Preparation Of Boronate Affinity Magnetic Nanoparticles And Its Application For Solation And Enrichment Of Target Glycoproteins

This thesis is involved in preparation of boronate affinity magnetic nanoparticles and its application for isolation and enrichment of glycoproteins and sialic acid. It consists of five chapters.In chapter one, the general introduction to "click chemistry", distillation-precipitation polymerization technique, boronate affinity materials utilized in this theis and applications of functionalized-magnetite nanoparticles for separation and determination of proteins. Additonally, the aim, significance and innovation was briefly presented as well.In chapter two, a novel approach was developed to synthesize aminophenylboronic acid functionalized magnetic nanoparticles (NPs) via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC)’click’ chemistry. Firstly, azide-functional Fe3O4NPs were obtained by a two-step chemical modification process. Then, an alkyne-phenylboronic acid molecule was connected onto the surface of magnetite by the CuAAC reaction. The morphology, structure and composition of the synthesized nanocomposites were characterized by transmission electron microscopy (TEM), X-ray powder diffraction (XRD), vibrating sample magnetometer (VSM), Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectrometry (XPS). Five proteins, including ovalbumin (OVA), transferrin (Trf), as glycoprotein templates and lysozyme (Lyz), bovine serum albumin (BSA), horse heart cytochrome c (Cyt C) as nonglycoprotein templates are chosen as target proteins. The as-prepared click-Fe3O4@APBA NPs with a mean diameter of23.2nm showed a strong magnetic response to an externally applied magnetic field and exhibited a high adsorption capacity and excellent specificity towards glycoproteins in comparison with nonglycoproteins. The click-Fe3O4@APBA NPs showed the higher adsorption capacity towards glycoproteins than the nonclick-Fe3O4@APBA NPs which were synthesized through a common nucleophilic substitution reaction. The greatly enhanced adsorption capacity towards glycoproteins demonstrated that the ’click’ method presented the great superiority in ligand immobilization. Finally, the click-Fe3O4@APBA NPs could efficiently enrich glycoproteins from real egg white samples as well.In chapter three, we presented a facile and time-saving method to synthesize phenylboronic acid and copolymer multi-functionalized magnetic nanoparticles (NPs) using distillation-precipitation polymerization (DPP) technique. The polymer shell is consisted of the affinity ligand3-acrylaminophenylboronic acid (AAPBA) and an additional functional monomer (hydrophobic/hydrophilic/charged monomers). The resulting Fe304@p(AAPBA-co-monomer) NPs exhibited an enhanced binding capacity towards glycoproteins for an additional functional monomer was complementary to the surface presentation of the target protein. The effects of monomer ratio of AAPBA to nine additional co-monomers on the binding of glycoproteins through different interactions were systematic investigated. The morphology, structure and composition of all the synthesized microspheres were characterized by transmission electron microscopy (TEM), X-ray powder diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA) and vibrating sample magnetometer (VSM). Five proteins, including ovalbumin (OVA), transferrin (Trf), as glycoprotein templates and lysozyme (Lyz), bovine serum albumin (BSA), horse heart cytochrome c (Cyt C) as nonglycoprotein templates are chosen as target proteins. The as-prepared Fe3O4@AAPBA and Fe3O4@p(AAPBA-co-monomer) microspheres showed an excellent performace in the separation of glycoproteins with high binding capacity, strong magnetic response to an externally applied magnetic field,which allowed them to be easily separated from solution in the presence of an external magnetic field. Moreover, synthetic Fe3O4@pAAPBA and co-polymeric NPs showed good adsorption to glycoproteins in physiological conditions (pH=7.4). The Fe3O4@p(AAPBA-co-monomer) NPs were successfully utilized to selective capture and identify the low-abundance glycopeptides from the tryptic digest of horseradish peroxidase (HRP). In addition, the selective isolation and enrichment of glycoproteins from the egg white samples at physiological condition was obtained by Fe3O4@p(AAPBA-co-monomer) NPs as adsorbents. These materials exhibited great potential in the glycoproteomic analysis. In chapter four, a facile and efficient approach combining the distillation-precipitation polymerization (DPP) and click chemistry was developed to synthesize the boronic acid ligands-modified magnetic nanoparticles for the enrichment of glycoproteins. Due to relatively large amount of benzyl chloride groups introduced by DPP on the magnetic core, wh,ich easily can be tranferred into azide groups, the alkyne-phenylboronic acid ligands were immobilized onto the surface of Fe3O4with high efficiency via the Cu(Ⅰ)-catalyzed azide-alkyne cycloaddition (CuAAC)’click’ reaction. The morphology, structure and composition of the resulting Fe3O4@pVBC@APBA nanocomposites were characterized by transmission electron microscopy, X-ray powder diffraction, vibrating sample magnetometer, Fourier transform infrared spectroscopy, thermogravimetric analysis and X-ray photoelectron spectrometry. The Fe3O4@pVBC@APBA microspheres held a-50nm polymeric shell, and exhibited the high magnetic response to external magnetic field. The binding results demonstrated that Fe3O4@pVBC@APBA possessed the high adsorption capacity and remarkable selectivity to glycoproteins. Moreover, the glycoproteins in the egg white sample could be enriched at the physiological conditions (pH=7.4) as well, due to the lower pKa value of alkyne-phenylboronic acid ligand. The high stability and selectivity of Fe3O4@pVBC@APBA for the glycoproteins were retained over several separation cycles. These boronate affinity materials have the potential applications in the biomedical and biotechnological fields including drug delivery and biosensors.In chapter five, a kind of phenylboronic acid functionalized thermosensitive magnetic nanoparticles was fabricated for enrichment of sialic acid (SA) from human serum via one-step distillation-precipitation polymerization method (DPP). Firstly, the magnetite was stabilized with the citrate groups, which held a good dispersion in polar solvent. Then, vinyl groups were grafted onto the magnetite by a sol-gel process without tetraethyl orathosilicate (TEOS) pre-coating. Finally, the monomers3-acrylaminophenylboronic acid (AAPBA) and N-isopropyl acrylamide (NIPA) were copolymerized by DPP process. The synthetic Fe3O4@p(AAPBA-co-NIPA) NPs were characterized by transmission electron microscopy (TEM), X-ray powder diffraction (XRD), fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA) and vibrating sample magnetometer (VSM). The optimum monomer ratios, adsorption pH value and adsorption temperature were well investigated. The content of SA was measured by HPLC through a pre-column derivative reaction. The combination of Fe3O4@p(AAPBA-co-NIPA) NPs and SA took on a linear tendency, and the adsorbed SA were isolated by the changes of pH value and temperature. Furthermore, the Fe3O4@p(AAPBA-co-NIPA) NPs could enrich SA from human serum at the acidic environment with no interference by serum proteins and other carbohydrate biomolecules.