Preparation And Application Of Immobilized Multi-enzyme Microreactor Based On Capillary Column

Immobilized enzyme microreactor(IMER)is a kind of device obtained by combining enzyme immobilization and enzymatic reaction in microchannel.IMER combines the advantages of specific catalysis,the features of immobilized enzymes and the low reagent consumption of microchannel reactions,creating more possibilities for research in biomedicine,proteomics and pharmacy.In recent years,IMER based on capillary column has been widely used,and capillary electrophoresis(CE)has shown great potential in the field of enzyme analysis in combined with IMER due to its fast analysis,excellent separation efficiency,small amount of reagents consumption and various separation modes.To achieve multi-enzyme immobilization in capillary column and improve the catalytic efficiency,stability and reusability of IMER,the preparation method of IMER is crucial.Herein,a new method of immobilizing enzyme,DNA-directed immobilization(DDI)technology,is used to immobilize enzymes on the inner wall of a capillary,which modified with polyamidoamine(PAMAM)dendrimer,to construct a highly efficient and controllable dual-enzyme co-immobilized open tube-IMER.And the microreactor combined with CE was applied to the detection of glucose and screening of enzyme inhibitors,which proved the feasibility of this method to be popularized in the field of bioanalysis.(1)Two different probe DNA strands were anchored with glucose oxidase(GOx)and horseradish peroxidase(HRP),and a new type of open tube-IMER was constructed by co-immobilizing DNA-enzyme complexes through DDI technology on the inner wall of a capillary,which modified with PAMAM dendrimer.The introduction of PAMAM can effectively enhance the loading capacity of the IMER.The enzyme ratio can be conveniently controlled by adjusting the ratio of the DNA strands,and considering that DNA hybridization is reversible,the reactor can be regenerated and reused.According to the confocal laser scanning microscope characterization,it can be proved that the double enzymes have been successfully immobilized in the capillary by DDI technology.Glucose and 2,20-azido-bis(3-ethylbenzothiazoline-6-sulfonic acid)-diammonium salt were selected as enzymatic substrates,and in off-line mode,the enzymatic and separation conditions was optimized based on the changes in product peak area.The performance of the microreactor constructed by DDI was investigated,the results showed that its stability and reusability are significantly better than the IMER obtained by non-specific adsorption.This study established the foundation for the subsequent preparation of online CE-IMER.(2)Based on off-line analysis,trypsin and ?-glucosidase were co-immobilized on the inlet inner wall of the capillary by DDI,which modified with PAMAM,and the on-line CE-IMER that capable of screening of enzyme inhibitors was constructed.N-benzoyl-L-arginine ethyl ester hydrochloride and p-nitrobenzene-?-D-glucoside were chose as enzymatic substrates,enzymolysis and separation conditions were optimized based on changes in product peak area.The CE-IMER was used for screening of enzyme inhibitors.The constructed multi-enzyme microreactor can continuously complete the entire process of enzymolysis,separation and detection in one capillary,which shorten the analysis time,increased the degree of automation and established a foundation for on-line screening of enzyme inhibitor in the future.