Probing heterogeneous binding events at single molecule level by single nanoparticle tracking

Measuring molecular binding at the single-molecule level is crucial for both fundamental biological research and patient care. Single-nanoparticle tracking, utilizing optical imaging techniques, provides an important platform for detecting biomarkers and characterizing molecular interaction at a single-molecule level. Herein, we develop the single-molecule sensing platform that tracks single nanoparticles hovering over the sensing surface via a dark-field microscope. By digitally counting the individual nanoparticles, the detection limit achieves 7.5 ng/mL for neuron specific enolase. Additionally, quantifying the heterogeneous velocities of individual nanoparticles allows us to study the transient binding events and differentiate between specific and nonspecific binding events. The detection performance is improved by excluding the counts of nonspecific binding events. Furthermore, the precise trajectories of single nanoparticles switching between different molecular complexes reveal the heterogeneity of surface modifications at the single-molecule level.