Yuzhe Ding, Yicheng Heng, Ka‐Ying Wong, Qingyu Chen, Juewen Liu
Abstract Selection of high affinity aptamers is the basis for aptamer‐related applications. Most aptamer selections prioritize thermodynamic factors such as the target concentration but neglect interaction kinetics. Using ampicillin as a target, aptamer selections were carried out based on the DNA strand‐displacement reaction. A low‐affinity aptamer ( K d = 12.7 µM) dominated under the typical condition of gravity flow with a short interaction time of 1 to 2 min, whereas a high affinity aptamer ( K d = 1.8 µM) dominated the library with an additional 10 min incubation. These two aptamers differ only in three positions, and the strand‐displacement kinetics was determined to be responsible for the selection outcomes. Kinetic effects were also observed in aptamer selection for adenosine, highlighting the broad applicability of this finding. Additionally, aptamers for a challenging target voriconazole were successfully enriched by extending incubation time. To reliably obtain thermodynamically best aptamers, a combination of low target concentration and long incubation time is recommended. This work not only yields a high‐affinity and selective aptamer for ampicillin but also highlights the interplay between thermodynamics and kinetics in aptamer selection.