Single-Extracellular-Vesicle Detection with a Plasmonic Chip and Enhanced Fluorescence Microscopy

Area of Science:

• Biotechnology
• Nanotechnology
• Medical Diagnostics

Background:

• Extracellular vesicles (EVs) are crucial biomarkers for disease prediction.
• Conventional fluorescence microscopy struggles to visualize sub-100 nm EVs due to optical diffraction limits.
• Accurate detection and quantification of single EVs are essential for reliable diagnostics.

Purpose of the Study:

• To develop a method for visualizing and quantifying single extracellular vesicles (EVs) below the optical diffraction limit.
• To enable sensitive detection of EVs for improved disease prediction.
• To differentiate single EVs from non-specific binding and aggregates.

Main Methods:

• Utilized a plasmonic chip to capture single EVs.
• Employed plasmon-field enhanced fluorescence with fluorescently labeled antibodies for detection.
• Analyzed bright spot characteristics (FWHM, peak value) and used transmitted light microscopy to distinguish EVs.

Main Results:

• Successfully detected and quantified single EVs in the picomolar to femtomolar range (1.4 pM–95 fM).
• Demonstrated detection of EVs by labeling two different membrane proteins simultaneously.
• Showcased the ability to detect multiple targets at multiple wavelengths, confirming specificity and sensitivity.

Conclusions:

• Plasmon-enhanced fluorescence offers a powerful, label-free (for the EV itself) approach for single EV detection.
• The method overcomes optical limitations, enabling visualization of nanoscale EVs for biomarker discovery.
• This technique holds promise for advancing sensitive and specific diagnostic tools based on extracellular vesicle analysis.