Super-Resolved Single Small Extracellular Vesicle Assay enabled by a Plasmonic Nanohole Array

The accurate quantification of biological nanoparticles, such as small extracellular vesicles (sEVs), is fundamentally hindered by a resolution-coincidence trade-off in digital assays. While physical confinement can isolate single particles, conventional optical readouts remain diffraction-limited, causing multi-particle occupancy to be miscounted as single events and thereby restricting the analytical dynamic range. Here, we report a nanoplasmonic platform that overcomes this limit by introducing a geometry-defined interface that uniquely unifies nanoscale compartmentalisation and near-field-assisted super-resolution imaging. Utilising a gold plasmonic nanohole array, the strict geometric periodicity of the lattice simultaneously serves as a template for single-vesicle confinement and a deterministic grid that generates an array of localised surface plasmon resonance near-field hotspots. This position-deterministic illumination pattern imposes known geometric priors on the excitation field, shifting high-spatial-frequency information into the detectable bandwidth to achieve sub-100 nm lateral resolution. This dual-purpose geometric determinism enables high-fidelity digital readout of individual vesicles with significantly fewer sub-images than stochastic, speckle-based metasurface structured illumination microscopy approaches. The assay achieves an analytical limit of detection of 143 sEVs/µL, matching the performance of state-of-the-art single-EV counting technologies. It successfully differentiates distinct sEV subpopulations based on surface biomarker expression, establishing a clear pathway for future clinical liquid biopsy applications. By replacing stochastic loading and illumination with geometric design, this work establishes a robust framework for precise vesicle interrogation with broad implications for emerging translational applications and fundamental biology.