Chiang Mai Journal of Science

       An amperometric bi-enzyme biosensor based on α-chymotrypsin (CHY) and alcohol oxidase (AOX) was developed for detection of aspartame in a flow injection analysis (FIA) system. The CHY and AOX were separately immobilized on bead supports using an adsorption technique. The cleavage of aspartame was catalyzed by CHY to produce methanol that was converted by AOX to formaldehyde and hydrogen peroxide. The formed hydrogen peroxide was detected amperometrically at a platinum electrode. The biosensor performance was optimized with respect to enzyme immobilization and operating conditions. The most suitable conditions for enzyme immobilization were 250 U/mL CHY and 100 U/mL AOX with 60 min immobilization time. The optimal conditions for operating the developed aspartame biosensor were a flow rate of 0.5 mL/min and pH 8.0 at an applied potential of +0.70 V versus Ag/AgCl. Under the optimal conditions, the developed biosensor showed a linear response over the aspartame concentration range 0.15–1.0 mM (coefficient of determination (R²) = 0.9941) with a sensitivity of 5.52 μA/mM·cm² and a detection limit of 0.10 mM. The developed biosensor was successfully applied in the determination of aspartame in commercial samples.