Amperometric biosensing of ethanol based on integration of alcohol dehydrogenase with a Pt/PPy–PVS/MB electrode

A novel amperometric ethanol biosensor with alcohol dehydrogenase (ADH) and nicotinamide adenine dinucleotide (NAD^+) on polypyrrole–polyvinylsulfonate (PPy-PVS) film was accomplished. Meldola’s blue (MB) was used as a mediator; it can facilitate the electron transfer that involves a chemical interaction between the electrode surface and NADH. Determination of ethanol was carried out by the oxidation of MB at –0.072 V vs. Ag/AgCl. The effects of pH and temperature were investigated and optimum parameters were found to be 7.0 and 40 °C, respectively. There are 2 linear parts at 1.0 µM–10.0 µM (R ² = 0.993) and 0.01 mM–0.1 mM (R ² = 0.996). The storage stability and operational stability of the enzyme electrode were also studied. The results showed that 95.7% of the response current was retained after 17 activity assays. The ethanol biosensor gave perfect reproducible finding with 4.6% relative standard deviation. The prepared biosensor retained 55.8% of initial activity after 24 days when stored in 0.1 M phosphate buffer solution at 4 °C. Effects of the several possible interfering substances such as ascorbic acid, citric acid, and methanol on the ethanol biosensor were investigated and the developed biosensor was tested in alcoholic beverages.