Scheme 1.

Workflow of the EV single‐molecule array (eSimoa) framework. The eSimoa framework combines EV isolation with high‐throughput Simoa technology to profile EV protein biomarkers with exceptional sensitivity and specificity. The eSimoa framework comprises three complementary pipelines that enable the spatial decoding of EV proteins. Pipeline (i) focuses on surface eSimoa, capturing and detecting two EV surface proteins. Pipeline (ii) focuses on luminal eSimoa, analyzing EV luminal proteins. Pipeline (iii) focuses on surface‐luminal eSimoa or pulldown eSimoa, integrating the surface and luminal eSimoa approaches by selectively targeting a subpopulation of EVs with a specific surface protein using pulldown beads, followed by the analysis of luminal proteins within this subpopulation.