Development of an Alcohol Dehydrogenase Biosensor for Ethanol Determination with Toluidine Blue O Covalently Attached to a Cellulose Acetate Modified Electrode

Abstract: In this work, a novel voltammetric ethanol biosensor was constructed using alcohol dehydrogenase (ADH). Firstly, alcohol dehydrogenase was immobilized on the surface of a glassy carbon electrode modified by cellulose acetate (CA) bonded to toluidine blue O (TBO). Secondly, the surface was covered by a glutaraldehyde/bovine serum albumin (BSA) cross-linking procedure to provide a new voltammetric sensor for the ethanol determination. In order to fabricate the biosensor, a new electrode matrix containing insoluble Toluidine Blue O (TBO) was obtained from the process, and enzyme/coenzyme was combined on the biosensor surface. The influence of various experimental conditions was examined for the characterization of the optimum analytical performance. The developed biosensor exhibited sensitive and selective determination of ethanol and showed a linear response between 1 × 10⁻⁵ M and 4 × 10⁻⁴ M ethanol. A detection limit calculated as three times the signal-to-noise ratio was 5.0 × 10⁻⁶ M. At the end of the 20^th day, the biosensor still retained 50% of its initial activity.