Single-Extracellular-Vesicle Detection with a Plasmonic Chip and Enhanced Fluorescence Microscopy

Endocytosis-derived extracellular vesicles (EVs), which can be as small as 100 nm, are useful for disease prediction. However, very small EVs are below the optical diffraction limit and are difficult to visualize with conventional fluorescence microscopy. In this study, single EVs captured on a plasmonic chip, where fluorescently labeled antibodies were bound over the EV surface, were detected as bright spots using plasmon-field enhanced fluorescence without any pretreatment of isolating labeled EVs, followed by analyzing the full width at half-maximum and the fluorescence peak value for each enhanced fluorescence bright spot. Bright spots smaller than the threshold determined by the observation of the fluorescent nanospheres were attributed to single EVs. The number of single EVs was quantitatively evaluated against the concentration of EV solution injected in the 1.4 pM–95 fM range. Furthermore, single EVs were detected by labeling two different membrane proteins. A molecularly imprinted polymer was applied to a capture interface on a plasmonic chip, and it is found that nonspecific adsorption of aggregates was suppressed. To accurately distinguish single EVs from aggregates of labeled antibodies, the fluorescence microscopy with transmitted light was superior to the epifluorescence method. Finally, single EVs were successfully detected with multiple targets at multiple wavelengths by using different fluorescently labeled antibodies.