Enhanced CRISPR/Cas12a-based quantitative detection of nucleic acids using double emulsion droplets

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Microfluidics

Background:

• Droplet microfluidics combined with CRISPR/Cas12a offers sensitive nucleic acid detection.
• Traditional water-in-oil single emulsion droplets lack stability for accurate digital assays.
• Water-in-oil-in-water double emulsion droplets provide improved stability and flow cytometry compatibility.

Purpose of the Study:

• To compare the performance of single emulsion (SE) and double emulsion (DE) droplets for CRISPR/Cas12a-based nucleic acid quantification.
• To evaluate the stability of SE and DE droplets under various conditions.
• To demonstrate the utility of DE droplets for high-throughput, accurate quantification of HPV18 DNA.

Main Methods:

• Comparative analysis of SE and DE droplet stability (size variation, merging, content interaction).
• CRISPR/Cas12a assay for quantitative detection of human papillomavirus type 18 (HPV18) DNA.
• Integration of DE droplets with flow cytometry for high-throughput analysis.

Main Results:

• DE droplets exhibited superior stability compared to SE droplets.
• The DE droplet platform coupled with CRISPR/Cas12a and flow cytometry enabled high-throughput and high-accuracy quantification of HPV18 DNA.
• Demonstrated the feasibility of using DE droplets for absolute quantification of nucleic acid biomarkers.

Conclusions:

• Water-in-oil-in-water double emulsion droplets are a more stable and reliable platform for CRISPR/Cas12a-based nucleic acid detection compared to single emulsion droplets.
• The developed DE droplet-CRISPR/Cas12a-flow cytometry system provides a robust method for high-throughput, absolute quantification of nucleic acid biomarkers.
• This technology holds promise for sensitive and accurate molecular diagnostics.