Preparation of a carbon paste/alcohol dehydrogenase electrode using polyethylene glycol-modified enzyme and oil-soluble mediator

A carbon paste (CP)/enzyme electrode was prepared from a mixture of CP, NAD (+), polyethylene glycol-modified alcohol dehydrogenase and an oil-soluble mediator, 7-dimethylamine-2-methyl-3-beta-naphtamido-phenothiazinium chlori de (3-NTB) by the ratio of 7:1:0.8:0.7 in weight. The current response for the electrode was proportional to the ethanol concentration up to 1 mM, and the response time was ca. 1 min. Lower detection limit of ethanol was 50 m u M (S/N = 5). The use of 3-NTB, an oil-soluble mediator, was highly effect ive for obtaining the CP-based sensor with high stability: the magnitude of the ethanol response on the 3-NTB-incorporated electrode did not change du ring 2 weeks, whereas the CP/enzyme electrode incorporated with Toluidine b lue (TB), an oil-insoluble mediator, showed much lower stability. Within 2 days, the magnitude of ethanol response for the TB-based electrode became h alf of the initial value. The enzyme electrode was applied to the ethanol d etermination in food samples: the results obtained agreed well with those o btained by the F-kit method. The enzyme electrode would be used as an ethan ol biosensor. (C) 2000 Elsevier Science S.A. All rights reserved.