Microfluidic Nano-Plasmonic Imaging Platform for Purification- and Label-Free Single Small Extracellular Vesicle Counting.

Tumor-derived circulating small extracellular vesicles (sEVs) is a promising non-invasive biomarker for disease diagnosis. However, their quantitative detection remains challenging due to their small size and the complexity of blood plasma. Typically, sample preparation like purification is required. This study presents a purification-free approach using a microfluidic chip integrated with PANORAMA (Plasmonic nano-aperture label-free imaging) for label-free single sEV counting in plasma. CD63, CD9, and CD81 antibodies, specific biomarkers for most sEVs, are functionalized on AGNIS (arrayed gold nanodisks on invisible substrate) for selective capture. The automated microfluidic platform minimizes manual errors and allows precise programming of flow rates, directions, and media for optimization. Only 20 µL of plasma is required, and the analysis is completed within 60 minutes. This platform shows great potential as a sensitive and efficient tool for detecting circulating sEVs without purification or labeling.