High-Throughput Single-Cell, Single-Mitochondrial DNA Assay Using Hydrogel Droplet Microfluidics.

Area of Science:

• Biotechnology
• Genomics
• Cell Biology

Background:

• Understanding single-cell heterogeneity and mitochondrial DNA (mtDNA) heteroplasmy is crucial but limited by current analysis scales.
• Droplet microfluidics enhance single-cell analysis throughput but face challenges in sub-cellular organelle analysis.

Purpose of the Study:

• To develop a scalable microfluidic method for single-cell, single-mtDNA analysis.
• To overcome limitations in analyzing mtDNA at the single-cell level.

Main Methods:

• An agarose-based droplet microfluidic approach encapsulating single cells in hydrogel beads for mtDNA retention.
• A parallelized device design to mitigate agarose viscosity and achieve high throughput (>700,000 drops/minute).
• Multiplexed rolling circle amplification (RCA) for analyzing specific single-mtDNA regions, compatible with microscopy and flow cytometry.

Main Results:

• Simultaneous processing of hundreds of mtDNA molecules within >10,000 individual cells.
• ~95% mtDNA retention achieved using the specialized microfluidic chip and agarose beads.
• Demonstrated utility through RCA analysis and compatibility with digital counting and high-throughput flow cytometry.

Conclusions:

• The developed microfluidic method significantly advances the scale and efficiency of single-cell mtDNA analysis.
• This approach facilitates deeper investigation into mitochondrial DNA heteroplasmy and cellular heterogeneity.
• The technology supports both detailed molecular analysis and high-throughput screening of mtDNA.