Aptamer-based Organic-silica Hybrid Affinity Column Prepared Via "Thiol-ene" Click Reaction For Enrichment And Detection Of Thrombin

Organic-silica hybrid monolithic column is a novel monolith material with many merits such as facile preparation, a wide pH stability and good mechanical strength, etc. Aptamer is a single-stranded DNA or RNA oligonucleotide, which can specifically bind to targets, showing advantages over antibodies such as strong affinity, good stability and ease of chemical modification. In recent years, aptamers-based affinity monolith has attracted much attention and been widely used in liquid chromatography, capillary electrophoresis, solid phase micro-extraction, etc.In this work, a novel method to prepare the aptamer-based affinity column has been developed. First, tetramethoxysilane-co-y-methacrylic acid trimethyl silane (TMOS-y-MAPS) hybrid silica monolithic capillary column was prepared by sol-gel method. Then,5’-SH-modified aptamer for human thrombin was covalently bonded on hybrid column via "thiol-ene" click chemistry to obtain the aptamer-based affinity hybrid column. Last, it was applied in the enrichment and detection of human α-thrombin employing the enzymatic reaction.1. Preparation and characterization of aptamer-based organic-silica hybrid affinity column via "thiol-ene" click reactionThe chromatographic performance of a hybrid monolithic column denpends on its morphology, structure and surface properties. In order to obtain the desired aptamer-based affinity column, a hybrid column was prepared by sol-gel method with tetramethoxysilane (TMOS) and y-methyl methacrylate trimethoxysilane (y-MAPS) as the precursors. Then,5’-SH-modified aptamer for human a-thrombin was immobilized on the hybrid monolith via "thiol-ene" click chemistry. By changing the ratio of the precursors (TMOS/y-MAPS), the amount of porogen, catalyst and other factors, we optimized permeability and porosity of the monolithic column and the optimized affinity monolith possessed a large surface area and good specificity. Last, the aptamer-based affinity hybrid column was further characterized by infrared spectra (FT-IR) and elemental analysis (EA), etc. Results demonstrated the aptamer was covalently bonded on the hybrid monolithic column, and the average coverage density of aptamer was 420 pmol/μL.2. Applications of aptamer-based affinity hybrid column in enrichment and detection of human a-thrombinWe applied the optimized aptamer-based affinity monolith for the enrichment and detection of proteins. In this work, we chose human a-thrombin as a model compound and achieved the rapid and sensitive detection of trace protein by taking advantage of sample enrichment and enzyme amplification. By changing pH of the eluent, temperature and catalysis condition, we achieved an effective elution and high sensitive detection of thrombin. Under optimal conditions, the enrichment factor for human a-thrombin in the affinity column was 3 and assay of human a-thrombin spiked in human serum showed the recovery of 91.8%. These results indicated that the developed "thiol-ene" click chemistry provided an effective method for preparation of aptamer-based hybrid monolithic column and it might be applied to selectively recognize trace proteins.