Preparation Of Surface Molecular Imprinted Polymers Based On Nanoparticles Carriers And Study Of The Recognition Property For The Proteins

Molecular imprinting technique(MIT) is a kind of separation and analysis technique used for specific recognition of template molecule, which mimics the naturally occurring antigen-antibody reaction mechanism. The molecularly imprinted polymer(MIP) prepared by MIT not only has the favorable features of predetermined molecular structure, specific recognition and extensive practicability, but also has the advantages of easy to be synthetic, low cost, good stability and reusability, when compared with the natural antibody. Therefore, MIT has attracted the research attention of many scholars in the fields of separation, extraction, biomimetic sensors and simulated antibodies and enzymes. At present, MIT for small molecule compounds has made significant progress. However, the MIT for the separation and analysis of bio-macromolecules particularly protein still has many difficulties with slow progress.This is mainly due to the nature of protein itself, like big size, complex structure and functional groups, flexible conformations and water solubility, which make traditional small molecular imprinting technology difficult to promote to the protein and other biological macromolecules. Surface imprinting technology is a emerging method in recent years which can address a number of challenges for imprinting of protein.In this paper, surface imprinting technology was combined with nanotechnology for the application of protein separation. We respectively synthesized three kinds of protein surface imprinted polymers on silica nanoparticles and modified chitosan nanoparticles, which were both used as carrier materials. The recognition performance of MIPs foe template proteins were investigated by adsorption experiments. The study contents are mainly as follows:(1) Preparation of surface molecularly imprinted polymer based on silica nanoparticle carrier for the recognition of lysozymeIn this work, silica nanoparticles with average diameter of about 500 nm were synthesized by modified St?ber method and used as imprinted polymerization carrier,lysozyme(Lyz) was used as template protein. Functional monomer 3-aminopropyl triethoxysilane(APTES) and crosslinker tetraethoxysilane(TEOS) were polymerized and formed new shell layer of imprinted polymer on the silica nanoparticles by sol-gel method. The chemical composition and morphology of the MIP nanoparticles were investigated by Fourier transform infrared spectrometry(FT-IR), transmission electron microscope(TEM) and thermogravimetric ana Lyzis(TGA). The effects of volume radio of APTES/TEOS on the adsorption capacity of MIP was investigated and finally we select the best volume ratio of 1: 2 to carry out the subsequent study. The results of adsorption experiments showed that Lyz-MIP came to equilibrium within 2 h under the adsorption equilibrium concentration of 0.8 mg/m L with the maximum theoretical adsorption of 89.29 mg/g and the imprinting factor ? of 2.53. In the selective adsorption experiment, MIP showed an excellent selectivity towards the template protein Lyz, because all the three selectivity factor ? of MIP to different reference proteins were above 2. So, the surface imprinted polymer prepared in this work could satisfactorily recognize the template protein and had a broad application prospect in the separation and analysis of proteins.(2) Preparation of bi-functional monomers surface molecularly imprinted polymer based on chitosan nanoparticle carrier for the recognition of bovine serum albuminWith acrylamide and acrylic acid as dual functional monomers, C=C double bond modified chitosan nanoparticle as carrier, we prepared bovine serum albumin(BSA)imprinted polymer shell layer on the carrier by surface molecular imprinting technique,then its physicochemical properties and adsorption performance were systematically investigated. FT-IR and TEM were used to characterize the chemical composition and morphology of the MIP. FT-IR spectra showed that, C=C was successfully introduced to chitosan and functional monomers were also successfully polymerized onto the chitosan carrier. TEM figures showed that, the diameters of modified chitosan were only 30 to 40 nm, but the diameters of MIP was between 150 and 160 nm, so we can speculate the thickness of imprinted polymer layer was about 100 nm. Adsorption experiments showed that the kinetics equilibrium time of MIP was 10 h, the thermodynamic equilibrium concentration of MIP was 1.0 mg/m L, and the adsorption capacity of MIP for BSA was significantly higher than the non-imprinted polymer(NIP)with a imprinting factor of 2.04. Maximum adsorption capacity 59.52 mg/g of MIP in theory was got by Langmuir adsorption model. MIP showed a higher adsorption for template protein BSA than reference protein bovine hemoglobin(BHb) and ovalbumin(OVA) in selective adsorption experiment, with selectivity factors of 2.02 and 2.43,respectively. This indicated that the presence of imprinted recognition sites capable of specifically recognizing for template protein on imprinted polymer shell layer of MIP particles. After three adsorption-desorption cycles, MIP still had 64.61% of the first adsorptiona capacity.(3) Preparation of polyacrylic acid surface imprinted polymer based on chitosan nanoparticle carrier for the recognition of bovine serum albuminIn this study, we still used C=C double bond modified chitosan nanoparticle as carrier and N,N- methylene-bis-acrylamide as crosslinker, but selected acrylic acid(AA) as the only functional monomer to prepre the BSA-MIP. The recognition performance of MIP was studied afterwaards. As chitosan surface contained a large number of amino and hydroxy groups, the introduction of acidic monomer AA with carboxyl functional groups expanded the types of functional groups which coulde interact with template proteins. Compared with chitosan nanoparticle carrier, MIP appeared-COOH carbonyl absorption peak at 1728 cm-1in FT-IR spectra and successfully proved occurrence of polymerization of AA onto chitosan nanoparticle.The results of adsorption experiments showed that BSA-MIP came to equilibrium within 2 h under he adsorption equilibrium concentration of 1.0 mg/m L and the adsorption thermodynamic behavior well fitted Langmuir adsorption mode with a maximum theoretical adsorption of 373.13 mg/g. MIP showed excellent selectivity to template protein BSA at the same adsorption conditions when chose BHb and OVA as reference proteins. MIP could still maintain 66.5% of absorption capacity after three adsorption-desorption cycles. It was also found that the p H buffer had great influence on the adsorption capacity for MIP, indicating electrostatic interactions play a decisive role in the absorption and recognition process.