amperometric responses of the system increase as the concentrations of lactate are elevated, and an electron-transfer turnover rate of 350 s-1 between the biocatalyst and the electrode is estimated. As the PAn2+ oxidizes the NADH units generated by the biocatalyzed oxidation of lactate, the PAn/PAn2+ steady-state ratio in the film is controlled by the concentration of lactate. Accordingly, the SPR spectrum measured upon electrochemical oxidation of the film is similar to the spectrum of PAn2+ at low lactate concentration, whereas the SPR spectrum resembles that of PAn at high concentrations of lactate.

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