Preparation And Evaluation Of Aptamer Modified POSS-based Organic-silica Hybrid Affinity Monolithic Column

Aptamer has attracted great attentions due to the merits of wide range of targets,high specificity and selectivity for the target,high stability,and easy chemical modification.As a type of newly emerging functional material,aptamer modified affinity monolithic column has become one of the hot spots in the research of affinity chromatography micro-separation.It is of great scientific significance to develop new preparation methods of aptamer affinity monoliths for improving the affinity monolithic column analysis technology.Polyhedral oligomeric silsesquioxanes(POSS)organic-silica hybrid monolithic columns showed advantages of easy fabrication,high mechanical stability and diversified functional monomers.Thus this paper developed a series of novel aptamer modified POSS-based affinity monolithic columns and studied their properties.Main points of this thesis are listed as follows:Chapter 1,literature review.In this part,the classification and development of monolithic columns were introduced.And the characteristics,the immobilization technology and the applications of aptamers were reviewed.Based on the classification,progress and applications of the aptamer affinity monolithic columns,research scheme of this study using OTA as the research object were briefly put forward.Chapter 2,the POSS-PEI organic-silica hybrid monolith with abundant amino groups on its surface was prepared by "one-pot" approach via "epoxy-amino"ring-opening polymerization,with branched polyethyleneimine(PEI)as functional monomer and polycyclic oligomeric silsesquioxane(POSS-epoxy)as cross-linking agent.The effects of different reactant compositions on the structure,permeability,electroosmotic flow and chromatographic properties of monolithic column were studied in detail.The prepared POSS-PEI organic-silica hybrid monolithic column has a controllable and porous 3D-network skeleton,appropriate permeability,high mechanical stability,hydrophilicity,and excellent reproducibility.Moreover,the rich amino active sites on the surface provide a good support for subsequent high-capacity immobilized aptamers for the preparation of novel affinity monolithic columns.Chapter 3,based on the aldol condensation reaction,a novel glutaraldehyde-coupled aptamer modified POSS-PEI affinity monolithic column(Apt@GA@POSS-PEI)was inovatively prepared by chemically bonding amino aptamers on the POSS-PEI matrix stationary phase.The conditions of the immobilization procedure were optimized.And the morphology,energy spectrum,mechanical stability,chromatographic performance,aptamer coverage density,binding capacity and specificity of affinity monolithic column were evaluated.This section also investigated the influence of the matrix’s permeability on the performance of the affinity monolithic column.And the mechanisms of interaction between the affinity monolithic column and the targets were discussed.The prepared affinity monolithic column has a unique skeleton structure,uniform pore distribution,excellent mechanical stability and higher selective recognition ability for the target OTA,achieving highly specific detection of OTA in the actual samples.Chapter 4,based on the nucleophilic reaction of 2,4,5-trichoro-1,3,5-triazine with amino groups,amino-modified aptamers were immobilized on the POSS-PEI matrix monolith to obtain a novel aptamer modified POSS-based organic-silica hybrid affinity monolithic column(Apt@TCT@POSS-PEI).The covalent interaction of TCT and amino-modified aptamers,the optimization of the modification parameters and the influence of permeability on the specific recognition of the affinity monolithic column were systematically studied.The mechanisms of the interaction between the affinity monolith and the targets were investigated.The morphology,energy spectrum,spectral properties,mechanical stability,reproducibility and specificity of affinity monolithic column were evaluated.The conditions of the nucleophilic reaction of 2,4,5-trichoro-1,3,5-triazine with amino groups were mild and controllable.This modification method was quick and efficient,with less damage to the biomolecules.A new technique for the chemical modification of amino aptamers has been established,of which the grafting yield of aptamer was 75.3%and the aptamer coverage density was 1799 pmol/μL.The binding capacity and specificity for target OTA was excellent,with high recoveries of OTA in the mixture ranged from 91.8 ± 1.9%to 90.0 ± 1.7%(n = 3).Chapter 5,by using gold nanoparticles(AuNPs)as intermediary,thiol-modified aptamers were immobilized on the POSS-PEI matrix monolith.A novel aptamer modified POSS-based organic-silica hybrid affinity monolithic column(Apt@AuNPs@POSS-PEI)with a high aptamer coverage density was innovatively developed.The effects of amine reagents and skeletal structures of the hybrid monoliths on the modification efficiency of Apt@AuNPs and the specific performance of the affinity monolith were studied.The column morphology,energy spectrum,mechanical stability,aptamer coverage density,binding ability to target and specificity were characterized,and the interaction mechanisms between the Apt@AuNPs@POSS,PEI affinity monolith and targets were explored.The prepared Apt@AuNPs@POSS-PEI affinity monolithic column was of a highly stereoscopic skeletal structure and ultra-high aptamer coverage density up to 1413 pmol/μL,which was 4～5 times more than the coverage density of the Apt@AuNPs modified monolith reported previously.Applying on the enrichment,separation and detection of OTA in real beer samples,the column can achieve a recovery rate in the range of 94.5±2.7%～92.5 ± 2.1%(n = 3).The above data indicated that this paper provided a novel aptamer-specific recognition material for high-specific enrichment,separation and detection of OTA.