Design And Preparation Of Protein Imprinted Polymer With Maintaining The Structure Stability Of Template

To date,molecular imprinting technology against small molecules has been well established.However,the imprinting of macromolecules,especially for protein and enzymes poses many challenges.The most difficult problem was the flexible structures of protein,which can easily denatured and unfolded during the preparation of molecular imprinted polymers(MIPs).Therefore,how to maintain the structural stability of protein during the MIPs preparation process is the key issue in protein imprinting technology.The object of this work is to achieve the effective imprinting and accurate recognition of protein,and this work is focus on maintaining the structural stability of protein in order to solve the long plagued problem of variability in protein structure during the preparation of MIPs.In order to achieve the above goal,the strategies of using ionic liquids act as protein stabilizer and utilizing macromolecular monomer to imprint protein steadily were carried out respectively and the work have yielded important information.The main research contents and results are listed as followed:(1)Thermal preparation of lysozyme imprinted microspheres was firstly investigated by using biocompatible ionic liquid(IL)as a thermal stabilizer.The effect of IL dosage on the secondary structure,tertiary structure and activity of lysozyme was investigated,and the results showed that 5 wt% IL as the stabilizer,75 °C as the reaction temperature could reserve the structure of template protein as much as possible.Furthermore,the imprinted microspheres made by IL could obtain the good recognition ability to template protein,whereas the imprinted polymer synthesized in the absence of it had poor selectivity.The study results demonstrated that IL could effectively maintain the stability of lysozyme,and thus could achieve the effective imprinting and accurate recognition of protein.(2)Through consulting the Hofmeister series,a novel biocompatible and polymerizable ionic liquid(IL)was designed and used as stabilizer and co-monomer to prepare bovine serum albumin(BSA)imprinted hydrogels.The research results demonstrated that IL could effectively enhance the structural stability of BSA.Compared to pure BSA solution,the BSA solution containing IL would reserve more integral structure of protein after incubation for 48 h.In contrast with traditional organic small monomers that could destroy the conformation and structure of protein,the imprinted hydrogels made by IL obtained much better selectivity and recognition ability to BSA.The strategy of applying biocompatible and polymerizable IL in imprinting technology will provide a new idea for effective macromolecule imprinting.(3)In this study,an assumption that a micromolecular monomer could easily permeate into the inside of protein and alter their conformation,while an inflexible macromolecular monomer may interact with the surface of protein and thus maintain the integrity of the template protein’s structure was firstly proposed and confirmed by using circular dichrosim and synchronous fluorescence spectroscopy.The adsorption experimental results demonstrated that the imprinted hydrogels prepared by the macromolecular monomer exhibited much better specific recognition ability to the template protein.Therefore,the strategy of using macromolecule to imprint could effectively overcome the mutability of protein during the preparation of imprinted polymers,and consequently would promote the development of imprinting technology.(4)Based on the structure of BSA,the macromolecular functional monomer(MFM)was designed and synthesized,and utilized in the preparation of BSA imprinted nanoparticles on the surface of Si O2.Results from circular dichroism and synchronous fluorescence experiments reflect the MFM tendency to interact with the protein surface instead of permeating it and destroying the hydrogen bonds that maintain the protein’s structural stability,therefore stabilizing the template protein structure during the preparation of MIPs.The selective and competitive adsorption experiments demonstrated the imprinted nanoparticles made by MFM possessed much better selectivity and specific recognition ability for template protein.Furthermore,the results also showed that it was effective way to decrease the mass transfer resistance of protein in MIPs by using surface imprinting technology.The strategy combining surface imprinting technology and using macromolecular monomer to steadily imprint protein would effective overcome the difficulties of mutable,huge and complicated properties of protein during the preparation of MIP.Therefore,it could be suggested as an important research idea for the future protein imprinting technology.