In Vitro Isolation of Quick-Response High-Affinity Aptamers for Continuous and Reagentless Detection of Thrombin

Continuous and reagentless biomolecular detection technologies are bringing an evolutionary influence on disease diagnostics and treatment. Aptamers are attractive as specific recognition probes because they are capable of regeneration without washing. Unfortunately, the affinity and dissociation kinetics of the aptamers developed to date show an inverse relationship, preventing continuous and reagentless detection of protein targets due to their low dissociation rates. Here, we describe an in vitro aptamer isolation strategy that enriches quick-response, high-affinity bivalent protein-binding aptamers. The method is general, as evidenced by the isolation of aptamers targeting thrombin and human serum albumin. We then demonstrated the excellent regeneration capability of the isolated thrombin aptamers using biolayer interferometry. The sensors instantly responded to alternating concentration changes of thrombin at nanomolar levels (200-500 nM), reaching highly consistent equilibrium signals within 10 s. In contrast, the well-known thrombin-binding aptamers, TBA-15 and TBA-29, were not capable of regeneration. Our study provides a simple means to obtain quick-response, high-affinity protein-binding aptamers. It can also be used for the isolation of aptamer pairs, which has been demonstrated to be quite challenging. Our study also provides insights into the rational design of aptamers to control their binding thermodynamics and kinetics.