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-β-naphtamido-phenothiazinium chloride (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 μM (S/N=5). The use of 3-NTB, an oil-soluble mediator, was highly effective for obtaining the CP-based sensor with high stability: the magnitude of the ethanol response on the 3-NTB-incorporated electrode did not change during 2 weeks, whereas the CP/enzyme electrode incorporated with Toluidine blue (TB), an oil-insoluble mediator, showed much lower stability. Within 2 days, the magnitude of ethanol response for the TB-based electrode became half of the initial value. The enzyme electrode was applied to the ethanol determination in food samples: the results obtained agreed well with those obtained by the F-kit method. The enzyme electrode would be used as an ethanol biosensor.