Preparation Of Core-shell Magnetic Nanoparticles And Its Study On Protein Separation And Purification

Applications of protein in therapeutics and advances in genetic and proteomic engineering have generated an ongoing need for recombinant proteins. Thus efficient separation analysis method has become the focus of attention. Magnetic nanoparticles?MNPs? were widely used in biomedical field, due to their highly specific surface area, good dispersibility, controlled size, unique magnetic responsiveness, easy separation and other characteristics. The protein-binding efficiency of magnetic nanoparticles was much higher than that of commercial micrometer-scale packing, with a remarkable decrease in nonspecific proteins adsorption of the surface. However, magnetic nanoparticles were easy to aggregate, surface unstable and even easy to lose magnetism when being used in complicated systems, which severely limited their applications. Therefore, it was quite worth exploiting well-established magnetic composite nanoparticles which integrated with good stability and solubility, higher enrichment and separation efficiency, and the lower nonspecific protein adsorption in bioseparation process.In this paper, the research content included two parts. The first part was the preparation of core-shell magnetic nanoparticles. Firstly, uniform iron oxide nanoparticles around 185 nm were synthesized by solvent hot method. Secondly, to improve their stability, Fe₃O₄@SiO₂ microspheres which exhibited a very narrow particle size distribution around 245 nm were prepared through the coating of the silica layer onto the magnetite Fe₃O₄ microspheres by a Stober method. Next, a typical procedure for the synthesis of Fe₃O₄@SiO₂@PHEMA trilayer composite microspheres by distillation precipitation polymerization?DPP?was completed. Thus, the thickness of the outer PHEMA polymer shell was ?37 nm. And then, to optimize the suspensions qualitatively, derivatizations of the polymer brushes including reaction with SA, activation of the resulting-COOH groups with EDC and NHS, and subsequent reaction with aminobutyl NTA were taken. Hydration radius of Fe₃O₄@SiO₂@PHEMA-SA-NTA by the light scattering measurements was 255 nm, the saturation magnetization?Ms? values of the multilayer microspheres was 18.58 emu/g and the amount of chelating metal ion Ni²⁺ was 34.37 ?g/mg.In the second part, we evaluated the protein separation and purification performance of magnetic nanoparticles. Firstly, to measure the binding capacity of His-tagged protein on the beads, we determined BSA binding capacity. In this case, the protein binding likely occured through interaction of BSA histidine residues with NTA-Ni²⁺ complexes. Based on a Bradford assay, the binding capacity was 124.87 mg of BSA per gram of MNPs. Afterwards, we used routine HepG2 cell lysate to demonstrate the ability of decreasing nonspecific protein adsorption. Furthermore, to demonstrate the utility of the product for protein purification, we isolated His-tagged CAV1 protein from a cell lysate. The results of SDS-PAGE and Western Blot indicated that Fe₃O₄@SiO₂@PHEMA-SA-NTA-Ni²⁺MNPs not only can separate and enrich His-tagged protein but also can resist some nonspecific protein adsorption.