Silica-based Monolith And Polymer-based Monolith For Preconcentration And Capillary Electrophoresis Of Biomolecules

Capillary electrophoresis (CE) is increasingly important for biomolecular analysis with the advantages of high resolution, short separation time, minimal sample and reagent requirement, and ease of automation. To meet the development of clinical and biomedical applications, the preconcentration and analysis of low volume of biological sample with complex matrices are generally required. The in situ prepared monolithic materials, which have the advantages of easy preparation, excellent permeability, low backpressure and large capacity for samples, were proposed for the preconcentration purpose in CE. In this thesis, a silica monolith was prepared for efficient DNA extraction and in-line coupling with CE for DNA separation. The preparation and application of an affinity polymer monolithic capillary for CE of protein were also included in this work. Main points of this thesis are listed as following:1. The preparation process of silica-based monolith and polymer-based monolith, the development and application of CE in the analysis of DNA and proteins, and monolith-based solid phase extraction (SPE) preconcentration coupling with CE were reviewed in Chapter 1.2. An amino silica hybrid monolithic column was prepared in situ by sol-gel method with the polymerization of tetraethoxysilane (TEOS) and N-(β-aminoethyl)-y-aminopropyltriethoxysilane (AEAPTES) in the presence of ethanol aqueous and porogen. The self catalyzed sol-gel reaction mechanism of the amino silica monolith was expounded. Several factors affecting the polymerization of the amino silica monoliths were investigated, including the content and the ratio of TEOS and AEAPTES, the amount of water and ethanol, the type of porogen. The optimized process was established to ensure the monolithic capillary columns having porous and uniform morphology. The total surface area and mesopore size distribution of the amino silica hybrid monolith were characterized. 3. The amino silica hybrid monolithic column was applied for DNA extraction successfully. The main extraction mechanism was attributed to Coulombic force between DNA and the monolith. DNA extraction conditions, such as pH, ion concentration and type, and loading capacity, were optimized online by CE set-up with laser-induced fluorescence detection. Under the optimal conditions, the prepared amino silica monolithic column can provide high capacity and excellent extraction reproducibility. The DNA extraction process was applied to purify PBE2 plasmid and remove of proteins from the crude Bacillus subtilis lysates. Since low ion concentration buffers in the extraction procedure were used, the extracted PBE2 plasmid can be amplified by polymerase chain reaction, which demonstrated that this monolithic column is a new attractive solid-phase support for DNA extraction.4. An in-line SPE precocentration method coupled to CE was developed the first time for the analysis of DNA. The amino silica hybrid monolith was prepared at the inlet end of a fused silica capillary, and the remaining part of the capillary was used as separation channel. The procedure for this in-line SPE-CE method was constructed on the basis of investigation on operational conditions such as the introduction mode of sieving matrix, the composition of elution solvent and the elution time. Under optimum conditions of the proposed method, a comparison with conventional CE was made by the analysis of DNA marker DL 2000 (100,250,500,750,1000,2 000 bp). It is proved that the in-line SPE-CE method provides excellent detection sensitivity as well as competitive separation efficiency. We also describe an application of this method for the study of plasmid DNA from Escherichia coli crude lysate. It could be shown that the in-line SPE-CE is an effective approach for the analysis of trace DNA in complex biological samples, which might be of great significance in many biological and biomedical applications.5. An in-line immunopreconcentration-CE method using glycidyl methacrylate-ethylene dimethacrylate (GMA-EDMA) polymer monolith immobilized with antibo-dy was designed and evaluated for the preconcentration of proteins. The polymer monolith was synthesized in situ at the inlet end of a fused-silica capillary by co-polymerization of GMA with EDMA in the presence of the initiator 2,2-azobis- isobutyronitrile (AIBN) and a mixture of porogens including cyclohexanol and dodecanol. Monoliths with various compositions of monomers and porogens were prepared and evaluated for permeability and morphology characterization to optimize the preparation process. The electroosmotic flow (EOF) was investigated in detail for different types of capillary columns, including capillaries containing different proportions of monolith, monolithic capillary immobilized with IgG, etc. Cathodic EOF could be observed for all of these columns studied but with different extent. Furthermore, anti-IgG FITC conjugate was used as the test analyte to demonstrate the effectiveness of the monolithic capillary immobilized with IgG for immunopreconcentration, the non-specific adsorption on monolith and the possibility of monolith applied to immobilize different antibodies. Additionally, the application of this affinity monolithic capillary for the in-line immunoproconcentration-CE analysis of anti-IgG was attempted successfully. This in-line system offers the possibility of the analysis of analyte for which an antibody was available. Therefore, it should be very useful for the analysis of biomarkers presented at low concentrations in complex biological samples.