UC Merced

This study addresses the challenge of determining how surface immobilization and crowding affect the binding affinity of DNA/RNA aptamers used in nanoparticle-based biosensors. Binding affinity is a critical determinant of biosensor performance. We employed isothermal titration calorimetry (ITC) to directly measure the binding interactions between thrombin and aptamer-functionalized gold nanoparticles. We found that binding affinity improves with increasing aptamer density due to entropic compensation, up to a critical threshold. Beyond this point, steric hindrance diminishes target binding. These findings demonstrate the utility of ITC in characterizing aptamer-target interactions and offer insights for optimizing the sensitivity, limit of detection, and dynamic range of aptamer-based biosensing platforms.