Amperometric on-line sensor for continuous measurement of hypoxanthine based on osmium-polyvinylpyridine gel polymer and xanthine oxidase bienzyme modified glassy carbon electrode Original Research Article

Osmium-polyvinylpyridine gel polymer (Os-gel-horseradish peroxide (HRP)) and xanthine oxidase (XOD) bienzyme-based amperometric biosensor was developed for use in a thin-layer radial flow cell for on-line measurement of hypoxanthine. Glassy carbon electrodes (GCEs) were initially cast-coated with Os-gel-HRP with a surface coverage of 7.1 μl/cm2 to sense hydrogen peroxide produced from XOD catalytic enzyme reaction based on HRP catalytic reduction mediated by osmium. Then, bovine serum albumin (BSA)/XOD was cross-linked to the Os-gel-HRP-coated glassy carbon electrodes (GCEs) with glutaraldehyde vapor for the measurement of hypoxanthine at 0.0 mV (versus Ag/AgCl). The resultant HRP and XOD bienzyme-based sensors (Os-gel-HRP/XOD/GCEs) were tested with techniques of cyclic voltammetry (CV), on-line amperometry in a flow system and flow injection analysis (FIA). In continuous-flow amperometric experiments, the Os-gel-HRP/XOD/GCEs were quick to stabilize background current and show linear and sensitive response towards hypoxanthine in a concentration range from 0.5 to 80 μM with a detection limit of 0.2 μM (S/N=3). The specificity against ascorbic acid was enhanced by negatively shifting the operating potential to −200 mV versus Ag/AgCl and was demonstrated to be acceptable for on-line performance. The interference from higher than physiological levels of dopamine (DA), norepinephrine (NE), serotonin, their principal metabolites and uric acid was minimal. The dependency of sensor response on pH value of the buffer and operating potential was also tested and optimized.