composition and molecular profiles of EVs in clinical samples. However, their unique sizes (50-1000 nm) impose technical challenges in conventional analytical methods, which often lead to variable findings. Conventional methods for protein analyses (e.g., western blotting, enzymelinked immunosorbent assay) require large amounts of samples and involve time-consuming and extensive processing steps, making them impractical in the clinical settings. Developing new EV molecular profiling platforms is, thus, a pivotal mandate to ultimately translate EVs into clinically relevant biomarkers. [9] To address these challenges, we previously developed a nanoplasmonic sensing platform, termed nanoplasmonic exosome (nPLEX), based on transmission surface plasmon resonance (SPR) through periodic nanohole gratings. [7,8,10] In the previous studies, we showed that the nPLEX sensors could rapidly and sensitively detect tumor-derived EVs directly from clinical samples. Although promising, fundamental limitations remain in the current nPLEX system and other state-of-the-art EV-sensing technologies: 1) sensitivity limited to bulk analyses; 2) necessities of EV lysis for detecting markers inside of EVs; and 3) lack of multiplexed analysis in single EVs. Analyzing single EVs could reveal unique molecular profiles of cell-specific EVs, which will further promote clinical use of these vesicles and allow us to construct a comprehensive EV atlas per different biological parameters (e.g., cellular origin, cell state). Extracellular vesicles (EVs)-nanoscale phospholipid vesicles secreted by cells-present new opportunities for molecular diagnosis from non-invasive liquid biopsies. Single EV protein analysis can be extremely valuable in studying EVs as circulating cancer biomarkers, but it is technically challenging due to weak detection signals associated with limited amounts of epitopes and small surface areas for antibody labeling. Here, a new, simple method that enables multiplexed analyses of EV markers with improved sensitivities is reported. Specifically, plasmon-enhanced fluorescence detection is implemented that amplifies fluorescence signals using surface plasmon resonances excited by periodic gold nanohole structures. It is shown that fluorescence signals in multiple channels are amplified by one order of magnitude, and both transmembrane and intravesicular markers can be detected at the single EV level. This approach can offer additional insight into understanding subtypes, heterogeneity, and production dynamics of EVs during disease development and progression.
Extracellular vesicles (EVs), actively shed from a variety of neoplastic and host cells, are abundant in blood, and carry molecular markers from parental cells. For these reasons, EVs have gained much interest as biomarkers of disease. Among a number of different analytical methods that have been developed, surface plasmon resonance (SPR) stands out as one of the ideal techniques given its sensitivity, robustness, and ability to miniaturize. In this review, we compare different SPR platforms for EV analysis, including conventional SPR, nanoplasmonic sensors, surface-enhanced Raman spectroscopy, and plasmonic-enhanced fluorescence. We discuss different surface chemistries used to capture targeted EVs and molecularly profile their proteins and RNAs. We also highlight these plasmonic platforms' clinical applications, including cancers, neurodegenerative diseases, and cardiovascular diseases. Finally, we discuss the future perspective of plasmonic sensing for EVs and their potentials for commercialization and clinical translation.
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