Amperometric Flow Injection Analysis of Glucose and Galactose Based on Engineered Pyranose 2-Oxidases and Osmium Polymers for Biosensor Applications

In the present study, wild type and three mutants of pyranose 2-oxidase (PyOx-WT, PyOx-MT1, PyOx-MT2, PyOx-MT3), which showed improved properties for D-galactose oxidation, were investigated for their oxidising ability when immobilised on graphite electrodes. Four different flexible Os polymers with formal potentials ranging between -0.140 and 0.270V vs. Ag|AgCl0.1MKCl were applied together with the various forms of PyOx to wire graphite electrodes using polyethylene glycol diglycidyl ether as crosslinking reagent. The pH profiles for the electrodes modified with wild type and all PyOx mutants in combination with the Os polymers were investigated with both glucose and galactose, respectively, since the PyOx variants showed an improved catalytic activity for galactose. All modified electrodes showed highest response in the pH range between 8.5-10 and K-M, I-max values for both glucose and galactose were determined. To prove the catalytic activity, the biosensors were also characterized with cyclic voltammetry. A protein amount 0.26U was found optimum for PyOx-WT, 0.36U for PyOx-MT1, 0.41U for PyOx-MT2 and 0.28U for PyOx-MT3 and the analytical characterization of the enzyme electrodes was performed for glucose and galactose under optimized conditions.