Spatially Controlled Capture and Site-Resolved Analysis of Single Extracellular Vesicles

Quantitative fluorescence analysis of single extracellular vesicles (EVs) is often complicated by heterogeneous particle loading on continuous surfaces, obscuring where and how a signal should be counted. This study presents a simple, array-based method that couples site-specific capture into optically resolvable nanowells with mask-gated image analysis to obtain unambiguous, single-site fluorescence measurements. EVs settle onto nanowell arrays, and excess particles on the inter-well surface are cleared by a PDMS translation step, resulting in the capture of single EVs in discrete nanowells and accurate detection of in-well signals that are registered to a bright-field nanowell mask. This format yields > 99% EV capture at the designed location in grids and enables the accurate detection of EVs' fluorescence signals with a low background. Compositional analysis on a single substrate accurately tracks programmed EV mixture ratios with near-unity slopes and HER2 positivity in breast cancer EVs. Finally, the patterning method is readily adapted to plasma-derived EVs, expanded to multi-channel fluorescence imaging, and applied to mixed cargos of co-patterned EVs and nanoparticles. These results establish a reliable pathway for array-based EV detection and precise manipulations of EVs and other nanoparticles.