Intrinsic Kinetic Control Enables Scalable and Ultrasensitive Single-Molecule Sensing.

Dynamic single-molecule sensing (DSMS) enables real-time monitoring of molecular interactions with exceptional sensitivity and kinetic resolution, offering significant potential for ultrasensitive biomarker detection. However, existing DSMS platforms often require probe redesign or external force modulation to tune binding kinetics, which limits system simplicity and scalability. Here, we report an intrinsically regulated DSMS platform engineered through systematic optimization of nanoparticle size and buffer ionic strength. We first established a theoretical model describing two dominant kinetic regimes─damping-dominated and entropic-confinement-dominated dynamics─and identified a critical inflection point where sensitivity and specificity are balanced. While this model provides insights into kinetic tuning, practical challenges such as nanoparticle heterogeneity and matrix complexity limit its direct application for sensor design. To address this, we empirically optimized a previously developed DSMS system using average binding dwell time and total binding events as two key performance indicators. The optimized platform, featuring 150 nm polystyrene nanoparticles under 150 mM NaCl, achieved femtomolar detection of thrombin and HIV-1 p24 antigen with limits of detection of 213.9 fM and 4.3 fM, respectively. Notably, the platform maintained excellent specificity in diluted serum through dwell-time filtering, highlighting its robustness in complex biological matrices. This work establishes a novel DSMS strategy that enables efficient single-molecule sensing without probe modification or external actuation, paving the way for scalable, high-performance biomarker detection in clinical diagnostics and point-of-care applications.