Automated enzymatic assays in a renewable fashion using the multisyringe flow injection scheme with soluble enzymes

In this paper, a novel flowing stream scheme based upon the multisyringe flow injection (MSFI) technique is presented as a powerful tool to perform automated enzymatic assays. The exploitation of enzymes in homogeneous phase circumvents typical drawbacks associated with the commonly used packed-bead or open tubular permanent columns, namely, malfunctions of the reactor, carryover effects, flow resistance, loss of binding sites, large reagent consumption, and use of harmful organic solvents during immobilization procedures. The proposed MSFI system is able to handle minute volumes of soluble enzymes and accommodate reactions with divergent kinetic and pH demands, as demonstrated via the indirect chemiluminescence determination of trace levels of glucose. The procedure is based on the on-line glucose oxidase-catalyzed oxidation of beta-glucose in homogeneous phase to beta-glucono-delta-lactone and hydrogen peroxide. Subsequently, the generated oxidant merges downstream with an alkaline slug of 3-aminopthalhydrazide and a metal-catalyst zone (viz., Co(II)) at a total flow rate as high as 72 mL/min aiming to warrant maximum light collection from the fast CL reaction. Under optimum conditions for both sequentially occurring reactions, a glucose concentration as low as 90 microg/L may be easily detected at a 1000-fold photomultiplier gain. A second-order polynomial regression equation of light emission versus substrate concentration is found over the range 90 microg/L-2.7 mg/L glucose, although a maximum concentration of 180 mg/L may be determined by suitable gain selection without requiring manifold reconfiguration. An injection throughput of 20 h(-1), a repeatability better than 2.5% at the 1 mg/L level, and a 3sigma detection limit of 72 microg/L are the analytical features of the designed analyzer. The proposed approach was applied to the analysis of ultralow glucose content soft drinks as well as fruit juices suitable for diabetic consumers. The accuracy was assessed using the spectrophotometric batch glucose-Trinder method as an external reference methodology for the determination of the target species in parenteral solutions.