Dual functionalities of 4-aminodiphenylamine in enzymatic assay and mediated biosensor construction

4-Aminodiphenylamine (N-phenyl-1,4-phenylenedi-amine, CAS 101-54-2) and its water-soluble HCl salt (CAS 2198-59-6) were demonstrated to be efficient mediators for glucose oxidase, lactate oxidase, xanthine oxidase, and lysine oxidase. Using cyclic voltammetry, single oxidative peak potentials were observed for scans ranging from 0 to 0.5 V vs Ag/AgCl. The half-wave potential for both preparations was 0.11 V vs Ag/AgCl at pH 7 and decreased 59 mV per unit pH increase. Peak current data were analyzed to estimate diffusivities of 0.8 × 10^-5cm²/s for soluble 4-ADPA HCl, and 2.36 × 10^-5cm²/s for 4-ADPA solubilized in 2.5 mM2-hydroxypropyl-β-cyclodextrin. The overall second-order kinetic constants (k) for the reaction of reduced glucose oxidase with oxidized 4-ADPA HCl and 4-ADPA in cyclodextrin were estimated to be 1.8 × 10⁵and 1.7 × 10⁵M^-1s^-1, respectively, using cyclic voltammetry measurements at varied scan rates and enzyme concentrations. Both preparations proved to be suitable electron acceptors for horseradish peroxidase, as indicated by changes in absorbance spectra upon oxidation or reduction. The electrochemical and spectral behavior of the preparations were applied in conjunction with glucose oxidase to devise mediated amperometric and hydrogen peroxide-coupled spectrophotometric assays for glucose. The results of both assays compared favorably with the hexokinase reference method.