Alcohol biosensors based on coupled oxidase-peroxidase systems

Amperometric alcohol biosensors were constructed by co-immobilising commercially available alcohol oxidase (AOD, EC 1.1.3.13) from various sources (Candida boidinii, Pichia pastoris and Hansenula polymorpha) with the hydrogen peroxide reducing enzyme, horseradish peroxidase, in a carbon paste matrix. Biosensors were built based on two different approaches, i.e. direct and mediated electron transfer. Previously shown efficient activators/stabilisers, such as polyethylenimine and lactitol were added to the non-mediated systems. Electrode characteristics were compared with those obtained for similarly constructed biosensors, based on mediated reduction of hydrogen peroxide. An osmium containing three-dimensional redox hydrogel, (poly[l-vinyl imidazole osmium (4,4ʹ-dimethyl-bipyridine)2Cl])+2+, was used to “wire” horseradish peroxidase. After preliminary screening, two compositions were found to yield the best characteristics (sensitivity, selectivity, operational and storage stability, ethanol conversion). These were AOD from Candida boidinii coupled with horseradish peroxidase and stabilised with polyethylenimine and AOD from Pichia pastoris coupled with “wired” horseradish peroxidase. The electrodes were operated in flow injection mode at a working potential of −0.05 V vs. AgAgCl (0.1 M KC1).