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. The method makes use of the photo reaction between a fluorophore (thionine) and NADH. The latter is generated during an enzymic reaction between ethanol and alcohol dehydrogenase (ADH) in the presence of nicotinamide adenine dinucleotide (NAD+) cofactor. An empirical relation is used to analyse the quenching, and a quenching constant of 27.2 (+/-2.8) M-1 is obtained for the substrate induced quenching (SIQ) of thionine 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 with the experimental results. Various applications of the reaction are discussed, including its use to a fibre optic biosensor for ethanol.