Quantification and Mitigation of Site-Preferred Nonspecific Interactions in Single-Nanoparticle Biosensors

Understanding the origin and behavior of nonspecific interactions is essential for advancing biosensing technologies. In this study, we investigate nonspecific interactions between a functionalized single nanoparticle (NP) and a sensor surface. The NP, tethered by a single DNA molecule, exhibits flexible motion that allows it to interact with the surface. Using surface plasmon resonance microscopy (SPRM) with nanometer precision, we tracked the motion dynamics of the NP and revealed that nonspecific binding leads to repeated transient trapping at the surface. The NP shows a preference for interacting with a particular site, indicating site-preferred nonspecific interactions. This behavior mimics specific binding events, emphasizing the need to mitigate such effects in biosensors. By systematically varying NP size, ionic strength, solution viscosity, blocking agents, and applying external forces, we identified external force as the most effective factor in reducing such nonspecific interactions. We hope these insights can provide strategies for designing next-generation single-NP and single-molecule biosensors with minimal nonspecific signals, thereby enhancing detection reliability.