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

Abstract

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_on and k_off), the binding kinetics (e.g., τ) and thermodynamics (K_D 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_on and k_off 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 Mg²⁺ exhibiting a significant increase of both k_on and k_off 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.