Constant Pressure-Regulated Microdroplet Polymerase Chain Reaction in Microfluid Chips: A Methodological Study

Area of Science:

• Biotechnology
• Analytical Chemistry
• Microfluidics

Background:

• Digital polymerase chain reaction (PCR) in microfluidic systems faces challenges with bubble formation post-amplification.
• Bubble formation leads to microdroplet fragmentation and reduced detection accuracy, hindering precise nucleic acid quantification.

Purpose of the Study:

• To develop and validate a constant pressure regulation method for microfluidic digital PCR systems.
• To prevent bubble formation during PCR amplification and maintain microdroplet integrity for enhanced detection accuracy.

Main Methods:

• Investigated air solubility in water under various pressures and temperatures to establish an ideal pressure reference value.
• Modeled air saturation solubility against pressure for different temperature scenarios.
• Employed a high-efficiency constant pressure device for precise modulation of microfluidic chip pressures, ensuring unsaturated air solubility during PCR.

Main Results:

• The constant pressure method successfully prevented bubble precipitation during PCR amplification.
• Quantitative analysis of the human epidermal growth factor receptor (EGFR) exon 18 gene showed a strong linear relationship (R² = 0.999) between detection signal and DNA concentration (10^1-10^5 copies/μL).
• Maintained microdroplet integrity and enhanced PCR efficiency.

Conclusions:

• Constant pressure regulation is an effective strategy to overcome bubble formation issues in microfluidic digital PCR.
• This methodology significantly improves microdroplet stability, PCR efficiency, and nucleic acid quantification accuracy.
• The approach holds substantial potential for sensitive nucleic acid detection and trace analysis.