| Boronate affinity chromatography is a chromatography method that uses boronic acid or boronate derivatives as the affinity ligands for the separation and enrichment of cis-diol-containing molecules such as nucleosides, nucleotides, carbohydrates, glycoproteins and glycopeptides. In recent years, with the development of proteomics, glycomics and metabolomics, boronate affinity chromatography has played an increasingly important role, and become a powerful tool for scientific research. Due to its merits of easy preparation, high mass transfer rate, low cost and high column efficiency, monolithic column is regarded as the fourth-generation separation medium, and has been widely used in the separation of various compounds. Boronate affinity monolith chromatography which combines boronate affinity chromatography with monolithic column will promote the researches in "-omics". In conventional boronate affinity chromatography, affinity interaction has to be carried out under alkaline conditions, which may cause the degradation of biological samples or protein denaturation, limiting the application of this technology. This work aimed at preparing some novel boronate affinity hybrid monolithic columns which were able to capture cis-diol biomolecules under neutral/weakly acidic conditions and reduce the nonspecific adsorption. The main contents of the research are described as follows:1. A novel boronate affinity capillary monolithic column with synergistic co-monomers was prepared by "one-step" approach which used tetraethoxysilane (TEOS),3-trimethoxysilyl propylmethacrylate (y-MAPS) and N-(?-aminoethyl)-y-aminopropyltriethoxysilane (AEAPTES) as alkoxysilane precursors,2,2'-azobisiso butyronitrile(AIBN) as initiator,4-vinylphenylboronic (VPBA) as functionalized monomer. Through the optimization of experimental conditions, a "one-step" method was developed for the preparation of the monolith. The method was time-saving, convenient and simple. Due to the intermolecular B-N coordination between the amino group and the boronate molecule, the esterification reaction for binding the cis-diol-containing compounds could proceed under lower pH conditions..The characterization results by various instruments indicated that the prepared boronate affinity hybrid capillary monolith had typical double pore structure, large surface-to-volume ratio and high loading capacity. The affinity of the monolith to the cis-diol-containing compounds was demonstrated by the specific capture of catechol.2. The nucleosides in urinary sample was captured and enriched by the boronate affinity monolithic column prepared above, and then analyzed by the reversed phase high performance liquid chromatography. The nucleosides in the real urinary sample could be enriched, separated and identified by using boronate affinity chromatography in combination with HPLC. The determination of urinary nucleosides was very important for early clinical diagnosis of cancer. The developed method was verified to be an effective way for monitoring nucleosides in urine.3. Another novel boronate affinity capillary monolithic column was prepared by "one-pot" approach which used tetrabutyl orthotitanate (TBOT), TEOS, y-MAPS and AEAPTES as co-precursors, AIBN as initiator, VPBA as functionalized monomer. The prepared monolith was characterized by X-ray diffraction (XRD), FT-IR spectrometer (FT-IR), scanning electron microscope (SEM), nitrogen absorption/desorption, energy dispersive X-ray spectrom (EDX) and X-ray photoelectron spectroscopy (XPS). Due to the introduction of TiO2, the prepared monolith was more hydrophilic, and could capture both small molecule nucleosides and macromolecules such as glycoproteins and antibodies at neutral even weakly acidic conditions.4. A novel boronate affinity monolithic material was prepared via a sol-gel method, in which TBOT, TEOS, AEAPTES were used as co-precursors, and boric acid (H3BO3) was used as functionalized monomer. After calcinating at900?, the SiO2/TiO2/B2O3inorganic monolith was obtained. The prepared material was characterized by XRD, FT-IR, SEM, EDX, XPS and Raman spectrum. Since inorganic boric acid functioned as the ligand, the nonspecific adsorption due to hydrophobic effect of the organic ligands was reduced. The inorganic material could bind the cis-diol-containing molecules such as nucleoside, glycoproteins and antibodies at physiological conditions. Nucleosides from a human urinary sample could be specifically captured by the inorganic material. |
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