Study Of Molecularly Imprinted Nanoparticles For Enrichment Of Low-abundance Protein From Cell Extract And Recognition Of Tumor-associated Characteristic Protein

In recent years, molecularly imprinted polymers (MIPs) have attracted increasing interests because of their good stability and highly specific recognition ability. However, MIPs applied for proteins with important physiological significance are developed slowly. In this doctoral thesis, a lot of meticulous research on protein imprinted polymers are performed based on our group’s previous work. The thesis contains two parts. The first part investigated the separation and enrichment of immunoglobulin binding protein (BiP) from cell extract by using molecularly imprinted polymer nanoparticles with silica nanoparticles as carriers. The second part studied the molecularly imprinted polymer nanoparticles for recognition of erythroblastosis B2(ErbB2) of tumor-associated transmembrane protein.In the first part of the work, in order to obtain more specific molecular imprinting materials with higher adsorption capacity, we used surface double-bonded silica nanoparticles as carriers and assistant recognition polymer chains (ARPCs) with randomly distributed carboxyl and pyridine groups as additional monomers. On the basis of that, core-shell protein imprinted polymer nanoparticles were synthesized by using surface grafting imprinting technique with cloned protein BiP as templates, and were applied in the recognition and enrichment of natural low-abundance protein. The combination of ARPCs and nano carrier could enhance the efficiency of MIPs with more specific recognition ability and larger adsorption capacity to the target protein. Also because of its unique core-shell structure, the MIP material was easier to reach adsorption kinetic equilibrium. With the application of the BiP imprinted nanoparticles, the content of BiP in natural endoplasmic reticulum (ER) extracts was improved from0.059%to6.25%, with an enrichment factor of116, which was higher than the conventional macro porous resin based imprinted materials with an enrichment factor of45. Moreover, the adsorption capacity of the core-shell imprinting nanoparticles were much higher. This work will be of great significance for the application of molecular imprinting technique in the purification of natural low-abundance proteins.The purpose of the second part of the work is to synthesis hydrophilic fluorescent molecularly imprinting nanoparticles capable of specifically recognizing ErbB2protein. It could be applied to the detection of trans-membrane protein ErbB2in order to effectively recognize, track and treat of ErbB2-related tumors. During the research, we applied epitope imprinting technique with a special polypeptide of ErbB2in the surface of cells as imprinting template. The template was first fixed to the solid phase (glass slides or glass beads) after several modification steps, and then ARPCs and monomers were introduced around the polypeptide to polymerize MIP naoparticles. We synthesized water-soluble chitosan-polyacrylic acid nanoparticles by template polymerization with different initiating methods. The obtained nanoparticles had a good dispersibility. The molecular imprinted nanoparticles were applied in the recognition and testing of target peptides fixed on the polyvinylidene fluoride (PVDF) membrane. The result showed a certain specific detection effect to the target peptides. This work laid a foundation to the recognition of the surface protein in tumor cells.