Online immobilized enzyme microreactor for the glucose oxidase enzymolysis and enzyme inhibition assay

Abstract: An online immobilized glucose oxidase (GOx) capillary microreactor was developed based on an enzymatic redox reaction with 1,4-benzoquinone as an acceptor of electrons, replacing the molecular oxygen typically used in a GOx reaction to achieve direct ultraviolet detection without derivation. A high efficiency of enzymolysis was obtained at 1mgml−1 1,4-benzoquinone for 5min of incubation at 25°C, and baseline separation of the substrate and product could be achieved with a resolution of 3.85 by employing 20mM phosphate buffer (pH8.0) containing 40mgml−1 sulfated β-cyclodextrin as an additive, a constant voltage of 15kV, and a detection wavelength of 220nm. In addition, an online enzyme inhibition study was performed on the immobilized GOx microreactor with metal ions Ag+ and Cu2+ used as model inhibitors. The results indicate that Ag+ (IC50 =69.16μM) has a markedly higher inhibitory effect than Cu2+ (IC50 =1.33mM). The protocol described can be applied in high-throughput screening of enzyme reactions and inhibitors. [Copyright &y& Elsevier]