| Biologically active protein with high purity is the base of proteome research. Therefore, efficient purifying technology is important for proteome research. Immobilized metal ion affinity chromatography (IMAC) has many advantages such as high selectivity,inexpensive,the stability of chemical and physical properties, high capacity of the column, easy eluting and easy regeneration. So, it has been widely used in the purification of protein, especially for His-tag protein and phosphorylated protein. However, it also has some limitations: not easy to scale separation system, difficult to purify trace protein, a long time for operation and low separation efficiency. The magnetic separation which is based on the magnetic materials has the advantages of enlarging the system easily, suitable for trace protein separation and purification, easy operation, high efficiency and mild condition. So it can be a good make up for deficiencies of IMAC. Therefore,the particles which have property of IMAC and magnetic separation will greatly shorten the separation time and simplify the separation process for purification of protein. SiO2 particles have many advantages, such as excellent biocompatibility, hydrophilicity, easy modification and good chemical and colloidal stability. Therefore, silica-coated magnetic composite particles can be well applied in the field of bio-separation.In this article, firstly, we modify the SiO2 particles'surface with amino, aldehyde, carboxyl and chelating ligand through silane. For each group, we established qualitative and quantitative methods.And then we modify the silica magnetic composite particles with chelating ligand by the method established above.Though adjusting the state of chelating ligands, we can get magnetic composite particles with different surface properties. For their different surface properties, the magnetic composite particles can be applied in purifying positively charged protein, His-tag protein, phosphorylated protein respectively. The major research of the paper is as follows: Chapter II:Modify the surface of silica particles and establish the qualitative and quantitative methods. The active groups which can react with biological molecules are necessary for the silica magnetic composite particles which are used in protein's separation and purification. Therefore, the particles are needed for surface modification. In the biological area, the commonly used reactive groups are amino, aldehyde, carboxyl, chelating ligands and so on. Therefore, in this chapter, we use St?ber method to prepare 60 nm SiO2 particles and then modify the surface with amino, aldehyde, carboxyl and chelating ligand by silane. The amount of suface groups is usually not a lot by modifying with silane. Therefore, suface groups are difficult to detect by conventional methods. Therefore, for each group, we established simple and practical qualitative and quantitative methods. Through the quantitative method, we research the stability of the chelating ligand on the particles. The research shows that the particles have good stability and is very suitable for biological application.Chapter III: Prepare Fe3O4/SiO2-IDA magnetic composite particles and use them in purifying the positive charged protein. About 75% protein purification scheme use the ion-exchange chromatography. Therefore, ion-exchange chromatography is the most common method for protein separation and purification. This method is based on the electrostatic interaction between the particles and the protein. Therefore, the surface charge density of the particles determines the adsorption capacity and purification capability. In order to improve the particles'surface charge density, different with conventional single-carboxyl-modified, we modify the Fe3O4/SiO2 magnetic composite particles with double-carboxyl groups and use it in purification of a positive charged protein. The adsorption capacity of the particles is 10 mg BSA per /g. It achieves the adsorption capacity of import magnetic beads of the same size. Compared with a single carboxyl-modified ion-exchange chromatography, the Fe3O4/SiO2-IDA magnetic composite particles have higher purification effect and shorter separation time.Chapter IV: Apply the Fe3O4/SiO2-IDA magnetic composite particles in His-tag protein's purification and research the stability of the particles. The most important application of the immobilized metal ion affinity chromatography (IMAC) is purifying His-tag protein. Therefore, IMAC magnetic composite particles have begun to attract attention. Usually, the purification effect of these particles is good, but the reusability is poor. Therefore, the synthesis of IMAC magnetic composite material with good purificated effect and repeatability is significate. In this chapter, immobilized metal ions Fe3O4/SiO2-IDA magnetic composite particles are directed to purify the target protein in lysate solution of E. coli. The Cu2+, Ni2+, or Zn2+ fixed magnetic composite particles are used to purify His-tag protein, respectively. The result showed that the protein puritied by Fe3O4/SiO2-IDA-Ni2+ magnetic composite particles had the highest amout and the best purity. We studied the stability and reusability of the particles. The result shows that the reused particles are infected by bacteria easily. The particles'collocation points will be shielded by bacterial and can not immobilize the metal ions. Therefore, the particles lose the affinity with His-tag protein. Though controlling the storage condition of the used particles, the particles have good stability. After repeated using six times, the particles'purification effect is not affected.Chapter V : Prepare Fe3O4/poly (St-co-MPS)/SiO2-IDA-Fe3+ magnetic composite nanoparticles and their application in purifying phosphorylated protein. Phosphorylated protein has specific interaction with Fe3+ ions. Therefore, immobilized Fe3+ ion affinity magnetic composite nanoparticles can be used for separation and enrichment of phosphorylated proteins. Phosphorylated protein is an important low abundance protein. So its separation and enrichment is significant. To improve the efficiency of separation and enrichment, IMAC matrix has been studied. The commonly used substrates are porous resins and zeolite nanoparticles. Both of them have shortcomings. The porous resin has hole-effect, which will make detection difficult. The zeolite nanoparticles need high-speed centrifugal and will produce precipitation effect. Therefore, it is significate to find suitable IMAC matrix material. Therefore, in this chapter, we choose magnetic composite nanoparticles which have a high adsorption capacity as IMAC matrix, and use them for separation and enrichment of phosphorylated proteins. Fe3O4/poly(St-co-MPS)/SiO2-IDA-Fe3+ magnetic composite nanoparticles have been synthesized and characterized. The results show that the size of the particles is about 100-200 nm and it has a strong magnetic responsiveness. When the coordination between IDA and Fe3+ ion is ensured, the efficiency of the particles'enrichment is increasing with the pH value decreases. Compared with strong anion-exchange chromatography, Fe3O4/poly (St-co-MPS) / SiO2-IDA-Fe3 + magnetic composite nanoparticles'efficiency is 20-fold higher and their operating time is shorter.
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