Research On Capillary Enzyme Microreactor Based On DNA-directed Immobilization

Capillary electrophoresis has become one of the most important tools for enzyme analysis because it has the advantages of low consumption,fast analysis,simple operation,easy separation and quantification,and can be combined with various detection methods.Therefore,the preparation of a capillary enzyme microreactor with high enzyme binding capacity and renewability is the main objective in the development of new immobilized techniques.In this thesis,the trypsin was immobilized on the aminated capillary wall based on a DNA-directed immobilization(DDI)technique.Combined with the characteristics of polyamidoamine(PAMAM)dendrimers,new types of capillary enzyme microreactor have been established.Firstly,an off-line capillary enzyme microreactor have been explored.Then,the feasibility of online capillary enzyme microreactor based on DDI technique have been demonstrated.(1)A novel type of trypsin capillary microreactor was developed based on a DDI technique applied to a fused-silica capillary modified with PAMAM.Trypsin binding to the inner wall of the capillary was confirmed by confocal laser scanning microscopy.The properties of the trypsin-DNA conjugated,PAMAM-modified capillary microreactor were investigated by monitoring hydrolysis of Nα-benzoyl-L-arginine ethyl ester.Through the hybridization and dehybridization of the DNA,the inner wall of the capillary functionalized with trypsin can be regenerated,thus indicating the renewability of this enzyme microreactor.In addition,these results demonstrated that introduction of PAMAM enabled higher amounts of trypsin to be immobilized,markedly improving the enzymolysis efficiency,compared with traditional modified capillaries.The digestion performance of the trypsin capillary microreactor was further evaluated by digesting cytochrome C,and a peptide numbers of 8,and a sequence coverage of 59%were obtained.This renewable and efficient immobilized trypsin capillary microreactor combines advantages of both DDI technology and PAMAM,and is potentially adaptable to high-throughput enzyme assays in biochemical and clinical research.(2)Trypsin was immobilized in the inlet end of capillary as a plug and a new type of online immobilized trypsin capillary enzyme microreactor was developed based on a DDI technique.The optimum enzymolysis conditions for BAEE in the online microreactor were evaluated.The enzymatic hydrolysis,electrophoretic separation and detection could be accomplished simultaneously.The results indicate that the online capillary enzyme microreactor has a good performance,and provide the possibility of online protein digestion.