Dual electrode signal-subtracted biosensor for simultaneous flow injection determination of sucrose and glucose Original Research Article

A multi-enzyme electrode obtained by a two-step immobilization of the enzymes glucose oxidase, mutarotase and invertase was developed for the determination of sucrose. Glucose oxidase was entrapped in a poly-1,3-diaminobenzene film on a platinum electrode by electrochemical polymerization and a combination of mutarotase and invertase was cross-linked over the electrode via bovine serum albumin and glutaraldehyde. The sucrose concentration was determined from hydrogen peroxide oxidation at +0.7 V vs. Ag/AgCl. This immobilization method minimized interference from ascorbic acid. A second electrode, for glucose only, was constructed containing inactive invertase. This gave an almost identical glucose response to the sucrose sensor and hence could be used for signal subtraction. In this manner, sucrose could be determined in the presence of glucose at higher concentrations. The influence of enzyme content, pH, temperature, and optimum operational conditions for use in a flow injection analysis (FIA) system were determined. When used in FIA, the sensor responded to sucrose in the range 1–300 mmol l−1. The sensor was stable for 15 h of continuous use (150 assays) and retained 70% activity after 30 days. When used to analyze the sucrose and glucose contents in a number of different soft drinks, the sensor showed good agreement with the standard liquid chromatography (LC) method.