Study And Application Of Protein Imprinting Technique Based On Magnetic Nanoparticles And Thermosensitive Hydrogel

Proteome refers to all protein expressed by a genome, a cell or tissue. Proteomicsis made a study of biosome at the protein level with quantitative, dynamic, holistically.It forces on the characteristics of the protein, which includes the expression,translation, modification, conformation of protein and the protein interaction and so on.The study of Proteomics is the need to explore the mysteries of life, and it also makes acontribution to the human health. With the increasing progress of proteomics, theseparation, enrichment and rapid detection of protein have been a great concern.Currently molecularly imprinting technique is a mature separation method for smallmolecule compounds. Molecularly imprinted polymers have three the property ofpredetermined molecular structure, specific recognition and widely practicability. Withthe deeply study of proteomics molecularly imprinting technique has made someprogress in proteomics research, which has a focus on purification, separation andenrichment of protein in recent years.In this work the protein is the subject investigated. We synthesized proteinimprinted polymers with specific recognition and high adsorption capacity incombination with Protein molecularly imprinting technique and magnetic nanomaterialand thermosensitive material. The main study was carried out mainly with thefollowing:1、Synthesis and Characterization of the silica-modified Fe₃O₄nanoparticlesIn this work, the average diameter of60nm Fe₃O₄magnetic nanoparticles wasprepared by the improved co precipitation method. Among this process, hydrazinehydrate was used as deoxidant, precipitant and reductant. And then the silica modifiedFe₃O₄nanoparticles were synthesized with a so gel process at the surface of Fe₃O₄nanoparticles with the hydrolysis of tetraethoxysilane （TEOS）. The results show thatthe mass loss of silica modified Fe₃O₄nanoparticles was about7%. And the averagediameter of Fe₃O₄magnetic nanoparticles was60nm. The surface modification processdid not change the crystal structure of particles. The silica modified Fe₃O₄nanoparticles have a good dispersion and stability, and its saturation magnetization is10.25emu/g. It is slightly lower than the Fe₃O₄magnetic nanoparticle which is38.16emu/g. The saturation magnetization of silica modified Fe₃O₄nanoparticles is enoughto have good magnetic behavior. 2、Polydopamine-based molecular imprinting on silica-modified magneticnanoparticles for recognition and separation of bovine hemoglobinSurface molecular imprinting, especially on the surface of silica modifiedmagnetic nanoparticles, has proposed as a promising strategy for protein recognitionand separation. Inspired by self polymerization of dopamine, we synthesized apolydopamine based molecular imprinted coating on silica Fe₃O₄nanoparticles forrecognition and separation of bovine hemoglobin （BHb）. Magnetic molecularlyimprinted nanoparticles （about860nm） possess a core shell structure. Magneticmolecularly imprinted nanoparticles （MMIP） show a relatively high adsorptioncapacity （4.65±0.38mg/g） and excellent selectivity towards BHb with a separationfactor of2.19. MMIP with saturation magnetization （10.33emu/g） makes it easy toseparate the target protein from solution an external magnetic field. After threecontinuous adsorption and elution processes, the adsorption capacity of MMIPremained at4.30mg/g. Our results suggest that MMIPs are suitable for the removal ofhigh abundance of protein and the enrichment of low abundance of protein inproteomics.3、 Thermosensitive Molecularly Imprinted Hydrogel cross-linked withN-Malely Chitosan for recognition of Bovine Serum AlbuminThe water soluble N maley chitosan （N MACH） cross linker was synthesized bythe acylation of chitosan with maleic anhydride. Because N maley chitosan containscarboxyl group, amino group and double bond, it can be used as the cross linker inprotein imprinting. Using radical polymerization，the thermosensitive molecularlyimprinted hydrogel （MIH） was obtained in aqueous solution using bovine serumalbumin （BSA） as the template molecule, N isopropyl acrylamide （NIPAM） andacrylamide （AAm） as functional monomers and N MACH as cross linker. The lowercritical solution temperature of thermosensitive molecularly imprinted hydrogel was ataround34℃. The absorption capacity of MIH was5.72mg/g, which was higher than4.8times for the non molecularly imprinted hydrogel. The adsorption process of MIHfit in with Langmuir Adsorption model. The Separation factor of MIH was4, whichindicated that the MIH had a specificity and selectivity to template proteins. The studyprovided a new water soluble cross linker for protein imprinting: N MACH, which hasexcellent water soluble and biodegradable properties. It is a kind ofenvironment friendly cross linking agent, which could be applied as cross linker in protein imprinting for the separation and enrichment of protein.