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The surface plasmon resonance (SPR) technique has been widely applied to biosensing technologies for the rapid quantification of biomolecules without enzyme and fluorescent labeling. However, the conventional prism-coupling SPR method generally has a detection area of a few mm2, and the large contribution of the background signal forms a barrier to highly sensitive detection. Based on a highly spatially resolved SPR method, the present study constructed a scanning GC-SPR imaging instrument using an objective lens with a high numerical aperture and a plasmonic chip that could be used for grating-coupled SPR. Focusing light on the diffraction limit can suppress background signals and improve detection sensitivity. SPR imaging can also be performed by scanning a focal spot. Using this method, the refractive index of a mixture of water and dimethyl sulfoxide was measured with a detection accuracy of 2.43 × 10−3 RIU. Polydopamine films prepared with a thickness of <5 nm were also measured, and each film thickness was evaluated with high sensitivity from the effective refractive index detected in a small area of <1 µm2. [ABSTRACT FROM AUTHOR]

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., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)