Application And Technique Development Of Capillary Electrophoresis In Drug Screening

Capillary elcotrophoresis (CE) is an analytical technique of high efficiency, fast speed and microscale, which is widely used in bioanalysis. In this article, according to its characteristics, CE was applied to drug screening, and new explorations were carried out in view of the key problems in drug activity screening, for example, developing fast and high efficiency screening methods and searching for new drug targets.Firstly, development of new method of drug activity screening. The study of interaction between compounds and a drug target is critical in drug screening. Affinity capillary electrophoresis is a common method used to determine their binding, but bad reproducibility, caused by temperature change, electroosmotic flow fluctuation and so on, limits its development. Based on the techniques discussed above, a pressure-mediated affinity capillary electrophoresis method was developed in this article. By using a special sequence of pressure and electrophoresis, the influence of factors was effectively avoided and the analysis time was greatly reduced. Using this method, the binding of eight drugs with different pKa and binding abilities and bovine serum albumin (BSA) was evaluated and binding constants were calculated. By comparison with results determined by traditional fluorescence spectrophotometry, the feasibility and accuracy of this method were verified. The results were shown that this method was well suitable for studying weak interacting systems.Secondly, establishment of drug structure screening model. Up to now, activity screening is one of the most popular screening methods. However, using single screening standard is prone to result in misleading effect. Merely increasing the number of compounds to be screened makes it difficult to find active but unknown compounds. As a result, all of those lead to high screening cost as well as low efficiency. In this article, a newly structure screening method was explored, which depended on the specific recognition ability between antigen and antibody to screen compounds with similar structure to antigen. Using CE ligands separation method and laser induced fluorescence detection technology, the binding of anti-fungal Echinocandin B nucleus and its polyclonal antibody was studied, and a structure screening model based on electrophoretic mobility changes of fluorescence labeled antigen was preliminary established. This screening model can be used to screen compounds with special structure and potential anti-fungal activities.Lastly, application in discovery of drug targets. Differential proteomic is an important method to discover drug targets. Two-dimensional gel electrophoresis and mass spectrometry are its key technologies to separate and identify proteins. Prior to mass spectrometry analysis, proteins separated by two-dimensional gel electrophoresis need a long and complex process, which makes it difficult to meet the requirements of automation. In order to resolve this problem, based on the work of the formers, a capillary electrophoresis method involving gel protein transfer and on-line proteolytic digestion was developed. Bovine serum albumin present in SDS-polyacrylamide gel as the model protein can be transferred fast and effectively from the gel to capillary by three-electrode transfer method. A capillary monolithic column which immobilized trypsin by the sol-gel method was used to digest the transferred protein. After connecting the capillary monolithic column to transference capillary, on-line digestion and detection of transferred proteins was carried out. This technique significantly improved transference efficiency, and provided an efficient way for the connection of two-dimensional gel electrophoresis and mass spectrometry.