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Related Experiment Video

Updated: Jul 14, 2025

Rapid Fluorescence-based Characterization of Single Extracellular Vesicles in Human Blood with Nanoparticle-tracking Analysis
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Double Digital Assay for Single Extracellular Vesicle and Single Molecule Detection.

David E Reynolds1, Menghan Pan1, Jingbo Yang2

  • 1Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, 19104, USA.

Advanced Science (Weinheim, Baden-Wurttemberg, Germany)
|October 6, 2023
PubMed
Summary

A new assay precisely counts single molecules on extracellular vesicles (EVs), enabling sensitive cancer biomarker detection. This breakthrough aids in understanding EV heterogeneity and discovering novel diagnostic markers.

Keywords:
biomarker discoverydouble digital microfluidicsextracellular vesiclemicrowellstyramide signal amplification

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Area of Science:

  • Biotechnology
  • Molecular Diagnostics
  • Nanotechnology

Background:

  • Extracellular vesicles (EVs) show potential as disease biomarkers.
  • Current EV detection methods face challenges due to low biomarker abundance and complex EV properties.

Purpose of the Study:

  • To develop a highly sensitive assay for absolute quantification of molecules from single EVs.
  • To overcome limitations in detecting low-abundance biomarkers on individual EVs.

Main Methods:

  • A double digital assay integrated with tyramide signal amplification (TSA) for enhanced fluorescent readout.
  • Utilized microfluidic technology for single EV compartmentalization and digital partitioning.
  • Applied the assay to quantify PD-L1 proteins on single EVs from melanoma cells.

Main Results:

  • Demonstrated successful compartmentalization and digital partitioning of single EVs.
  • Achieved absolute quantification of individual molecules from single EVs.
  • Quantified approximately 2.7 PD-L1 molecules per EV in melanoma-derived EVs.

Conclusions:

  • The developed assay enables precise quantification of rare, low-abundance protein molecules from single EVs.
  • This technology is applicable for profiling cancer biomarkers, aiding in prognosis and therapy response assessment.
  • The findings facilitate a deeper understanding of EV heterogeneity and the discovery of novel EV-based biomarkers.