Chip Free Flow Electrophoresis Isoelectric Focusing For Protein Separation And Monolithic PH Gradient Immobilization

Proteomics has become a new frontier in life science. The analysis of complex protein mixture is one of the major contents of proteimics, and depends very much on separation technologies with high throughput, high sensitivy, fast speed and high integraty. Free Flow Electrophoresis (FFE) is a continuous and parallel separation method, and its miniaturization is especially applicable for pre-processing a small amount of protein samples rapidly and efficiently. How to make use of μ-TAS technique to achieve the miniaturization of FFE system and improve its performance is drawing more and more attention.In the first chapter, FFE and its application, especially the structure of FFE chip and FFE isoelectric focusing (FFIEF), were reviewed. Recent development on structure of FFE chip and major problems were discussed, and corresponding possible solutions were proposed. pH gradient immobilization method was also introducted, and the concept of establishing immobilized pH gradient in FFE chip was proposed for solving the problems caused by using carrier ampholytes (CAs) in free solution isoeletric focusing.In the second chapter, FFE chip fabrication was improved on the basis of the former works. A polyacrylamide gel gap was prepared and aligned in FFE chip by photopolymerization to isolate the electrode reservior and the separation channel. The inlet tubes, collection cells and the open electrode reserviors were all fabricated mechanically on the same PMMA cover plate taking advantage of the mechanical properties of PMMA. The PMMA cover plate and bottom glass plate were sealed with a double-faced adhesive spacer to form a hybrid FFE chip. Isotachophoresis (ITP) and IEF were performed with the FFE chip. Fluorescent dyes and acid-base indicators were used as probes for characterization, and the pH gradient established in the separation channel was characterized by fluorescence imaging and color change of the indicators.In the third chapter, dynamic coating and addition of sieving medium were found important for inhibiting adsorbtion of protein on the channel inner wall. Soybean trypsin inhibitor (pI4.6) and egg white lysozyme (pI11) were successfully separated and collected with the improved chip FFE-IEF system as characterized by capillary zone electrophoresis. In the forth chapter, introduction of immobilized pH gradient in the chip FFE separation channel was investigated by in-situ polymerizing of monolithic material, formation of pH gradient by free CAs and local immobilization of the CAs on the permeable material. Preliminary result showed the feasibility of this method.