Multi-marker profiling of extracellular vesicles using streaming current and sequential electrostatic labeling.

Area of Science:

• Biotechnology
• Nanotechnology
• Analytical Chemistry

Background:

• Small extracellular vesicles (sEVs) exhibit high heterogeneity in membrane protein expression.
• Current antibody-based bulk methods for sEV analysis are biased, capturing only specific subpopulations.
• Multiplexed protein analysis of a representative sEV population is needed but largely limited to fluorescence methods.

Purpose of the Study:

• To develop a novel, unbiased method for multiplexed membrane protein analysis of sEVs.
• To enable comparison of protein expression levels across different sEV subtypes.
• To introduce a microchip-based, all-electrical approach for sEV profiling.

Main Methods:

• Development of a novel electrostatic labeling strategy for sEV surface proteins.
• Implementation of a microchip-based, all-electric method for sEV capture and analysis.
• Utilizing alternating charge labels for multiplexed profiling of captured sEVs.

Main Results:

• Demonstrated proof of concept using both non-specific (unbiased) and anti-CD9 (biased) sEV capture.
• Successfully profiled multiple surface proteins on captured sEVs using charge-labeled antibodies.
• Validated the all-electrical method against a single-sEV fluorescence technique.

Conclusions:

• The novel electrostatic labeling and microchip-based method provides unbiased analysis of sEV membrane protein expression.
• The technique allows for comparison of expression levels in different sEV subsets and fractional estimation of subpopulations.
• This all-electrical approach overcomes limitations of existing biased, antibody-dependent methods for sEV characterization.