Amperometric bienzymatic biosensor in flow injection analysis system for determination of aspartame in foods

An amperometric bienzymatic biosensor was developed for the determination of aspartame in a flow injection analysis (FIA) system, consisting of two enzyme reactor columns packed with immobilized α-chymotrypsin (CHY) and alcohol oxidase (AOX) beads and a hydrogen peroxide electrode, connected in series. The CHY and AOX were separately immobilized on glutaraldehyde (GA)-activated beads through covalent bonding. The biosensor fabrication and operational conditions were optimized. The optimal fabrication conditions were: 2% GA with 120 min activation time; and 250 U/mL CHY and 100 U/mL AOX, with 180 min enzyme immobilization time. The optimal operational conditions were a flow rate of 0.5 mL/min and pH 8.0 at room temperature. The developed biosensor showed linearity over the aspartame concentration range 0.01-1.2 mM, with a detection limit of 0.005 mM. The developed biosensor was satisfactorily applied for detecting aspartame in beverage samples without any excessive pretreatments.