Dynamic Single-Binding Event Profiling With on-Chip Microlenses for Wash-Free Digital Biosensing

Single-molecule detection enables precise biomarker quantification, yet conventional endpoint assays suffer from long incubation and nonspecific binding. Kinetic assays resolve these via real-time monitoring of molecular interactions, but their reliance on complex optics has restricted their practical adoption, especially in point-of-care settings where speed, portability, and ease of use are paramount. We present a microlens-assisted platform that enables real-time tracking of single-binding events under conventional bright-field microscopy. An on-chip microlens array amplifies scattering from gold nanoparticle labels, enabling kinetic monitoring with low-magnification optics. Dynamic fingerprints of individual events distinguish specific from nonspecific interactions, achieving wash-free digital biosensing with high sensitivity and specificity. This approach not only preserves the analytical advantages of kinetic profiling but also enables an exceptionally compact and portable optical configuration. Using cardiac troponin I, a time-critical biomarker for acute myocardial infarction, we demonstrate an ultralow detection limit of 0.051 pg mL⁻¹ and validate clinical applicability in undiluted serum samples, paving the way for miniaturized, high-performance biosensors that can deliver laboratory-grade diagnostics at the point of need.