A multisyringe flow injection system with immobilized glucose oxidase based on homogeneous chemiluminescence detection

In this paper, enzyme containing reactors are for the first time implemented in the multisyringe flow injection analysis (MSFIA) technique interfaced with chemiluminescence detection for biochemical assays. The automated methodology is based on the on-line substrate conversion in an oxidase packed-bed reactor and the post-column chemiluminogenic catalysed-reaction of the generated oxidising species with an organic molecule (namely, 3-aminophthalhydrazide) in front of the photosensor module. Various catalysts in homogeneous phase are compared taking advantage of the benefits of the MSFIA concept. On one hand, mineral catalysts (namely, Co(II)) are assessed, on the other hand, minute and accurate volumes of soluble organic species (viz., horseradish peroxidase (HRP)) are readily handled without requiring further immobilization protocols. The potentials of the MSFIA–CL concept with immobilisation of the proper oxidase protein are demonstrated using glucose as a model of substrate. Despite the different pH and kinetic requirements for both the substrate conversion in the enzyme-reactor and the Co(II)/HRP-mediated luminol oxidation integrated in the flow system, the MSFIA approach warrants maximum yields owing to the independent optimisation of the physical and chemical parameters of the various reactions involved. Under the optimised configurations and experimental variables, dynamic working ranges from 2.5×10^-6 to 1.0×10^-3 mol l^-1 glucose may be obtained for both detection schemes by proper photomultiplier gain selection. The detection and determination limits calculated at the 3(sigma) and 10(sigma) level were 8.6×10^-7 and 2.0×10^-6 mol l^-1 glucose, respectively, for the Co(II)-luminol system, and 1.3×10^-6 and 2.3×10^-6 mol l^-1 glucose, respectively, for the HRP-luminol procedure. The repeatability (n=10) at the 1.0×10^-5 mol l^-1 level was slightly better for the Co(II)-catalysed reaction (2.5% versus 4.0%). The developed MSFIA–CL methodology was used for kinetic studies of the mutarotation reaction between (alpha) and (beta) anomeric forms of glucose, obtaining quantitative information of the specific glucose oxidase (GOD) anomer formed at prefixed intervals of time. It was also successfully applied to the determination of traces of glucose in complex matrices, namely, human urine, soft drinks and fruit juices, exploiting the Co(II)-mediated luminol oxidation.