Preparation And Properties Of A Graphene Oxide Functionalized Capillary Electrochromatography Microreactor

The capillary electrophoresis enzyme immobilized microreactor is a kind of micro chemical reaction device,which including biomolecular immobilization,micro enzyme reaction and high efficiency separation technology.Because of the advantages of immobilized enzyme and microanalysis,such as good stability,reusable,low consumption of reagent and easy separation,it is a kind of low cost,high efficiency and easy to automate enzyme catalyzed tool.It also has a unique advantage in protein enzymolysis,biotransformation and enzyme inhibitor screening etc..The capillary electrophoresis enzyme microreactor is mainly composed of immobilized enzyme microreactor and capillary electrophoresis.The activity of immobilized enzyme is closely related to the carrier matrix of the enzyme,and its separation performance is close to the separation mode.The research mainly includes the following contents:1.The recent development of techniques for immobilized enzyme was reviewed.We mainly introduced the method of physical adsorption,chemical bonding and embedding,and its application in enzymolysis,etc..2.A new method for preparing the capillary electrochromatography based on functionalized graphene oxide by using the physical adsorption and chemical covalent method was established.A series of characterization of the capillary electrochromatography were carried out,such as Raman spectrum,scanning electron microscope and thermal weight loss,and so on.The column's stability and separation performance were investigated.Compared to bare capillary,the separation degree of BAEE and BA determined by graphene oxide modified capillary electrochromatography increased from 3.7 to 4.7.The result indicated that graphene oxide modified capillary can significantly improve the separation performance,which can be used for the separation of complex compounds.3.Based on the different immobilization techniques,including physical adsorption and chemical covalent method,we have made a first attempt to obtain a capillary electrochromatography microreactor successfully.The enzyme kinetic study of capillary electrochromatography immobilized enzyme was carried out taking trypsin as the model.The results showed that the physical method was better than the chemical covalent one.Using the physical method,the Michaelis constant(Km)was 1.10 mM,the maximum reaction rate(Vmax)of 0.32 mM · s-1.Vmax was 3000 times higher than for the free enzyme solution,while the Kn was about 1/20 of the free enzyme solution.Compared with the immobilized enzyme on the unmodified capillary(Km 109.77 mM,Vmax 0.00046 mM·s-1),both the affinity and the catalytic rate were significantly increased.It indicated that graphene oxide can improve the catalytic activity of the immobilized enzyme.4.Rapid screening was prepared by micro reactor used in ten kinds of herbs in the crude extracts.the results showed that the Pseudo-ginseng and the Rheum Officinale containing trypsin inhibitor activity,and their inhibition of immobilized trypsin was 26%and 26.12%,respectively.The results show that the prepared graphene oxide modified enzyme immobilized microreactor is a potential rapid inhibitor screening tool.