MEASUREMENT OF ETHANOL USING FLUORESCENCE QUENCHING

A new assay procedure for the measurement of ethanol concentrations is described which is based on fluorescence quenching of an indicator. T he method makes use of the photo reaction between a fluorophore (thion ine) and NADH. The latter is generated during an enzymic reaction betw een ethanol and alcohol dehydrogenase (ADH) in the presence of nicotin amide adenine dinucleotide (NAD+) cofactor. An empirical relation is u sed to analyse the quenching, and a quenching constant of 27.2 (+/-2.8 ) M-1 is obtained for the substrate induced quenching (SIQ) of thionin e by ethanol. The reported method is suitable over a range of 0-40 mM with a detection limit of 0.15 mM. A theoretical model for the overall ethanol assay is developed, and its validity is shown by comparison w ith the experimental results. Various applications of the reaction are discussed, including its use to a fibre optic biosensor for ethanol.