High-throughput single-cell, single-mitochondrial DNA assay using hydrogel droplet microfluidics.

Area of Science:

• Mitochondrial biology
• Genomics
• Microfluidics

Background:

• Understanding single-cell heterogeneity and mitochondrial DNA (mtDNA) heteroplasmy is crucial but limited by current low-throughput single-cell mtDNA analysis methods.
• Droplet microfluidics has enhanced single-cell genomics, but its application to organelle analysis remains challenging.

Approach:

• Developed an agarose-based droplet microfluidic system for single-cell, single-mtDNA analysis, encapsulating cells in agarose beads for mtDNA retention.
• Engineered a parallelized microfluidic device to overcome agarose viscosity, achieving ~95% mtDNA retention at high throughput (>700,000 drops/minute).
• Utilized multiplexed rolling circle amplification (RCA) for targeted analysis of single mtDNA molecules.

Key Points:

• Simultaneously processes hundreds of mtDNA molecules within >10,000 individual cells.
• Agarose microbeads retain mtDNA after cell lysis, serving as a scaffold for multi-step analysis.
• Demonstrated compatibility with microscopy for digital counting and flow cytometry for high-throughput analysis of RCA products.

Conclusions:

• This scalable microfluidic approach significantly advances the capacity for single-cell mtDNA analysis.
• Enables deeper investigation into mitochondrial genetic variations and their biological implications at the single-cell level.