The Application Of Cryogels In Protein Imprinting

Cryogel is a polymer material synthesised by freezing polymerization, the distribution of its pore size is between microns and hundreds of microns, it gets more and more attention of people which possesses macroporous structure, good mechanical performance and stability. Now cryogel has broad applications in adsorption, immobilization, molecular imprinting and catalytic carrier and other fields. Protein imprinted polymers can be used as antibodies, enzymes, or other natural biological substitutes which show a broad application prospect in the field of medicine and biological engineering.This paper formed a novel method of preparation of molecularly imprinted polymer by combining technologies of molecular imprinting and cryogel, and prepared amphoteric polyacrylamide imprinted cryogel. The adsorption and recognition properties of protein imprinted cryogels were studies, as well as the recognition mechanism was discussed. The main contents are as follows:1. Synthesis and characterization of polyacrylamide cryogelsThe modified cryogels were polymerized with acrylamide as monomer, N, N-methylene-bis-acrylamide as crosslinker, acrylic acid, allyl amine as functionalmonomers.The effects of synthesis condition such as the ratio between monomer and crosslinker, types of solvent, polymerization temperature, and polymerization time on the cryogels-performance were investigated. The results indicated that the prepared cryogels provide the good mechanical strength and permeability under optimal experimental conditions which were showed as follows, the mass ratio of 2:1 between monomer and crosslinker, solvent:lOmM PBS(pH=7.4), temperature:-20℃, time:24h. Then the synthesized cryogels was characterized with digital cameras, scanning electron microscopy, infrared spectroscopy, conductometric titration and thermogravimetric analysis.2. Study on synthesis and properties of amphoteric imprinted cryogelsUsing lysozyme, pepsin, ovalbumin, bovine hemoglobin, y-globulin as templates, five amphoteric imprinted cryogels were prepared and a series of different template of imprinted monolithic columns in situ polymerization were also prepared. Various proteins covering wide scopes of molecular weights and isoelectric points (pIs) were used to investigate and compare the imprinting capability of amphoteric polyacrylamide cryogels. Capillary electrochromatography experiments revealed higher retention time in imprinted columns than in none imprinted columns, regardless of the molecular weights or isoelectric points of the proteins. Lysozyme gave the highest imprinting factor 4.8, and pepsin the lowest 2.6, other IFs are in-between. Conclusively the amphoteric polyacrylamide cryogels are effective imprinting materials for various proteins, and could potentially be useful for protein separation and analysis.3. The strengthening of the charged groups on protein imprintingUsing bovine serum albumin (BSA) as the template, as well as acrylamide and N, N’-methylenebisacrylamide, acrylic acid and diallyl amine were added to get various modified molecular imprinted polymers. Imprinting factors of various polymers were measured by the adsorption experiment. The IF on the cryogel made from only AM and Bis AM is about 1.38. Either acidic or basic, the introduction of charged monomers into the polymers increased the IFs obviously. One of the basic cryogels gave the maximum IF (about 2.0) for this type. As both acrylic acid and diallyl amine were used, it further strengthened the affinity without exception. An amphoteric polymer with a suitable acid-base ratio gave a high IF of 3.7. Whatever used alone or both, too much added amounts of the acidic or basic monomer resulted into the reduction of IF. This could be attributed to the coulombic repulsion towards the template molecule from the redundant charged groups. MIP-BSA was used as chromatography stationary phase and filled into a stainless steel column(20mmx4.6mm I.D.). Separating BSA, BHB and mixed solution by MIP-BSA column and NIP column respectively. MIP column shows the specificity recognition ability towards template protein which makes amphoteric cryogel has a good application prospect in the field of complex sample pretreatment.