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Extracellular Vesicle (EV) Dot Blotting for Multiplexed EV Protein Detection in Complex Biofluids
Nikan Momenbeitollahi1, Roshan Aggarwal1, Gisela Strohle1
1School of Engineering, University of Guelph, Guelph, Ontario N1G2W1, Canada.
Analytical Chemistry
|May 9, 2022
Summary
We developed EV dot blotting, a sensitive method for detecting cancer-derived extracellular vesicles (EVs) directly in blood plasma. This technique offers multiplexed protein analysis with minimal sample use, improving early cancer detection.
Area of Science:
- Biotechnology
- Molecular Diagnostics
- Nanotechnology
Background:
- Extracellular vesicles (EVs) in biofluids hold potential for disease biomarker discovery.
- Current EV protein detection methods lack sensitivity, require large sample volumes, and often necessitate EV isolation.
- Sensitive and multiplexed detection of EV proteins is crucial for clinical applications like early cancer diagnosis.
Purpose of the Study:
- To develop a simple, sensitive, and multiplexed method for detecting EV surface proteins directly from complex biofluids.
- To overcome limitations of existing EV detection techniques, including sample consumption and the need for isolation.
- To establish a platform for low-cost EV protein quantification for diagnostic applications.
Main Methods:
- Developed 'EV dot blotting', a technique inspired by conventional dot blotting for multiplexed EV protein detection.
- Optimized critical assay parameters including antibody concentration, blocking reagents, 3D membrane types, and gold nanoparticle signal enhancement.
- Performed multiplexed assays using cancer-cell-derived EVs spiked into normal human plasma without prior EV isolation.
Main Results:
- Achieved high sensitivity for EV detection: Limit of detection (LOD) for CD9 was 3.1 × 10^5 EVs/mL, CD24 was 4.7 × 10^4 EVs/mL, and EpCAM was 9.0 × 10^4 EVs/mL.
- Demonstrated LODs up to 4 orders of magnitude lower than conventional ELISA.
- Successfully detected EV protein markers directly from plasma, showcasing the method's applicability to complex biofluids.
Conclusions:
- EV dot blotting provides a sensitive, multiplexed, simple, and cost-effective approach for EV protein detection.
- The method minimizes sample consumption and eliminates the need for EV isolation from biofluids like plasma.
- This platform offers a valuable tool for multiplexed EV protein quantification, supporting various diagnostic and research applications.
