Protein Detection And Interaction Of Capillary Electrophoresis Methods

It is of great importance to develop highly sensitive detection and interaction methodologies for proteins in the field of diseases diagnosis and therapy, such demands are expected to extensively promote the advancement of proteomics. In recent years, the development of analytical methods for functional proteins with high sensitivity has still attracted great attention in the life science research field. Novel capillary electrophoresis (CE)-based methods were established in this thesis, focusing on indirect, high-sensitive detection of proteins and the interaction between proteins and drug or ssDNA aptamers. The thesis is divided into four chapters.Chapter 1 summarized recent advances in high-sensitive detection and recognition on proteins, the developments of CE technology in the protein separation and interaction are highlighted.In chapter 2, combining high separation performance of CE and specific colorimetric reaction of proteins-bicinchoninic acid (BCA) method, a new rapid, sensitive method for protein determination was established, with a microwave-assisted incubation approach. Meanwhile, withβ-cyclodextrin as an inclusion additive, the complex of BCA-Cu+ can be effectively separated from free BCA molecule, which resulted in an enhanced sensitivity at low wavelength for the specific complex. This method was also successfully applied in the determination of some ricin samples in the first international proficiency test on the quantification of ricin. The derivation of our measured and real concentration of ricin is ranged from 0.4 to 6.8%, which is further confirmed the concept that the quantification of proteins can be indirectly substituted by determination of BCA-Cu+ complex.In chapter 3, the interaction between basic drug CBZ and HSA was investigated by CE-frontal analysis(CE-FA). When different concentration of CBZ and HSA were determined, a narrow range of R value was selected so as to fit the linear regression with the original nonlinear regression well. The binding constant and ratio are thus conveniently calculated. In this weak interaction system, the dissociation constant (Kd) is 7.09×10^-5M, the binding ratio between CBZ and HSA is approximately 1:1, and the binding percentage of CBZ to HSA is around 24%.In chapter 4, three modes of CE-FA method, combined with laser induced fluorescence detection and aptamer labeling technique, were thoroughly compared with regard to binding between thrombin and its F-29mer aptamer. These modes included R-Cf nonlinear regression, non-linear fitting of the change of plateau height vs concentration of analyte, and single-point clculated way. The scope of application for three modes can be successfully used was discussed.