Ethanol determination by an amperometric bienzyme sensor based on a Clark-type transducer

The aim of this work is to develop a method for ethanol determination in some alcoholic beverages, by using an alcohol dehydrogenase/peroxidase-based amperometric enzyme electrode. The enzymes alcohol dehydrogenase and horseradish peroxidase, as well as the coenzyme nicotinamide adenine dinucleotide (NAD+) were immobilized on a nylon (Biodyne A) membrane. The enzyme-membrane was consecutively attached to the polyethylene membrane of a Clark oxygen electrode, which functioned as a transducer. Ethanol is oxidized by NAD+ in the presence of alcohol dehydrogenase; the NADH produced is aerobically oxidized by horseradish peroxidase. The rate of molecular oxygen consumption, which is directly proportional to the alcohol concentration in the sample, is amperometrically monitored with the oxygen electrode-based biosensor. alytical characteristics of the biosensor (linear range, sensitivity, selectivity, response time, stability) were investigated. The value of the current intensity was monitored as a function of time, for different ethanol concentrations. The obtained calibration graph was linear within the range 10–80 mM. The values of the ethanol content obtained for the analysed beverages ranged between 4.56% (v/v) for Belheimer beer and 41.83% for Stalinskaya vodka.