Reusable ethanol sensor based on a NAD+-dependent dehydrogenase without coenzyme addition

An amperometric sensor for the detection of ethanol has been designed. The sensing layer consists of alcohol dehydrogenase (ADH), NADH oxidase and NAD+-dextran, entrapped together in a matrix of poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ). Measurements have been carried out at a low oxidizing potential (equivalent to 250 mV vs. SCE) to detect hexacyanoferrate(II), thus avoiding interferences due to presence of oxidizable compounds in real samples. The influence of the amount of polymer, enzymes and coenzyme in the sensing layer on the sensor sensitivity, linear range and operational stability has been studied. The sensitivity was close to 2 mA 1 mol−1, with a linear range 3 × 10−7 −2 × 10−4M and a response time <2 min. Good operational stability was observed, allowing more than 40 reproducible assays without NAD+ addition. Alcoholic beverages were analysed with the use of sensor and the results showed good correlation with those obtained using a standard spectrophotometric procedure.