Highly specific detection of thrombin using an aptamer-based suspension array and the interaction analysis via microscale thermophoresis

Abstract

A novel aptamer-based suspension array detection platform was designed for the sensitive, specific and rapid detection of human α-thrombin as a model. Thrombin was first recognized by a 29-mer biotinylated thrombin-binding aptamer (TBA) in solution. Then 15-mer TBA modified magnetic beads (MBs) captured the former TBA–thrombin to form an aptamer–thrombin–aptamer sandwich complex. The median fluorescence intensity obtained via suspension array technology was positively correlated with the thrombin concentration. The interactions between TBAs and thrombin were analyzed using microscale thermophoresis (MST). The dissociation constants could be respectively achieved to be 44.2 ± 1.36 nM (TBA1–thrombin) and 15.5 ± 0.637 nM (TBA2–thrombin), which demonstrated the high affinities of TBA–thrombin and greatly coincided with previous reports. Interaction conditions such as temperature, reaction time, and coupling protocol were optimized. The dynamic quantitative working range of the aptamer-based suspension array was 18.37–554.31 nM, and the coefficients of determination R² were greater than 0.9975. The lowest detection limit of thrombin was 5.4 nM. This method was highly specific for thrombin without being affected by other analogs and interfering proteins. The recoveries of thrombin spiked in diluted human serum were in the range 82.6–114.2%. This innovative aptamer-based suspension array detection platform not only exhibits good sensitivity based on MBs facilitating highly efficient separation and amplification, but also suggests high specificity by the selective aptamer binding, thereby suggesting the expansive application prospects in research and clinical fields.