New Continuous Electrophoresis Prefractionation Method For Small Amount Of Complex Sample

As one of the major contents of life science in the post-genomic era, protein has become the focus and hot research spots. At present, there are certain challenges in the study of protein. The main reason includes that protein composition in the biosamples is very complex, and the abundance of some proteins is too low. These problems make the analysis difficult. One of the ways to solve the above difficulties is to separate and concentrate the protein samples. Free-flow electrophoresis is a unique separation method. It is well suited for the pretreatment of proteins and other biological samples, since the separation conditions are very mild, and it can separate continuously. This article describes a new chip free-flow electrophoresis device. Ion concentration polarization mechanism was introduced by using ion-exchange membrane, thus electrophoretic separation and concentration could be performed at the same time.In the first chapter of this paper, free-flow electrophoresis separation principle and its application, micronanofluidic and vortex phenomena, ion exchange membrane and proteomics were introduced and reviewed.In the second chapter of this paper, we fabricated a free-flow electrophoresis chip system by polymethyl methacrylate (PMMA) substrate, and a series of chip structure improvements were made. The three fluorescent probe molecules were well separated using the improved chip. The factors related with the separation and concentration performance were characterized, including buffer concentration, voltage, power mode, the separation chamber height. The current-voltage curves as well as the conductivity and pH of the collected fractions were also measured. The experimental results show that the chip device exhibited good separation effect when a forward electric field (anode-cation selective membrane-anion selective membrane-cathode) was applied. However, depletion and vortex phenomena were observed at the ion exchange resin interface when a reversed electric field (cathode-cation selective membrane-anion selective membrane-anode) was applied. Certain concentration effect was showed in both the above two operation modes. Separation and concentration of a model protein mixture (pⅠ1.5-2.5pepsin, pⅠ6.9hemoglobin and pⅠ11.0lysozyme) was tried. The separation chamber was treated with hydroxyethyl cellulose to avoid protein adsorption. The collected fractions were characterized by SDS-polyacrylamide gel electrophoresis.This study showed that the device has the potential to separate and concentrate microscale complex protein mixture. It is expected that this prefractionation device may find wide application for online sample pretreatment of the low abundant charged components, especially biological macromolecules.