| Aptamers are often used as affinity ligands due to their high specificity and selectivity,and high stability,easy chemical modification,and wide range of target ligands.Thus,it has been widely used in environmental detection and food safety analysis.In this paper,the hybrid monolithic column was prepared by"sol-gel"method,and gold nanoparticles were used as the connecting medium to prepare a high coverage density of OTA aptamer modified affinity hybrid monolithic silica column was prepared,to realize high sensitivity recognition and detection of ochratoxin A.The main contents are as follows:In chapter 1,literature review.In this part,the types and research progress of capillary monolithic column were introduced.The research progress and application of AuNPs modified monolithic column were summarized.The characteristics of aptamers and their research progress and application in aptamer affinity monolithic column are reviewed in detail,Therefore,the research content and significance of this paper has been put forward.In chapter 2,by using tetramethoxysilane(TMOS)and 3-merraptnpropylt rimethnxysilane(MPTMS)as silane agents,the poly(TMOS-co-MPTMS)hybrid monolithic silica column was prepared.'Based on the poly(TMOS-co-MPTMS)monolithic column,using AuNPs with different sizes as an intermediary for a sandwich structure to realize the functional modification of the surface of the monolithic column and derived by post-column derivation by AuNPs and aptamer,the Apt@AuNPs@poly(TMOS-co-MPTMS)hybrid affinity monolith column was prepared.The composition,morphology,EDS,FTIR and mechanical stability of the monolithic column were investigated.The amount of aptamer on hybrid monolithic column was characterized.The effects of the permeability of polymer structure on the coverage intensity of Apt@AuNPs and affinity reaction of OTA were evaluated,and the influence of AuNPs with different sizes on the coverage density of Aptamer was also investigated.Then,the binding amount of aptamer on hybrid monolithic column was investigated.The results showed that the new type of Apt@AuNPs@poly(TMOS-co-MPTMS)hybrid affinity monolith column has the advantages of high aptamer coverage density,which could reach 3,494 pmol/uL,and strong stability.In chapter 3,based on the Apt@AuNPs@poly(TMOS-co-MPTMS)monolithic column and its specific selectivity and high affinity to the target(OTA)of the aptamer,the affinity performance to the target(OTA)was studied by using Apt@AuNPs@poly(TMOS-co-MPTMS)hybrid affinity monolithic column.The recognition of OTA and the specific selectivity of Apt@AuNPs@ poly(TMOS-co-MPTMS)hybrid affinity monolithic column were investigated.the recovery yield of the affinity hybrid monolithic column of OTA was investigated;and the elution parameters were optimized,the maximum binding capacity of target OTA in the Apt@AuNPs@poly(TMOS-co-MPTMS)hybrid affinity monolith column was also evaluated with the frontal chromatography method.In addition,the analysis and detection of the target in actual samples(beer and other wine samples),the stability and lifetime of the Apt@AuNPs@poly(TMOS-co-MPTMS)hybrid affinity monolithic column were investigated.The results showed that,the hybrid monolithic column with high specificity for OTA and the recoveries of OTA were carried out with high values level above 92.3±2.1%and 92.5±0.5%(n=3),respectively.The binding capacity of OTA in the hybrid monolithic column was therefore high and reached 9.75 ng,and the detection limit(LOD)for OTA was 0.025 ng/mL without large-scale injection enrichment.In the real samples,the affinity hybrid monolithic column still has high specific recognition ability to the OTA.Applied to beer and wine samples with different fortified levels of OTA(0.5 ng/mL-5 ng/mL),the average recoveries of OTA were gained in the range of 89.6±3.7-91,8±1.6%and 90.3±2,8-91.6±2.7%,respectively.In addition,the aptamer modified affinity silica hybrid monolithic column has high stability and its lifetime was more than 3 months. |
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