Single-Extracellular-Vesicle\nDetection with a Plasmonic\nChip and Enhanced Fluorescence Microscopy

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