High‐Throughput Single‐Cell, Single‐Mitochondrial DNA Assay Using Hydrogel Droplet Microfluidics
Abstract: There is growing interest in understanding the biological implications of single cell heterogeneity and heteroplasmy of mitochondrial DNA (mtDNA), but current methodologies for single‐cell mtDNA analysis limit the scale of analysis to small cell populations. Although droplet microfluidics have increased the throughput of single‐cell genomic, RNA, and protein analysis, their application to sub‐cellular organelle analysis has remained a largely unsolved challenge. Here, we introduce an agarose‐based droplet microfluidic approach for single‐cell, single‐mtDNA analysis, which allows simultaneous processing of hundreds of individual mtDNA molecules within >10,000 individual cells. Our microfluidic chip encapsulates individual cells in agarose beads, designed to have a sufficiently dense hydrogel network to retain mtDNA after lysis and provide a robust scaffold for subsequent multi‐step processing and analysis. To mitigate the impact of the high viscosity of agarose required for mtDNA retention on the throughput of microfluidics, we developed a parallelized device, successfully achieving ~95 % mtDNA retention from single cells within our microbeads at >700,000 drops/minute. To demonstrate utility, we analyzed specific regions of the single‐mtDNA using a multiplexed rolling circle amplification (RCA) assay. We demonstrated compatibility with both microscopy, for digital counting of individual RCA products, and flow cytometry for higher throughput analysis.
- Location
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Deutsche Nationalbibliothek Frankfurt am Main
- Extent
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Online-Ressource
- Language
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Englisch
- Bibliographic citation
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High‐Throughput Single‐Cell, Single‐Mitochondrial DNA Assay Using Hydrogel Droplet Microfluidics ; day:27 ; month:03 ; year:2024 ; extent:12
Angewandte Chemie / International edition. International edition ; (27.03.2024) (gesamt 12)
- Creator
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Park, Juhwan
Kadam, Parnika S.
Atiyas, Yasemin
Chhay, Bonirath
Tsourkas, Andrew
Eberwine, James H.
Issadore, David A.
- DOI
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10.1002/anie.202401544
- URN
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urn:nbn:de:101:1-2024032813044045841479
- Rights
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Open Access; Der Zugriff auf das Objekt ist unbeschränkt möglich.
- Last update
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14.08.2025, 11:01 AM CEST
Data provider
Deutsche Nationalbibliothek. If you have any questions about the object, please contact the data provider.
Associated
- Park, Juhwan
- Kadam, Parnika S.
- Atiyas, Yasemin
- Chhay, Bonirath
- Tsourkas, Andrew
- Eberwine, James H.
- Issadore, David A.
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Droplet microfluidics for single‐molecule and single‐cell analysis in research, diagnosis, and therapy
High‐throughput droplet‐based microfluidics for directed evolution of enzymes
Droplet‐Based Microfluidics Platform for the Synthesis of Single‐Atom Heterogeneous Catalysts
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