Preparation And Evaluation Of Aptamer-bonded Monolithic Columns Based On Epoxy Ring-opening Reaction

Aptamers which possess of the properties of good stability,easy chemical modification,reusability,high affinity and specificity,have attracted more research attentions.In this thesis,three types of aptamer-bonded monolithic columns were prepared via the epoxy ring-opening reaction.And the performances of the aptamer-bonded monolithic columns were studied by using ochratoxin A(OTA)as the model compound.The main contents are summarized as follows:In the first chapter,the research development of affinity chromatography and capillary monolithic columns were reviewed.And the characteristics and immobilization methods of aptamers were summarized.Then the thesis basis and research scheme were put forward for special detection of OTA.The second chapter discussed the preparation and evaluation of Aptamer@poly(GMA-co-POSS-MA)affinity monolithic column.The stationary phase with epoxy groups on the surface was prepared by applying glycidyl methacrylate(GMA)as a functional monomer and polyhedral oligomeric silsesquioxane(POSS-MA)as cross-linker.A 5’-amino aptamer-bound monolithic column was prepared by the ring-opening reaction between epoxy and amino groups.The morphology,composition,mechanical stability and reproducibility of the polymer monolithic column were evaluated.The electroosmotic flow and infrared spectra of the monolithic column were determined before and after modifying by the aptamer.The selective enrichment and separation of OTA from OTA standard samples and mixture samples containing OTA,ochratoxin B and aflatoxin B1 were investigated on the aptamer column,respectively.The prepared hydrophobic aptamer-bonded monolithic column can achieve specific recognition of OTA.The third chapter mainly focused on the preparation and evaluation of Aptamer@poly(epoxy-MA-co-POSS-MA)affinity monolithic column.Methacrylate epoxy cyclic siloxane(epoxy-MA)was used as an organic functional monomer.The morphology,composition,mechanical stability and reproducibility of the monolithic column were investigated.The electroosmotic,infrared and hydrophilic retention mechanisms of epoxy-based column and aptamer-bonded monolithic column were comparatively analyzed.The performance of the aptamer monolithic column for OTA enrichment and separation was tested by using both OTA samples and mixture samples with the optimized eluent.The results showed that epoxy-MA as an organic functional monomer effectively increased the epoxy group content of the monolith,leading to an increase of the aptamer modification efficiency.And the specific recognition of OTA by the Aptamer@poly(epoxy-MA-co-POSS-MA)affinity column was significantly improved。In the fourth chapter,a Aptamer@poly(epoxy-MA-co-PETA)affinity monolithic column prepared by using epoxy-MA as an organic functional monomer and hydrophilic pentaerythritol triacrylate(PETA)as a cross-linker.The morphology,composition,mechanical stability and reproducibility of the polymeric monolithic column were studied.The electroosmotic flow and infrared spectrum were compared for polymeric monolithic columns with and without aptamer modification.OTA were separated and enriched from the OTA standard samples and mixture samples by the aptamer column.The-results showed that pentaerythritol triacrylate as an hydrophilic cross-linker was introduced to effectively reduce the hydrophobicity of the hybrid monolith,and the non-specific adsorption of OTA was effectively attenuated by modifying the aptamer on the surface of the stationary phase.