The Design, Preparation And Application Of Novel Affinity Monolithic Columns

is-diol containing biomolecules (ccs-diols), which have exhibited great biological significance, are an important class of compounds in life science, including saccharides, catechols, nucleosides and glycoproteins. Many of these biomolecules have been biomarkers for disease diagnosis. However, these biomarkers are usually in complex biological samples with very low abundance. Therefore, how to selectively enrich and separate these target cis-diols is a great challenge in analytical chemistry for life science. Boronate affinity monolith has been an important tool for the analysis of cis-diols based on its reversible pH-controllable capacity. However, there are still some issues for boronate affinity monoliths, such as poor hydrophilicity, low binding strength towards cis-diols, etc. Although boronate affinity monoliths have exhibited excellent pH-controllable ability for the analysis of glycoproteins, the analytes are only limited to glycoproteins. In porous affinity separation media, the confinement effect of pores was usually considered as a detrimental factor. However, favorable nanoconfinement effects in separation science have been continuously unveiled recently. Based on the fact that molecular interactions confined in nanopores were significantly enhanced as compared with those under non-confined conditions, nanoconfinement effect might be used as a new element for the rational design of unique protein affinity materials. To resolve the issue of poor hydrophilicity of boronate affinity materials, we prepared a boronate affinity organic-silica hybrid monolith for the good hydrophility of silica materials. Meanwhile,3-acrylamidophenylboronic acid (AAPBA) was chosen as the functional monomer for two important reasons. First, AAPBA is more hydrophilic than conventional vinylphenylboronic acid (VPBA). Second, as boronate affinity chromatography usually requires that the binding pH≥pKa of the boronic acid ligand, the relative low pKa value of AAPBA (8.2) avoids the use of high basic pH condition at which silica may hydrolysis. As a result, the prepared AAPBA-silica hybrid monolithic column will be more hydrophilic, which is good for eliminating unfavorable non-specific interactions without adding organic solvents. The hybrid monolith could bind cis-diols at pH as low as6.5, very suitable for the treatment of physiological samples. The binding capacity of the hybrid monolith was the highest among reported boronate affinity monolithic columns, which could be attributed to its large specific surface area. In addition, the hybrid monolith exhibited apparent secondary separation capability, allowing two dimensional (2D) separation in a single column. An off-line hyphenation of boronate-silica hybrid monolith based2D capillary liquid chromatography and capillary electrophoresis was established, to demonstrate the feasibility for application to the analysis of modified nucleosides in human urine.For the low binding strength of boronate affinity materials towards cis-diols, we chose a boronic acid with high affinity towards cis-diols for the preparation of boronate affinity monolith. The compound2,4-difluoro-3-formyl-phenylboronic acid (DFFPBA) was chosen as the functional ligand, because it exhibited not only ultrahigh boronate affinity but also a relatively low pKa value (6.5). As expected, the prepared DFFPBA-functionalized monolithic columns exhibited an ultrahigh boronate affinity towards cis-diols. Meanwhile, the DFFPBA-functionalized monolith with appropriate spacer arm can capture cis-diols at pH as low as6.0. Additionally, the length of spacer arms could strongly influence the boronate affinity towards cis-diols. With spacer arm length increasing the boronate affinity reduced; what is more, boronate affinity towards glycoprotein was even eliminated with inappropriate spacer arm length. A possible mechanism was proposed to explain such a unique phenomenon. Finally, a DFFPBA-functionalized monolithic column was applied for selective enrichment of modified nucleosides in human urine.Because of the excellent pH-controllable capacity of boronate affinity materials and its limited binding targets (only glycoproteins), and further understanding of nanoconfinement effect by us, a nanoconfining strategy was proposed for the development of new affinity materials. Two nanoconfinement affinity materials (NCAMs) that allow for pH-mediated protein capture/release have been developed. The NCAMs provided forceful complementary tools to boronate affinity materials, and extended analytical targets from glycoproteins to non-glycoproteins. The NCAMs also exhibited other features and advantages:First, the NCAMs could bind proteins with molecular weight more than18kDa when the surrounding pH was≥6.0, while the captured proteins could be reversibly released upon switching the environmental pH<3.0. The dissociation constants for three test proteins were measured in the range of10-5to10-7M. Second, since the conformation and activities of captured proteins could be well retained, the NCAMs could provide a general supporting material for facile fabrication of protein-functionalized chiral separation columns and immobilized enzymatic reactors. Third, the NCAMs have exhibited a great promise for the depletion of high-abundance serum proteins for proteomic analysis. Although the current NCAMs could not provide target specificity, which could be available via molecular imprinting polymers (MIPs), a significant advantage of NCAMs over MIPs is that no template molecules are needed. If base materials with narrowly dispersed nanopores are available in future, size-specific NCAMs can be possible. This strategy opens up new avenues for the rational design of unique functional materials.