Quantification of serum glucose by glucose oxidase and peroxidase co-immobilized on free and affixed alkylamine glass beads in a beaker

J. Arora, K. Nashier, K. Rani, V. Kalia and C. S. Pundir*

Biochemistry Research Laboratory, Department of Bio-Sciences, M. D. University, Rohtak - 124 001 (India)

ABSTRACT: Glucose oxidase from Aspergillus niger and per­oxidase from horseradish peroxidase have been co-immobilized on free and affixed alkylamine glass beads in a glass beaker through glu­taraldehyde coupling. A colorimetric method for determination of serum glucose was devel­oped employing co-immobilized enzymes onto both free and affixed alkylamine glass beads. The method is based upon measurement of H2O2 generated from serum glucose by coimmobilized glucose oxidase with a colour reaction consisting of 4-aminophenazone and phenol and coimmobilized peroxidase. The minimum detection limit was 1.8 mg/dL and 3.6 mg/dL and the percent recovery of added glucose in serum was 92.16 mg/dL and 95.32 mg/dL and within / between batch coefficient of variation for glucose determination in six serum samples were 9.4%/7.36% and <5%/<12% for the method employing free and affixed glass bead bound enzymes respectively. A good correlation, r1 = 0.999 for enzymes bound to free and r2 = 0.905418 for enzymes bound to affixed glass beads was obtained with enzymic colorimetric commercial method employing free glucose oxidase and peroxidase. The method is unaffected by various serum substances. The enzymes bound to free and affixed glass beads were reused for 125 and 100 times respectively, without any considerably loss of activity when stored at 40C in distilled water. The enzymes coimmobilized onto free glass beads showed better stability than that on affixed glass beads.

KEYWORDS: Glucose; serum; glucose oxidase; peroxidase; coimmobilization; alkylamine; glass beads

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