Immobilized Enzyme-Based Microtiter Plate Assay for Glucose in Foods

Abstract

The method presented is based on the use of glucose oxidase (GOD), peroxidase (POD), and 2,2‘-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS). Enzymes were separately immobilized on the inside surface of each well of a 96-well microtiter plate. Prior to the immobilization, the surface was activated with glutaraldehyde. The activation conditions remarkably affected both the activity and the stability of immobilized enzymes. A solution containing H2O2 (20 μL) was added into the well with immobilized POD (POD-well) containing 180 μL of 2 mM ABTS (pH 6.5) and the absorbance change at 415 nm for 10 min was measured with a microplate reader. A sample solution containing d-glucose was first added into the well with immobilized GOD. After 30 min, an aliquot (20 μL) was transferred into the POD-well. The calibration curves for H2O2 and d-glucose were linear up to 1 and 1.5 mM, respectively. The glucose concentration in fruit juices obtained by the present method agreed well with that by the F-kit method. Keywords: Glucose; glucose oxidase; peroxidase; immobilized enzyme; microtiter plate assay; citrus; fruits juice