The enzyme requires O2 to oxidize the ethanol and the products formed are acetaldehyde and hydrogen peroxide. Since, AOX enzymatically converts all primary alcohols and formaldehyde [12], it suffers from a lack of selectivity to ethanol. However, this should not be a problem in the use of such a biosensor for analysis of ethanol in fermented beverage samples, since ethanol is present at much higher levels. The main problem of AOX-based biosensors is their limited stability. For this reason, several ways of stabilizing AOX in the dry state using a combination of polyelectrolytes and sugar derivatives have been studied [13,14].In this work the development of a novel and simple visual ethanol biosensor based on AOX immobilized onto a polyaniline (PANI) film is reported.

PANI is a polymer that changes conductivity and colour with changes in pH or redox reactions as a result of changes in the degree of protonation of the polymer backbone, making it useful as an optical or a visual sensor. PANI film itself acts both as a matrix support compatible with biomaterial (e.g., enzyme) and as the indicator, and can be easily be fabricated [15,16]. Furthermore, PANI has already been reported as a polymeric matrix in chemical sensors [17�C19] and biosensors [20�C24] developments. In the case of a PANI-based biosensor, most employ a class of enzymes known as oxido-reductases, mainly oxidases and dehydrogenases. In the case of oxidases, they are mainly based on peroxidase, glucose oxidase, or cholesterol oxidase [25]. A few of them used lipase [26], invertase [27] and polyphenol oxidase [28], and very few of them have used AOX.

Here, we used AOX as enzyme catalyst for ethanol detection, coupled with the optical properties of PANI as a visual sensor, so that it the presence of the alcohol could be detected by the naked eye due to a colour change from green to blue. For quantitative detection, the colour change of the films towards ethanol has been scanned and analysed using image analysis software (i.e., ImageJ). Optimisation of experimental conditions has been carried out and the analytical parameters of the biosensor have been determined. The operational and storage stability of the biosensor were also evaluated.2.?Experimental2.1. Reagents and SolutionsAniline (AR-grade), alcohol oxidase (AOX) (A2404, EC1.1.3.

13, 10�C40 units/mg protein, from Pichia pastoris), ascorbic acid (A5960), gallic acid (G7384) and Drug_discovery l-cysteine (W326305) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Absolute ethanol (>99.5%), methanol, orthophosphoric acid 85% and sodium hydroxide (pellets) were delivered by Merck (Nottingham, UK). All chemicals were of analytical reagent grade. The Milli-Q water used was obtained from a Millipore Direct-QTM 5 purification system. Stock solutions of ethanol was prepared daily in 0.