3D projection electrophoresis for single-cell immunoblotting
Samantha M Grist1, Andoni P Mourdoukoutas1,2, Amy E Herr3,4,5
1Department of Bioengineering, University of California, Berkeley, USA.
Nature Communications
|December 5, 2020
Summary
We developed 3D single-cell immunoblots for high-throughput protein analysis. This novel projection electrophoresis method overcomes previous limitations, enabling faster and more comprehensive single-cell protein profiling.
Area of Science:
- Biotechnology
- Molecular Biology
- Microfluidics
Background:
- Immunoassays and mass spectrometry are key single-cell protein analysis tools.
- Current methods face bottlenecks in interfacing and throughput.
- Limited ability to analyze both cytosolic and nuclear proteins simultaneously.
Purpose of the Study:
- Introduce a novel 3D single-cell immunoblot technique.
- Enable simultaneous detection of cytosolic and nuclear proteins.
- Overcome throughput and interfacing limitations in single-cell protein analysis.
Main Methods:
- Utilized a 3D microfluidic device with a photoactive polyacrylamide gel and microwell array.
- Employed "electrophoretic projection" to move single-cell lysates into the 3rd dimension (z-axis) for size separation.
- Photo-captured separated analytes within the gel for immunoprobing and imaging.
- Design and analysis guided by the physics of 3D diffusion.
Main Results:
- Achieved electrophoresis throughput >2.5 cells/s (70x faster than serial sampling).
- Obtained a density of 25 immunoblots/mm² (10x increase over previous methods).
- Synchronized analysis of hundreds of cells, reducing inter-cell analysis delays.
Conclusions:
- Projection electrophoresis significantly enhances single-cell protein analysis capabilities.
- The 3D microdevice provides a high-throughput solution for protein profiling.
- This method can augment existing genomic and transcriptomic single-cell atlases with crucial protein-level data.
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