Study And Application Of Porous Layer Open-tubular Capillary Column In Immobilized Enzyme Microreactor And Capillary Electrochromatography

The immobilized enzyme microreactors(IMERs)is a kind of enzyme reaction system which combines immobilization technology of biological enzyme molecules with biological enzyme reaction in microchannels.The immobilized enzyme microreactor based on the open-tube capillary column has advantages of high selectivity catalysis of enzyme,reusable and low reagent consumption and easy separation,and the microreactor is simple in preparation,easy operation and easy connected with other analytical equipment and is widely used in life sciences such as enzyme reaction kinetics,screening inhibition,proteomics and biocatalysis.For how to improve the performance of immobilized enzyme microreactor,including catalytic efficiency and stability,the enzyme immobilization technology in the capillary column is very important,and it is also the research direction that has attracted much attention in related research fields.Corresponding to the capillary immobilized enzyme microreactor,the preparation of capillary chromatographic column is the core part of the capillary electrochromatography efficient separation and analysis and it is also the focus of electrochromatography theory and application research.It plays an important role in the development of capillary electrochromatography and at present has become a rapid development of emerging technology.The study of this work focused on the research and application of a novel open-tube capillary column in immobilized enzyme microreactor and capillary electrochromatography and carry out the following research work:1.We report a robust capillary-IMERs based on a three dimensional porous layer open tubular(3D-PLOT)column and successfully applied for analysis of tryptic digests of proteins,or HeLa cells.The porous layer on the inner surface of the silica capillary is easy-to-control surface modification strategy via single-step in situ biphasic reaction.The 3D-PLOT column with highly uniform porous geometry and narrow distribution of porosity can greatly enhance the surface-area-to-volume ratio of the micro-channels,and increases the enzyme loading.Compared with the unmodified capillary microreactor,the 3D-PLOT modification improves the efficiency of the enzyme reaction,shortens the analysis time,and exhibits excellent stability and reproducibility.Taking trypsin as an example,we systematically analyzed the properties of the prepared 3D-PLOT-IMER.By coupling the 3D-PLOT-IMER with a nano-LC-MS/MS system for online digestion of standard proteins,cell extraction and living HeLa cells,demonstrating the surface modification with the proposed 3D-porous layer is a simple and effcient approach for enzyme immobilization and could be widely suitable for different kinds of IMERs.2.We report a robust capillary electrophoresis-immobilized enzyme microreactor(CE-IMER)with efficient surface modification for enzymatic assays of acetylcholinesterase(AChE)and detection of organophosphorus pesticides in vegetables.With the aid of a single-step in-situ biphasic reaction,a highly uniform porous-layer was modified on the inner surface of the capillary tip,and the IMERs is then fabricated by layer-by-layer assembly of AChE on the produced porous-layer open tubular column.The remainder of the capillary is used as a CE separation analysis channel.When the substrate of the enzyme reaction flows through the enzyme reactor,on-line enzymatic reaction generate product,and the substrate is then separated from the immobilized enzyme by electrophoresis to terminate the reaction,and the product and substrate are separated and detected in the subsequent electrophoresis.The proposed CE-IMER exhibits excellent stability and inter/intra-day reproducibility,ensuring the reliability and accuracy of on-line enzyme assays.The micro-porous layer can greatly enhance the effective surface area which serves as an ideal support for enzyme immobilization to enhance reaction efficiency and shorten analysis time.The IMERs are successfully applied for detection of organophosphorus pesticides in pakchoi juice samples,showing the feasibility for potential application in real sample analysis.Our study provides a simple and efficient approach for on-line CE assays with micro-reactors,which could be of value for extensive application in bioanalysis.3.A new capillary electrochromatography(CEC)method based on capillary column with highly uniform porous surface modification was developed.Based on the capillary inner wall porous layer modification method proposed above,producing homogeneous porous-layer modified surface with ~240 nm thickness in a 50 μm-id capillary.Taking the mixture of naphthalene and biphenyl as test samples,the chromatographic performance of the prepared PLOT column as a capillary electrochromatography column was systematically studied,which proved that the PLOT column showed good separation efficiency,stability and reproducibility.The column has been successfully applied for CEC analysis of three different types of samples without any further modification of the columns,including complicated peptide products from tryptic-digestion of proteins(lysozyme and BSA),-blockers(basic samples)and polycyclic aromatic hydrocarbons(neutral samples).The results showed that the PLOT column is simple to prepare and has high column efficiency.The porous layer modification improves the comparison and column capacity,and increases the interaction between the stationary phase and the sample in the electrochromatographic analysis.Our study show that the present PLOT column is a promising approach that can significantly enhance CEC separation efficiency and could be of potential value in analysis of various different samples.4.A novel method for dispersive liquid-liquid microextraction using ionic-liquid was applied for detection drugs in urine samples,by coupling with high performance capillary electrophoresis.We use ionic liquid dispersion liquid phase microextraction technology,1-butyl-3-methylimidazolium hexafluorophosphate ionic liquid as extractant,combined with capillary electrophoresis technology to achieve trace-amounts drugs(ephedrine and ketamine)in urine with high-efficient pre-concentration and separation detection.Under the optimum conditions,the method shows good linearity for detection of ephedrine and ketamine in the range of 0.10-10 mg/L,the LODs(S/N=3)of 0.21 mg/L for ephedrine and 0.39 mg/L for ketamine,and the spiked recoveries in the range of 79%-90%.The method has properties such as cheap,easy-to-operate,high enrichment,sensitive detection and little interference,and would have valuable potential application in detection of trace-amount drugs in the biological samples.