The Development And Applications Of Dopamine-Assisted Capillary Electrophoresis-based Enzyme Microreactors

In recent years,capillary electrophoresis(CE)has been widely used in the field of enzyme analysis because of its high efficiency,high sensitivity,low sample consumption and easy automation.The CE based immobilized enzyme microreactor(IMER)has gradually become a powerful platform for on-line enzyme analysis.It has many important advantages,such as enhancing the stability of enzyme,reducing the use of enzyme and reusable of enzyme,and no complex purification procedure is required when separating the reaction solution.In this study,three CE based IMERs were constructed based on the unique physical and chemical properties and good biocompatibility of the polydopamine(PDA)membrane formed by the self polymerization of dopamine(DA).They were used in the study of enzyme kinetics and the evaluation of inhibitor activity.This thesis mainly includes five chapters.In the first chapter,the enzyme and methods of enzyme immobilization,the advantages and characteristics of immobilized enzyme,as well as the?-D-glucosidase(?-Glu),xanthine oxidase(XOD),thrombin(THR)and factor Xa(factor Xa,FXa)used in this study were introduced.Furthermore,the research progress of IMER based on CE in recent years was reviewed.In addition,the characteristics and applications of DA and PDA formed by self aggregation were briefly introduced.Finally,the content and significance of this study were proposed.In the second chapter,the CE based?-Glu-IMER was successfully constructed using the adhesion of PDA membrane,which was used for the study of enzyme kinetics of?-Glu and the screening of inhibitors.The Michaelis constant(K_m)of immobilized?-Glu in the IMER was 2.79 m M,and half-maximal inhibitory concentration(IC₅₀)and inhibition constant(K_i)of the known inhibitor castanospermine were 69.9 and 8.14?M,respectively.The method was applied to evaluate the inhibitory activity of 12 flavonoids on?-Glu.The results showed that genistein,baicalein,epicatechin gallate and epigallocatechin gallate had good?-Glu inhibitory activity,and their binding mode and binding energy with enzyme were further studied by molecular docking analysis.The established?-Glu-IMER is a reliable method for screening?-Glu inhibitors from natural products.It has the advantages of simplicity,reusability,low sample consumption,high stability,and can be used in the study of other enzymes.In the third chapter,PDA membrane was optimized to polydopamine/graphene oxide(PDA/GO)membrane by increasing specific surface area of GO,so that the amount of immobilized enzyme could be increased.A CE based XOD-IMER was successfully constructed,and it was used in XOD enzymatic reaction kinetics research and inhibitor activity evaluation.The K_m value of the immobilized XOD was determined as 0.39 m M,and the IC₅₀ and K_i value of the inhibitor4-aminopyrazolo[3,4-d]pyrimidine on XOD were determined as 11.9 and 5.2?M,respectively.The IMER was used to determine the inhibitory activity of 10 flavonoids on XOD.The results showed that dihydroquercetin,quercetin,biochanin A and epicatechin had obvious inhibitory effect on XOD,and their binding mode with enzyme was further studied by molecular docking analysis.In the fourth chapter,a CE based dual-enzyme(thrombin and factor Xa)co-immobilized microreactor(THR-FXa-IMER)was constructed for studying enzyme kinetics and screening dual-target inhibitors against THR and FXa with the aid of the PDA/GO coating.The K_m values of immobilized THR and FXa in THR-FXa-IMER were calculated to be 187.26 and 48.80?M,respectively.The K_i values for 2 known inhibitors,argatroban and rivaroxaban,on THR and FXa were determined to be 14.73and 0.406 n M,respectively.The developed method was successfully applied to screening dual-target inhibitors against THR and FXa from 30 small molecular compounds.Among them,10 compounds such as salvianolic acid C and epigallocatechin gallate have dual-enzyme inhibitory activity,and 2 compounds named saikosaponin A and oleuropein have single THR inhibitory activity,5 compounds such as rosemary acid and salvianolic acid B have single FXa inhibitory activity.Finally,the molecular interaction between enzyme and potential inhibitors was further verified via the molecular docking analysis,and a new compound with a theoretically good THR/FXa inhibitory effect was designed by scaffold hopping study.The fifth chapter is the summary and prospect of this thesis.CE-based IMER has unique advantages in the field of enzyme analysis,such as quick and simple operation,small sample consumption,and easy automation.Various enzyme immobilization methods developed in this study provide more options for the applications of IMER,while the study of dual-enzyme IMER can further improve the efficiency of enzyme analysis and provide a simple method for the screening of dual-enzyme inhibitors.