Preparation Of Magnetic Composite Microspheres By Template Mini-emulsion Polymerization And Their Application In Protein Purification

In recent years, the organic/inorganic composite polymer microspheres with magnetic properties have attracted more and more attention. Because these magnetic hybrid microspheres not only exhibit high magnetic susceptibility to an external magnetic field, but also easily further functionalized and surface-modified by the attachment of various bioactive molecules, they can conveniently separate the objective biomolecules from the medium with the help of magnets. These merits make magnetic hybrid microspheres a powerful tool for application in biomedicine and biochemistry fields. However, when diagnostics are considered, an ideal magnetic microspheres used in these applications should be exhibit the following properties: (1) a narrow size distribution allowing homogeneous particle behavior, (2) a high iron oxide (superparamagnetic) content for rapid separation under a magnetic field, (3) surface functionality for the covalent binding of biomolecules. However, either most of existing methods failed to produce uniform-sized hybrid microspheres with high magnetite content to enhance their susceptibility to magnetic field or the processes used to making such materials were too complicated. In addition, the obtained magnetic carriers possess the lower adsorption capability and binding efficiency. Based on the problems mentioned above, systematical study has been carried out on the preparation, surface modification and application of magnetic microspheres in protein separation and purification in this thesis. The main results are listed as follows:(1) Hybrid Fe₃O₄@Poly(St/DVB) microspheres with controllable magnetite content and narrow size distribution, composed of Fe₃O₄ nanoparticles encapsulated in a crosslinked polystyrene matrix, were synthesized by magneto-template (Fe₃O₄ minidroplets) miniemulsion polymerization, the synthetic procedure for fabrication of high-quality magnetic hybrid microspheres is simple and easily scaled up, which have a potential application in industry. In this process, firstly, the magneto-template (magnetic nanoparticles cluster) was fabricated by the ultrasonic emulsification. Secondly, monomer St and crosslinked agent DVB was added and polymerized. The DLS results confirmed that the formation process of the magnetic microspheres was dominated by miniemulsion polymerization that was initiated and polymerized in monomer-swollen magneto-templates (MSM). The formed magnetic microspheres had a reasonably narrow size distribution. The average particle size of the magnetic hybrid microspheres was in the range of 70-130 nm depending on the amount of the surfactant. The magnetite content of the magnetic hybrid microspheres can be effectively modulated in the range 40-70 wt % by feeding different amounts of ferrofluid. Based on the related parameters, for instance, a given St/DVB ratio, the amount of surfactant, hydrophobic agent, ultrasonic power, type of initiator, solid content of ferrofluid, the feed ratio of magneto-miniemulsion and monomer, an optimum recipe to prepare the magnetic hybrid microspheres were obtained. The magnetic hybrid microspheres are superparamagnetic, which allow them to serve as excellent candidates for biomedical applications. This simple procedure for preparation of uniform magnetic hybrid microspheres opens a new facile route for encapsulation of inorganic nanomaterials in polymer microspheres.(2) Fe₃O₄/Poly(St/DVB)@Poly(GMA/DVB) composite microspheres with core-shell structure were rationally fabricated by the seeded emulsion polymerization. TEM images show that the size of composite microspheres is uniform. The magnetite content of the magnetic composite microspheres can be effectively modulated in the range 23-43 wt % by polymerizing the different amounts of shell-monomer. The content of available epoxy groups was determined by Na₂S₂O₈ titration method and was found to be 0.126-0.190 mmol/g microspheres. By covalent coupling of iminodiacetic acid (IDA) chelator to the surface of the magnetic microspheres, a new immobilized metal affinity magnetic carrier was achieved. The amount of chelated Cu²⁺ in magnetic carriers to was 0.068-0.072 mmol/g and 0.091-0.110 mmol/g as determined by UV spectroscopy and atomic absorption spectrophotometry (AAS), respectively.(3) When magnetic carriers were used to purify S-adenosylmethionine synthetase (SAMS), the purified SAMS protein can be separated conveniently and rapidly with an external magnet. Based on the detailed investigation on the purification condition such as the initial SAMS concentration, the concentration of imidazole elution, the ionic strength (NaCl concentration), pH, the type of metal ion, an optimum purification condition for SAMS protein was obtained. By the optimum purification condition, various initial concentrations of SAMS protein was purified by the magnetic carriers. The experimental results indicated that the maximum protein purification capacity was 50.0 mg/g marries as for the 3.0 mg/L initial SAMS concentration. SDS-PAGE results shown that purified SAMS protein had high purity and low nonspecific adsorption.