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

• We immobilize aptamer on hybrid monolithic column by facile thiol-ene click reaction.
• The coverage density of aptamer of thrombin on the monolith is high to 420 pmol μL−1.
• The affinity monolithic column retains thrombin with high specificity and capacity.
• We assay thrombin extracted by the affinity column using chromogenic substrate T1637.

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 μL−1. Human α-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 μM by spectrophotometry. Furthermore, the extraction recovery of thrombin at 0.15 μM 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.

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