Label-free biomolecular sensing via near-interface single-particle Brownian motion tracking

Label-free biosensors typically couple molecular recognition and signal transduction at the same interface, limiting reconfigurability and multiplexing. Here, we report a charge-sensitive sensing strategy based on near-interface Brownian motion tracking (ni-BMT) of single particles. In this architecture, molecular recognition is localized on replaceable particle probes, while the substrate serves as a common confinement and optical readout interface. Target-dependent changes in particle interfacial electrostatics are associated with measurable changes in the apparent lateral diffusion coefficient under near-wall hydrodynamic confinement. By combining a high-density microwell array with automated particle tracking, ni-BMT enables high-throughput analysis of approximately 7000 particles within a single field of view. Mechanistic studies support the electrostatic-hydrodynamic transduction mechanism, and quantitative detection of proteins, nucleic acids, and small molecules is demonstrated with sub-nanomolar limits of detection. Furthermore, size-encoded particle probes enable simultaneous three-channel detection on the same substrate. By decoupling molecular recognition from signal readout, ni-BMT establishes a reconfigurable label-free sensing architecture that integrates high-throughput single-particle analysis, real-time monitoring, and multiplexed detection, providing a versatile framework for biomolecular analysis.