Study On Preparation And Applications To Protein Separation Of Fe(Ⅲ)-immobilized Nanoparticle-modified Capillary

As proteins are a significant part of the living organisms, the study on this kind of macromolecules has received more and more attention and become a new science-proteome. Based on the current problems in the separation and concentration of low-abundance and post-translational proteins, the research interest of this work focused on the on-line separation and enrichment of proteins by combined functionalized microspheres,capillary electrophoresis and protein analysis.This dissertation consists of four chapters.In chapter 1, the basic knowledge and recent researches of functionalized microspheres,capillary electrophoresis and capillary coating were summarized. And detailed reviews of functionalized nanoparticles applying to capillary electrophoresis were introduced. In the end, the intention and meaning of this dissertation were described.In chapter 2, we synthesized epoxy-based magnetic silica nanoparticles, and characterized them with Fourier-transform infrared (FT-IR) spectroscopy and scanning electron microscope (SEM). The FT-IR spectrum and SEM image provided supportive evidence that epoxy-based magnetic silica nanoparticles with an average diameter of 160±(10～15) nm were obtained. Based on that, a novel coated capillary modified with metal-immobilized nanoparticles has been investigated for the capillary electrophoretic (CE) separation of phosphoproteins and nonphosphoproteins. The metal-IMAN capillary was achieved by covalently immobilising epoxy-based magnetic silica nanoparticles on the prederivatised 3-aminopropyl-trimethoxysilane (APTMS) fused-silica capillary (75-μm I.D.), followed by disodium iminodiacetate and metal. The chemically modified capillary was characterized by streaming potentials (SPs) and inverted fluorescence microscope. The evolution of the multilayer streaming potential of capillaries (i.e., the bare capillary, APTMS capillary, epoxy-based nanoparticle capillary, and Fe3+-IMAN capillary) over the pH range of 3 to 9 indeed demonstrated that metal-IMAN were bound to the surface. And the repeated measurement of streaming potential provided supportive evidence that the coated capillary was stable. An inverted fluorescence microscope was used to characterizing the surface features of the Fe3+-IMAN capillary derivatized with morin. And bright dots could be clearly distinguished on the Fe3+-IMAN capillary surface, indicating that each bright dot corresponds to morin-Fe3+-IMAN complexes in comparison with the epoxy-based nanoparticle capillary.In chapter 3, Use of the metal-IMAN capillary is essential to perform CE analysis of a simple protein mixture containing phosphoproteins (ovalbumin andβ-casein) and nonphosphoproteins (hemoglobin and BSA) and it was determined that the coated capillary was stable and performed efficient on-line separation of phosphoproteins and nonphosphoproteins by CE. And the proteins migrate out in the order of nonphosphoproteins (hemoglobin and BSA), phosphoproteins (ovalbumin andβ-casein) with the theoretical plate numbers per meter of 29000 (hemoglobin),42 000 (BSA),32000 (ovalbumin) and 233000 (β-casein), respectively. The separation repeatability of the capillary modified with Fe3+-IMAN has been investigated. Small variations in the migration times were observed for hemoglobin (0.72% RSD), BSA (1.48% RSD), ovalbumin (2.57% RSD), andβ-casein (0.89% RSD) for eight consecutive runs under the conditions used. In this study, a Fe3+-IMAN capillary was used for more than 150 runs with no observable changes in the separation efficiency and the run-to-run repeatability of the streaming potential measurements was satisfactory. It was determined that the coated capillary was stable and performed efficient on-line separation of phosphoproteins and nonphosphoproteins by CE.To further confirm the separation effectiveness of the coated capillary, a complex mixture of phosphoproteins and nonphosphoproteins-bovine milk proteins was analysised by CE. Five proteins were well-separated, which confirmed that the metal-IMAN capillary had enormous potential for CE separation of phosphoproteins and nonphosphoproteins. It is interesting that the migration order of nonphosphoproteins (α-lactalbumin andβ-lactoglobulin) and phosphoproteins (α-casein,β-casein andκ-casein) is the same as above-mentioned separation. The utility of the Fe3+-IMAN modified CE approach for analysis of the complex sample has shown great potential for phosphoproteomics applications.In chapter 4, conclusion and prospects are given.