Determination of ethanol based on oxygen consumption with an enzyme sensor using vitamin K₃ as a mediator

Abstract

NADH formed in the reaction of alcohol dehydrogenase (ADH) was regenerated to NAD with dissolved oxygen using vitamin K₃ and diaphorase (DI). The regeneration method makes it possible to construct an FIA system for ethanol by using a Clark oxygen electrode detector. The phosphate buffer (0.05 M, pH 8.0) carrier solution was pumped through a sample injection valve and a reactor packed with Sepharose on which ADH and DI were co-immobilized, and transported to a new type of flow-through cell in which the surface of the oxygen electrode was set upward into the carrier stream. The peak current was linearly related to ethanol in the range 4 × 10^-4 M to 2 × 10 ^-3 M. The detection limit was 5 × 10^-5M and the R.S.D. was 1.1% for 5 successive determinations at 2 × 10^-3 M level. The sampling frequency was 15 samples/h. When the system was applied to the determination of ethanol in wine, the results were in good agreement with those obtained by the F-kit method.