Plasmon‐Enhanced Biosensing for Multiplexed Profiling of Extracellular Vesicles

Abstract

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.

Advances in the analysis of single extracellular vesicles: A critical review

“…Min et al used a method they reported previously, nanoplasmonic exosome (nPLEX) [ 39 ], for multiplexed detection of surface biomarkers via fluorescently-labeled antibodies [ 105 ]. The antibodies are captured on a surface with gold nanoholes to enable plasmon-enhancement of the fluorescence.…”

High-Throughput Single Extracellular Vesicle Profiling

“…The labeled EVs are imaged, and their fluorescent intensities are analyzed. Therefore, biomarker distribution analysis could be performed on a single-EV level [25]. A localized surface plasmon resonance imaging (LSPRi) platform improves the limit of detection down to the single exosome limit.…”

Multiplexed Profiling of Extracellular Vesicles for Biomarker Development

“…In another recent study, Min et al explored long-range (> 100 nm) SPR-enhanced fluorescence on a periodic array instead of the localized "hotspot" surfaces (< 20 nm). This approach is more suitable for measurements with intact EVs given their dimension (30-1000 nm) [118,119]. Four fluorescent dyes (AF488, Cy3, Cy5, and Cy5.5) were conjugated with streptavidin separately in order to code biotinylated EV membrane proteins (CD9, CD63, CD81, GAPDH, EGFR, EGFRvIII) after their capture on neutravidin-PEG layers (Fig.…”

“…In summary, the binding events of EV components to the sensing interface can be monitored by surface sensitive techniques such as SPR in a label-free format or by labelling them with one or multiple reporters, i.e. using a combination of physico-chemical coding strategies [118]. The advanced nano-/micro-fabrication-assisted SPR arrays and their integration into microfluidic chambers have been increasingly employed to facilitate better sample manipulation and miniaturization.…”