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

ABSTRACT 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.