An aptamer-capture based chromogenic assay for thrombin

A simple chromogenic assay for human alpha thrombin is developed through aptamer affinity capture and a subsequent enzyme reaction. Thrombin is captured on the aptamer-modified magnetic beads, and catalyzes the conversion of chromogenic substrates to optically measured products. The measurement of the generated products by an absorbance spectrometer allows for the final quantification of thrombin. This assay shows high sensitivity by taking advantage of sample enrichment and enzyme amplification, and exhibits good specificity by involving the selective aptamer binding and the specific enzyme reaction. A concentration detection limit of 40 fM can be reached when the tripeptide substrate of tosyl-Gly-Pro-Arg-p-nitroanilide is used in a 24 h enzyme reaction, and the use of 2h enzyme reaction in the assay enables the detection of 400 fM thrombin for a rapid analysis. This assay can be applied to detect thrombin in dilute human serum.