Preparation Of Magnetic Polymer Microspheres, Characterization And Performance Study For The Immobilized Enzyme,

Now with the research development in field of biomedicine and bioengineering, people are exploring various functional polymer microspheres which can serve as smart or intelligent tools for drug delivery, biomacromolecules purification, biosensor and enzyme immobilization, etc. Magnetic polymer microsphere has been fully developed as a new functional material in recent decades. The hybrid microsphere which is composed of polymer and inorganic magnetic particles is magnetic property. The main advantage of magnetic polymeric microspheres over conventional polymeric micropsheres is that, because of their super-paramagnetic properties, they can be rapidly separated from solution under magnetic field, which proposes a new way for biological separation and detection, also brings new direction of development for researching functional materials.Based on the research background and the development trend of polymer microspheres, the research interest of this work focused in the preparation, characterization and properties for enzyme immobilization of magnetic polymer microspheres, which involves in four parts, i.e. the preparation and surface modification of magnetite nanoparticles, preparation of core-shell Fe₃O₄/SiO₂ particles, preparation and primary application for enzyme immobilization of dual-responsive polymer microspheres which possess magnetically responsive and pH-responsive properties. Preparation of new kind of magnetic polymer microspheres as support materials through combining nanotechnology and bio-immobilized technology offer the immobilized enzyme a bio-friendly microenvironment and improved activity. The results of each part are listed as follows:1.Magnetite nanoparticles were prepared by chemical co-precipitation process. The super-paramagnetic magnetite nanoparticles of about 8nm were obtained by controlling the reaction temperature increased stepwisely. The characterization results of X-ray diffraction (XRD), Fourier transform-infrared (FT-IR), Transmission electron microscopy (TEM) and Vibration sample magnetometer (VSM) confirmed super-paramagnetic spherical particles with an average size of about 8nm were prepared. They had cubic spinel structure nano-crystalline.2.By treating as-prepared magnetite nanoparticles with citrate acid aqueous solution, stable magnetic fluid (dispersion of magnetite nanoparticles in water) was prepared. Using the modified magnetite nanoparticles as seeds, core-shell structure silica-coated magnetite particles (Fe₃O₄/SiO₂) were prepared via a modified St(?)ber method. It was found that the morphology of resulted Fe₃O₄/SiO₂ particles is related with the amount of tetraethyl orthosilicate (TEOS) by TEM. Furtherly, the composite particles of the composition and magnetic properties were characterized by FT-IR, XRD and VSM.3. Using Fe₃O₄/SiO₂ particle as seed, a dual-responsive chitosan-coated Fe₃O₄/SiO₂ microsphere was prepared from layer by layer approach. Up to now, using the microsphere support for pectinase immobilized has not been reported to our best know. In this study, pectinase was immobilized onto a new type of magnetic polymer microsphere support from layer by layer approach. Various characteristics of immobilized pectinase such as the pH stability, thermal stability and storage stability were valuated. The immobilized pectinase revealed acceptable pH stability over a broad experimental range. The colloidal stability is not impeded by the adsorbed proteins despite the fact that up to 284.7 mg of enzyme is adsorbed per gram of the carrier particles. The activity of immobilized pectinase adsorbed onto these particles is analyzed in terms of the Michaelis-Menten parameters. The Michaelis constant Km differs only slightly from the Km value of the native enzyme when the amount of adsorbed enzyme is raised despite the high local concentration of immobilized enzymes.4. The polyelectrolyte brush (polystyrene sodium) was grafted onto the surface of Fe₃O₄/SiO₂ composite particles by atom transfer radical polymerization (ATRP). The prepared super-paramagnetic Fe₃O₄/SiO₂-g-PSStNa microsphere has good biocompatibility. The idea of preparing the surface of natural polymer chitosan layer pre-absorbed on the "spherical polyelectrolyte brush" via layer by layer approach receives special relevance in immobilized enzyme technology. The colloidal stability was not impeded by the adsorbed proteins despite the fact that up to 318.6 mg of enzyme was adsorbed per gram of the carrier particles. The immobilized pectinase revealed acceptable pH stability over a broad experimental range of 3.0-4.5. The activity half lives for native and bound states of enzyme were found as 13.5 d and 30 d, respectively. Enzyme activity was found to be approximately 49.7% for immobilized enzyme after storage for 1 month.