Immobilization of Uricase onto a Hydrogen Peroxide Permeable Membrane and Application for an Enzyme Electro d e

Abstract

A rapid, specific, and simple enzyme electrode method for the analysis of uric acid was developed. A cellulose acetate membrane with asymmetric structure which has the permeability toward hydrogen peroxide was prepared. Uricase (EC 1.7.3.3) was immobilized onto the porous side of the membrane by entrapping with chitosan. The enzyme electrode was constructed with combination of the immobilized uricase membrane and a polarographic electrode sensing hydrogen peroxide. The enzyme electrode responded linearly to concentration of uric acid over zero to 50 mg/d/. Response time was within 20 s. The enzyme electrode was applied to the analysis of uric acid in human blood. Only 25 μl of human serum or human whole blood was required for analysis. Analytical precision was O.6% at 6.4 mg/dl on human plasma and 2.2% at 2.3 mg/dl on human whole blood. Analytical recovery was 99.9% on controlled human serum. Correlation was established with enzyme colorimetric method; r = 0.985, y=0.977 x + 0.05 (x: Enzyme colorimetric). The immobilized uricase membrane was sufficient ly stable to perform 1000 assays (17 days operation) for the determination of uric acid in human blood. The storage shelf life of dried membrane was more than 14 months at 4°C. The present enzyme electrode method offers an excellent assay procedure for uric acid in clinical analysis.