Enzymatic determination of ethanol based on the intrinsic fluorescence of alcohol dehydrogenase

A fluorimetric enzymatic method for the determination of ethanol is presented. The method is based on the intrinsic fluorescence changes caused in alcohol dehydrogenase (ADH) during the enzymatic reaction. This variation is due to the different inner filter effects of the co-enzyme (NAD+) and its reduced form (NADH). The latter is generated during the enzymatic reaction. A study is made of the origin of the ADH fluorescence signal using a mathematical model, which makes it possible to explain the linear response between the enzymeʹs analytical signal and the concentration of ethanol ranging from 5.5 × 10−5 to 3.2 × 10−3 M. The 3σ limit of detection is 3.1 × 10−6 M, and the relative standard deviation for 6 determinations at 1.0 × 10−4 M ethanol is 2.8%. The method is applied to low ethanol concentration drink samples and vinegar without pre-treatment.