Aptamer-based organic-silica hybrid affinity monolith prepared via thiol-ene click reaction for extraction of thrombin

A novel strategy for preparing aptamer-based organic-silica hybrid monolithic column was developed via thiol-ene click chemistry. Due to the large specific surface area of the hybrid matrix and the simplicity, rapidness and high efficiency of thiol-ene click reaction, the average coverage density of aptamer on the organic-silica hybrid monolith reached 420 pmol mu L-1. Human alpha-thrombin can be captured on the prepared affinity monolithic column with high specificity and eluted by NaClO4 solution. N-p-tosyl-Gly-Pro-Arg p-nitroanilide acetate was used as the sensitive chromogenic substrate of thrombin. The thrombin enriched by this affinity column was detected with a detection of limit of 0.01 mu M by spectrophotometry. Furthermore, the extraction recovery of thrombin at 0.15 mu M in human serum was 91.8% with a relative standard deviation of 4.0%. These results indicated that thiol-ene click chemistry provided a promising technique to immobilize aptamer on organic-inorganic hybrid monolith and the easily-assembled affinity monolithic material could be used to realize highly selective recognition of trace proteins. (C) 2015 Elsevier B.V. All rights reserved.