Preparation Of Protein Molecularly Imprinted Nanocomposites And Its Applications

With the accomplishment of the gene sequencing of Human Genomic Project (HGP), increasing attention is focused on the proteomic research. Currently, one of the major bottlenecks encountered in proteomic research is to deplete high abundant proteins and enrich bwabundant proteins from complex biological samples. Developing some novel separation methods and preparation of novel pretreatment materials is the best way to solve themMolecularly imprinted nanocomposites, which combined the high selectivity of molecularly imprinted technique (MIT) in molecular recognition and the characteristics of na no materials with large surface area to volume ratios, are regarded as a promising technology and one of research hot spots. The most interest in this thesis is aimed at developing some novel methods for preparation of protein-template molecularly imprinted nanocomposites and its application in selective separation of target protein.The thesis consists of four chapters. In chapter 1, the general introduction including the principle and historical retrospect of MIT and nanomaterials, protein molecularly imprinted nanocomposites, the difficulties and preparation methods, the potential application of protein molecularly imprinted polymer(MIP) nanocomposites were described in detail. In addition, the aim and significance of this thesis were also briefly presented.In chapter 2, highly monodisperse and uniform-sized silica nanoparticles (NPs) with average diameter of-400 nm were synthesized by using tetraethoxysilane(TEOS) as a single precursor, and then vinyl groups were introduced onto the surface of silica NPs by chemical modification of γ-methacryloxypropykrimethoxysilane (γ-MAPS). Subsequently, the molecularly imprinted polymer (MIP) coating was copolymerized and anchored onto the surface of vinyl modified silica NPs dispersed in aqueous media with lysozyme (Lyz) as a template. Themorphobgy and structure property of the resultant MIP-coated silica NPs were characterized by scanning electronic microscopy (SEM), transmission electronic microscopy (TEM) and Fourier transform infrared spectroscopy (FT-IR).The polymerization and adsorption conditions were investigated in detail in order to obtain the highest selectivity and binding capacity. Under the optimized conditions, the imprinted nanoparticles showed higher binding affinity toward the template than non-imprinted (NIP) nanoparticles, and the corresponding imprinted factor (a) reached 1.68. The specificity for Lyz recognition was evaluated with competitive experiments, indicating the imprinted nanoparticles had a higher selectivity for the template. In addition, the stability and regeneration were also investigated, which indicated theimprinted silica NPs had excellent reusability.In chapter 3, a facile method was developed for synthesis ofpolydopamine-coated molecularly imprinted silica nanoparticles (PDA-coated MIP silica NPs) based on self-polymerization of dopamine (DA) on the surface of silica NPs in the presence of template protein. Transmission electronic microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR) and thermogravimetric analysis (TGA) showedthatPDA layers were successfully attached on the surface of silica NPs and the corresponding thickness was about 5 nm, which enabled the MIP silica NPs to acquire fast binding kinetics and high binding capacity. Under the aqueous media, the imprinted silica NPs showed much higher binding affinity toward template than non-imprinted (NIP) silica NPs. The protein recognition properties were examined by single-protein or competitive batch rebinding experiments and rebinding kinetics study, validating that the imprinted silica NPs have high selectivity for the template. The resultant BHb-MIP silica NPs could not only selectively separate BHb from the protein mixture, but also specifically deplete high-abundance BHb from cattle whole blood. In addition, the stability and regeneration were also investigated, which indicated that the imprinted silica NPs had excellent reusability.In chapter 4, based on the principle of boronate affinity interaction, a new molecularly imprinted SiO2 nanocomposites were synthesized for glycoprotein recognition, in which nano-sized SiO2 was used as supporting matrix, horseradish peroxidase (HRP) as glycoprotein template, and 3-acrylamidophenylboronic acid (AAPBA) as functional monomer forpre-assembling template. Well defined core-shell structure of SiO2 nanocomposites can be obtained after coating silane by sol-gel process. The synthetic conditions were investigated in detail in order to obtain the highest selectivity and binding capacity. Under the optimized conditions, the imprinted nanoparticles showed higher binding affinity toward the template than non-imprinted (NIP) nanoparticles, and the corresponding imprinted factor (a) for HRP reached 2.53. The specificity for Lyz recognition was evaluated with competitive experiments, indicating that the imprinted nanoparticles have a higher selectivity for the template. In addition, the stability and regeneration were also investigated, which indicated the imprinted silica NPs had excellent reusability.