Studies On Synthesis Of Functionalized Magnetic Composite Particles And Application In Biomedicine

Functional magnetic composite particles are superpararamagnetic colloid composites that composed of ferrofluid and other materials coatings (such as organic polymer and inorganic matrix). The magnetic particles not only have the magnetic property of solid, but also have the fluidity of liquid. The bioactive molecules such as enzymes, antibodies (monoclonal or polyclonal), nucleic acids or oligonucleotides may be coupled on the magnetic particle surface via the functional linker or the functional groups such as amino, carboxyl, thiol etc. Therefor, magnetic composite particles coupled with biomolecue could be used for cell sorting, enzyme immobilization, immunossay examination, drug delivery, purification, detection, and hybridization of nucleic acids because of its high efficiency, low toxicity and simple manipulation. Silanized magnetic particle is kind of functional magnetic particle with good magnetic response, biocompatibility, superparamagnetic property and abundant functional groups immobilizing for biomolecule. Based upon the above properties, they have been used in cytology, immunology, molecular biology and other fields.In China, some research works have been done in the synthesis of magnetic polymer microspheres fields, however, the development of application and related products are less advanced. The magnetic particles have been first commercialized in 1980s by Dynal company in Norway, furthermore there are many inspect kits and apparatus in market in USA, Germay and other country, the kit or apparatus is very expensive. Magnetic particles have good properties of separation and enrichment for biomolecules, so our attentions should concentrated in developing kits and apparatus in our country. In this thesis, we focused on the chemical synthesis of frunctional magnetic composite particles and their applications in immobilization for biomedicine. The research work could be devided into four parts as the following:1. The magnetic particle was prepared by chemical co-precipitation of Fe(II) and Fe(III) ions in alkaline medium, and the Fe₃O₄ particle surface was modified by the -NH2 groups via silanization reagent (3-aminepropyltrimethoxysilane). Bifunctional reagent glutaricanhydride was used to react with the -NH2 groups on Fe₃O₄ particles, and magnetic particles with -COOH terminal groups were obtained. The reaction conditions were optimized too. Fourier transform infrared spectroscopy (FTIR), X-raydiffraction (XRD), transmission electron microscopy (TEM) and laser particle size assay (LPSA) were used to characterize these two kinds of magnetic particles. The linear potentiometric titration and conductometric titration were used to analyzed -NH2 groups and -COOH groups quantitatively. The results have shown that Fe3C?4 diameter was about 15 nm, and it has high magnetic response and superparamagnetism. The contents of amino and carboxyl groups were 400 + 50 umol/g and 380 + 50 umol/g respectively.2. Magnetic composite particles could be used to immobilize macrobiomolecules. BSA protein and rabbit anti-HIVIgG antibody were binded onto the magnetic particles by amino terminal groups by glutaraldehyde and 1,4-phenylene diisothiocyanate, HIVIgG antibody was coupled on the magnetic particles with carboxyl terminal groups via carbodiimide. The efficiency of coupling was analyzed by UV-Vis spectrophotometer, the active of molecules on magnetic particle surface was detected by Enzyme-Linked Immunosorbent Assay (ELISA). The results was shown that the efficiency of coupling BSA gets to 108-130 mg/g by different methods, and HIVIgG to 65-90 mg/g. The method of ELISA based on magnetic particles is simple and quick compared with conventional microtiter plate method, and there is extremely significant difference between positve and nagetive group samples. It could be used in antibody/antigen purification, cell isolation and clinic diagonosis, etc.3. Using 1,4-phenylene diisothiocyanate as a crosslinker, NH2 group labeled oligonucleotide probe was immobilized on magnetic paticles surface, and other oligonucleide sequence was designed to hybridize with the probe on magnetic particles surface, the fluorencence signal was intrduce to detect the immobileization efficiency. The results have shown that efficiency of immobilized oligonucleotide gets up to 120 nmol/mg, and hybridization result was very distinct compared with the contrast sample.4. Magnetic chitosan composite particles (MCCP) were attained in emulsion system, and nanometer-sized Fe3C>4 was bedded in the network structure that was formed by the reaction between crosslinker and chitosan. And this kind of particle could be used as a target drugs carriers, which were used to be tumour therapy with adsorbed Doxorubicin Hydrochloride. The morphological and magnetic properties of the MCCP were characterized by different techniques (i.e., FTIR, TEM). The results indicated that the MCCP have strong magnetic response, nanometer-sized distribution (200-300 nm), low immunocompetence, high medicament carrying capacity and...