Bi-enzymatic optode detection system for oxalate determination based on a natural source of enzyme

This work describes a simple and low cost methodology for oxalate determina tion in food samples, which employs a bromothymol blue-based pH optode for the determination Of CO2 generated in the enzymatic reaction between oxalic acid and oxalate oxidase. The enzyme was immobilised on barley seeds, toge ther with catalase enzyme, and placed in a stirring bar type enzymatic reac tor. The system showed a linear response range from 0.0080 up to 0.100 mol l(-1), when the measurements were carried out in 0.050 mol l(-1) succinate buffer at pH 4.0 and 25 degreesC. In these conditions, the lifetime of the system was about 120 h, with a relative standard deviation <2% (four measur ements of a 0.020 mol l(-1) oxalate solution). A value of 0.075 mol l(-1) w as obtained for the apparent Michaelis-Menten constant, with a maximum velo city of 1908 mu mol min(-1) for oxalic acid oxidation. No significant diffe rences were found at a confidence level of 95%, when the results were compa red with those obtained with the AOAC official Standard method (974.24) for oxalate determination in spinach. (C) 2001 Elsevier Science B.V. All right s reserved.