Measuring binding processes at the single-molecule level underpin significant functions in understanding biological events. Single-nanoparticle imaging techniques are providing a new concept for mapping the heterogeneous behaviors and characterizations of individual dynamics such as molecule-molecule interactions. Here, we develop the optical imaging techniques for directly counting and monitoring the binding and motion events of single nanoparticles linked to the substrate via the specific and reversible interactions between biomolecules. The one-step digital immunoassay realizes the biomolecular detection based on dynamic counting of the single nanoparticle binding event to substrate with the bright-field imaging. The detection limit achieves 8.4 pg/mL for procalcitonin with detection time of 14 min. Meanwhile, we map the accurate trajectory of single nanoparticle switching between different target molecules among the x-y plane with the total internal reflection imaging technique, which reveals the spatial coordinates of single target molecules on the substrate surface with high spatial and temporal resolutions.
Keywords: digital counting; dynamic tracking; optical imaging; single-molecule biosensing; single-molecule localization.