The generation of droplets is one of the most critical steps in the droplet digital polymerase chain reaction (ddPCR) procedure. In this study, the mechanism of droplet formation in microchannel structure and factors affecting droplet formation were studied. The physical field of laminar two-phase flow level was used to simulate the process of droplet generation through microfluidic technology. The effect of the parameters including flow rate, surface tension, and viscosity on the generated droplet size were evaluated by the simulation. After that, the microfluidic chip that has the same dimension as the simulation was then, fabricated and evaluated. The chip was made by conventional SU-8 photolithography and injection molding. The accuracy of the simulation was validated by comparing the generated droplets in the real scenario with the simulation result. The relative error (RE) between experimentally measured droplet diameter and simulation results under different flow rate, viscosity, surface tension and contact angle was found less than 3.5%, 1.8%, 1.4%, and 1.2%, respectively. Besides, the coefficient of variation (CV) of the droplet diameter was less than 1%, which indicates the experimental droplet generation was of high stability and reliability. This study provides not only fundamental information for the design and experiment of droplet generation by microfluidic technology but also a reliable and efficient investigation method in the ddPCR field.

A device and method for the constant pressure regulation of microdroplet PCR in microfluidic chips are developed to optimize for the microdroplet movement, fragmentation, and bubble generation in microfluidic chips. In the developed device, an air source device is adopted to regulate the pressure in the chip, such that microdroplet generation and PCR amplification without bubbles can be achieved. In 3 min, the sample in 20 μL will be distributed into nearly 50,000 water-in-oil droplets exhibiting a diameter of about 87 μm, and the microdroplet will be subjected to a close arrangement in the chip without air bubbles. The device and chip are adopted to quantitatively detect human genes. As indicated by the experimental results, a good linear relationship exists between the detection signal and DNA concentration ranging from 10¹ to 10⁵ copies/μL (R² = 0.999). The microdroplet PCR devices based on constant pressure regulation chips exhibit a wide variety of advantages (e.g., achieving high pollution resistance, microdroplet fragmentation and integration avoidance, reducing human interference, and standardizing results). Thus, microdroplet PCR devices based on constant pressure regulation chips have promising applications for nucleic acid quantification.

Digital polymerase chain reaction (PCR) technology in microfluidic systems often results in bubble formation post-amplification, leading to microdroplet fragmentation and compromised detection accuracy. To solve this issue, this study introduces a method based on the constant pressure regulation of microdroplets during PCR within microfluidic chips. An ideal pressure reference value for continuous pressure control was produced by examining air solubility in water at various pressures and temperatures as well as modeling air saturation solubility against pressure for various temperature scenarios. Employing a high-efficiency constant pressure device facilitates precise modulation of the microfluidic chip’s inlet and outlet pressure. This ensures that air solubility remains unsaturated during PCR amplification, preventing bubble precipitation and maintaining microdroplet integrity. The device and chip were subsequently utilized for quantitative analysis of the human epidermal growth factor receptor (EGFR) exon 18 gene, with results indicating a strong linear relationship between detection signal and DNA concentration within a range of 10¹–10⁵ copies/μL (R² = 0.999). By thwarting bubble generation during PCR process, the constant pressure methodology enhances microdroplet stability and PCR efficiency, underscoring its significant potential for nucleic acid quantification and trace detection.

The device and method in accordance with the constant pressure regulation of micro droplet PCR in microfluidic chips are developed to explore the optimization way for the micro droplet movement, fragmentation, and bubble generation in microfluidic chips in existing digital PCR technology. In the developed device, an air source device is adopted to regulate the pressure in the chip, such that micro droplet generation and PCR amplification without bubbles can be achieved. In 3 min, the sample in 20ul will be distributed into nearly 50,000 water-in-oil droplets exhibiting a diameter of about 87μm, and the micro droplet will be subjected to a close arrangement in the chip without air bubbles. The device and chip are adopted to quantitatively detect the human gene . As indicated by the experimental results, a good linear relationship exists between detection signal and DNA concentration ranging from 101~ 105 copies /μL (R2=0.999). The micro droplet PCR devices based on constant pressure regulation chips exhibit a wide variety of advantages (e.g., achieving high pollution resistance, micro droplet fragmentation and integration avoidance, reducing human interference, and standardizing results). Thus, micro droplet PCR devices based on constant pressure regulation chips have promising applications in nucleic acid quantification.