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ABSTRACT
GOD was covalently immobilized on polymer/silica gel hybrid support prepared by coating high surface area of silica gel with modified acrylonitrile co-polymer. The relationship between immobilization factors and enzyme activity were examined by the series of contour plots. The selections of the immobilization variable range were extremely precise in the 3-level-3-factor fractional design. The results indicated that the optimal conditions for GOD immobilization were: 0.1% enzyme solution, immobilization temperature- 4°C and immobilization time- 24 h. Immobilized GOD was applied to amperometric determination of glucose using flow-injection analysis. The optimal flow rate was determined as 4.0 ml/min when injecting 100 μl sample volumes. The linear response range for the on-line detection of glucose using immobilized GOD in column minibioreactor was estimated to be from 0.01 mM to 20 mM (a correlation coefficient of 0.985). Moreover, its experimental detection limit is 10 μM (S/N=3) and the apparent Michaelis-Menten constant was calculated to be 20.15 mM. The proposed method for glucose measurement was validated in real samples of fruit juices.