Plasmon-Enhanced Characterization of Single Extracellular Vesicles

Abstract

Extracellular vesicles (EVs) represent heterogeneous populations of membrane-bound vesicles shed from almost all kinds of cells. Although superior to conventional methods, most newly developed EV sensing platforms still require a certain number of EVs, measuring bulk signals from a group of vesicles. A new analytical approach that enables single EV analysis could be extremely valuable for understanding EVs' subtypes, heterogeneity, and production dynamics during disease development and progression. Here, we describe a new nanoplasmonic sensing platform for sensitive single EV analysis. Termed nPLEX-FL (nano-plasmonic EV analysis with enhanced fluorescence detection), the system amplifies EVs' fluorescence signals using periodic gold nanohole structures, enabling sensitive, multiplexed analysis of single EVs.

Keywords: Biosensing; Extracellular vesicles; Molecular analysis; Plasmon-enhanced fluorescence; Surface plasmon resonance.

Fig. 1.. Overview of nano-plasmonic EV analysis with enhanced fluorescence detection (nPLEX-FL).

A. Biotinalyated EV are captured on avidin-coated gold nanohole surface. The captured EVs are immunostained by antibodies for target EV surface proteins, followed by fluorophore-conjugated secondary antibody labeling. The fluorescently labeled EVs are imaged by a fluorescence microscope, and images are analyzed to detect individual EVs and measure target protein levels from fluorescence intensities. B. A scanning electron micrograph of gold nanoholes. The hole diameter is 200 nm, and the periodicity is 500 nm. Scale bar: 1 μm. Reproduced from ref. with permission from John Wiley and Sons.

Fig. 2.. Plasmon-enhanced fluorescence intensity in the nPLEX-FL system.

A. Four different fluorophore-conjugated streptavidin (streptavidin with AF488, Cy3, Cy5, or Cy5.5) coated on nPLEX-FL chips showed the distinguishing enhancement of fluorescence depending on the absorption and emission spectra. Scale bar: 20 μm. Au nanoholes are made in the 100 × 100 μm² sized square area highlighted by a white dashed box. B. The cross-sectional intensity profiles along the blue dashed lines of images in A. C. Enhancement factors of fluorescence intensity on nanohole area compared to flat Au area depending on the fluorophores. D. The overlaid spectrum of plasmon-supported light transmission on periodic nanoholes. Reproduced from ref. with permission from John Wiley and Sons.

Fig. 3.. A scanning electron micrograph of periodic nanohole patterns made in a Si wafer by interference lithography.

The hole diameter is 200 nm, and the periodicity is 500 nm.

Fig. 4.. A representative dual-channel nPLEX-FL fluorescence image.

Left: biotinylated EVs are labeled by Cy3-conjugated streptavidin. Right: EVs are labeled by anti-EGFR primary antibodies, followed by AF647-conjugated secondary antibodies.

Fig. 5.. ImageJ Comdet Plugin.

Particle size 4 and intensity threshold 3 are used as a default setting.