Abstract
A method for the determination of ethanol using alcohol oxidase (AOD), peroxidase (POD), and 2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS) was developed in which those enzymes were separately immobilized on the inside surface of each well of a 96-well microtiter plate. Prior to immobilization, the surface was activated with glutaraldehyde. The activation conditions remarkably affected the activity of the immobilized AOD. A sample solution containing ethanol was first added into the well with immobilized AOD (AOD-well). After 30 min, an aliquot (20 µl) was transferred into the well with immobilized POD containing 180 µl of 2 mM ABTS and the absorbance change at 415 nm for 10 min was measured with a microplate reader. The calibration curve was linear over the range 0.10 - 1.0 mM, and the relative standard deviation (n=5) was 0.8% for 1 mM ethanol. The ethanol concentration in alcoholic beverages, obtained by the present method, agreed well with that by the F-kit method. The AOD-well was reusable for 20 successive determinations.
