Establishment And Characterization Of Chip Free Flow Electrophoresis System

Human genome project has completed, and proteome research has become a new frontier of life science. Proteome analysis relies very much on the development of pretreatment method for reducing the complexity of the protein samples. An imminent merit of free flow electrophoresis (FFE), comparing to that of conventional slab gel electrophoresis and capillary electrophoresis, is that separation can be performed in a continuous manner. Miniaturization of FFE system is beneficial for further reducing joule heating problem often encountered in large scale FFE system, and compatible for fast separation and management of small amount of samples.In the first chapter, features and applications of the FFE and chip FFE in particular were reviewed. Main issues in the chip system were analyzed based on recent reports, and possible improvements are proposed in the light of potential applications in proteomics analysis.In the second chapter, a simple and fast chip FFE preparation method was described. First, the ITO electrodes on the ITO glass were prepared by photolithography, and the chip FFE was assembled by bonding with a cover glass sheet using a double tape as a spacer. Separation of fluorescein and rhodamine 6G was demonstrated and characterized by fluorescence imaging, and factors such as buffer concentration, flow rate, pH as well as electric field strength were also studied.In the third chapter, total glass FFE chip with a separation channel of 15μm deep and 10 mm wide was fabricated by photolithography and thermal bonding, and 2 Pt wire electrodes of 50μm were introduced into the channel with guide channels. The chip was characterized and compared with the ITO chip FFE.In the fourth chapter, the mobility of fluorescein and that of rhodamine 6G was measured by capillary electrophoresis and used to correlate some of the phenomenon found in the experiments, with more attention on the consequences of electro-osmotic flow.The chip FFE with large width depth ratio has been successfully demonstrated by free zone electrophoresis in this work, and could be the basis for the development of other separation modes.