Study On The Protein Imprinted Molecularly Core-Shell Microspheres

Molecular recognition is an important activity in life process. Molecular imprinting technique is an artifical method of synthesizing polymer with special selectivity by simulating recognition function of natural molecular. In this work, great advance has been made by effort. In specially, for small molecules, the various techniques have been perfect and applied to many aspects. For bio-macromolecules such as protein, however, the existed methods are not ideal. Experimental results have not good reproducibility. Recognition mechanism has not been completely understood under water environment and bio-macromolecules imprinted polymers have poor mass transport. Thus it is necessary to further study bio-macromolecules technique. This has a great significance for the application of bio-macromolecules imprinted materials in the domains of catalysis, separation, bio-pharmacy, analytical chemistry, sensor, and so on.In this paper, general principle, application and actuality of molecular imprinting technique were reviewed. Molecular imprinting technique originated from immunology. After the theophylline imprinted polymer was reported in Nature (1993), molecular imprinting technique has made breakthrough. The general principle of technique is polymerization process. Functional monomers form a complex with the template molecule. After the functional monomers are polymerized with the cross-linker, a polymeric network is formed around the template. The polymer liberates complementary binding sites by removing the template, and these sites can memorize the template. Molecular imprinting technique can be divided into tow distinct approaches: covalent (preorganization) and noncovalent (self-assembly). Molecularly imprinted polymers exhibit good behavior in the applications of chromatography separation, bio-mimetic catalysis, drug analysis and analytical sensor. The studies of molecular imprinting are transforming from small molecules to bio-macromolecules. At present, various proteins were used for molecular imprinting such as bovine serum albumin, bovine hemoglobin, ribonuclease and lysozyme etc. For protein molecular imprinting, the monomers commonly used are acrylic acid, methacrylic acid, acrylamide, N, N'-ethylenebis (acrylamide), N, N'-methylenebisacrylamide. The physical forms of protein molecular imprinting can conveniently be subdivided into either 3D (bulk) or 2D (thin film).The experiment mainly includes the preparation of polystyrene microspheres, the preparation of imprinted core-shell microspheres in aqueous solutions for proteins, and the studies of the specific binding ability and the recognition of the imprinted microspheres. The mechanisms of imprinting and recognition for protein were analyzed.Firstly, suspension polymerization was selected for the preparation of polystyrene microspheres from the approaches to synthesize microspheres. The effects of temperature, stirring speed, initial amounts of initiator and monomer, and surfactant concentration on morphology of polystyrene microspheres were investigated. The morphology, size and size distribution of the microspheres were characterized using scanning electron microscope (SEM). The experimental results indicate that polystyrene microspheres with the diameter of 50-60μm can be prepared by the method under appropriate conditions. The microspheres have uniform size and clean surfaces. The advantages of the method are that the speed of reaction is rapid, manipulation is convenient and product is easily separated.Secondly, the thin films of poly-3-amonophenylboronic acid in aqueous environment were grafted on polystyrene microspheres by oxidation for forming core-shell microspheres. The effects of acetic acid solution and sodium phosphate buffers on stability of grafted thin films were valued. The samples were characterized using transmission electron microscope (TEM), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS) and nitrogen adsorption/desorption methods. The results show that poly-3-amonophenylboronic acid can be grafted upon surfaces of the polystyrene microspheres by aromatic ring electron-pairing interaction, and the surfaces of core-shell microspheres have high stability in acetic acid solution and sodium phosphate buffers.Thirdly, based on the core-shell microspheres, protein molecular imprinting was implemented when protein was presented during grafting. For examining the feasibility of the strategy, lysozyme imprinted core-shell microspheres were first prepared. The 3-amonophenylboronic acid and lysozyme solutions of definite concentration were mixed for them interacting. Then definite polystyrene microspheres and the ammonium persulfate solution of definite concentration were added. After the polymerization was carried out at 22-24℃, the imprinted microbeads were separated from the solution and washed repeatedly with deioned water and dried for definite time. Finally, they were washed repeatedly with the acetic acid solution for removing template molecules and dried at room temperature. The batchwise adsorption experiments were carried out for detecting the time to reach equilibrium of adsorption, the abilities of specific adsorption for the template protein, and selective recognition for the template protein and the stability for the imprinted core-shell microspheres. The imprinted microspheres can reach equilibrium of adsorption in 60 min, and this proves the imprinted microspheres have good mass transport. The imprinted microspheres have higher specific adsorption than the nonimprinted microspheres when adsorption reaches equilibrium. The imprinted microspheres have higher adsorption ability for the template protein than non-template proteins. In additionally, the adsorption ability of imprinted microspheres didn't obviously decrease for template protein over two months, and this shows that the imprinted microspheres possess high stability.Finally, the imprinted microspheres for various proteins such as bovine hemoglobin, bovine serum albumin, trypsin and papain were prepared using the strategy and batchwise adsorption experiments were carried out for detecting the abilities of specific adsorption for the template protein, and selective recognition for the template protein, mass transport and the stability for the imprinted core-shell microspheres.In collusion, the protein imprinted core-shell microspheres prepared by us possess good mass transport, high ability of adsorption and good recognition for template proteins. The molecularly imprinted technique can be used in aqueous solution for protein imprinting under the mild conditions. The technique is convenient and has a certain extent generality.