Ultrahigh-throughput approach for analyzing single-cell genomic damage with an agarose-based microfluidic comet...

Area of Science:

• Genomics
• Biotechnology
• Cell Biology

Background:

• Traditional comet assays for genomic DNA damage are limited by low throughput and reproducibility.
• There is a need for more efficient and reliable methods for single-cell genomic analysis.

Purpose of the Study:

• To develop an ultrahigh-throughput microfluidic chip for simultaneous DNA damage assessment in thousands of individual cells.
• To improve the throughput and reproducibility of DNA damage analysis compared to conventional methods.

Main Methods:

• Fabrication of a microfluidic chip using agarose as both a structural material and an electrophoretic sieving matrix.
• Alignment of single cancer cells (HeLa or HepG2) in parallel microchannels using capillary effects.
• Single-cell gel electrophoresis and fluorescence detection for quantifying DNA damage via comet-shaped DNA morphology.

Main Results:

• The microfluidic chip achieved approximately 100-fold higher throughput than conventional comet assays.
• The method demonstrated improved reproducibility in assessing DNA damage across individual cells.
• Evaluation of DNA repair capacity validated the reliability of the microfluidic comet assay.

Conclusions:

• The agarose-based microfluidic comet array electrophoresis is a simple, highly reproducible, and high-throughput method.
• This technique provides a novel approach for highly efficient single-cell genomic analysis.
• The developed chip enables robust interrogation of DNA damage at the single-cell level.