A survey on manipulating association/dissociation rate constants of electrochemical aptamer-based biosensors for thrombin analysis.

The accurate analysis of protein plays a significant role in disease diagnosis and treatment. Electrochemical aptamer-based (EAB) sensors, known for their high sensitivity and cost-effectiveness, are good candidates for protein analysis. Despite of their advantages, there is a lack of the systematical study on optimized conditions for protein analysis. For instance, the association and dissociation rate constants (k and k), the binding kinetics (e.g., τ) and thermodynamics (K value) are of significance for sensing purpose, which are greatly impacted by several parameters including probe density, temperatures, or salt concentrations. Here, we employ thrombin as a test bed to investigate the sensing performance dependence on these three parameters. We achieved an optimal probe density of 10 nm to support a robust binding event across the probe distance range of 8 nm-62 nm. For temperature condition, we observed that at 45 °C sensors exhibited highest values for both k and k constants, resulting a moderate binding affinity. The optimal temperature for the overall sensor performance remains as 37 °C. Finally, we demonstrated that the protein analysis is greatly dependent of salt concentrations as well as the valency, with the addition of 20 mM Mg exhibiting a significant increase of both k and k values. The findings contribute to advancing biosensor technology and expanding its applications in biological research, holding promise for future developments in protein detection and analysis.