The accurate determination of ethanol is important in food technology, forensic laboratories, as well as in clinical
analysis. Methods of ethanol assessment include spectrometry, gas chromatography, high performance liquid chromatography,
but also biosensors, mainly with electrochemical or optical detection.
The purpose of this study is the investigation of the role and influence of alcohol dehydrogenase and horseradish peroxidase
amounts on the analytical characteristics of a bienzyme amperometric sensor for ethanol. The reduced form of the
coenzyme, NADH, resulted in the alcohol dehydrogenase-catalysed reaction, is oxidized by molecular oxygen, under the
influence of horseradish peroxidase. Both enzymes were coimmobilized with NAD+ on a nylon membrane, fixed on an
oxygen electrode. The oxygen consumption, proportional to the NADH amount, can be correlated to the ethanol concentration.
The analytical performances of the biosensor were studied at alcohol dehydrogenase loadings ranging between
100 and 900 units and horseradish peroxidase loadings ranging between 0 and 30 units. The improvement of the bienzyme
sensor allowed ethanol assessment in wine and beer.