Surface Crowding Effects in Molecular Recognition by Thrombin Binding Aptamers Conjugated to Gold Nanoparticles

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.