Abstract: A highly robust droplet digital polymerase chain reaction(ddPCR) chip with filtering bubbles and enhancing fluorescence is constructed to meet the core requirements of the stable preservation of droplets in the amplification process and the high-efficiency detection after the reaction. The chip could produce more than 2×10⁵ droplets with a radius of ca. 21 μm in 10 min. The droplet collection chamberis built independent of the chip material via "glass ceiling" method, provided a stable environment for droplets. At the same time, the filter structure is constructed to effectively filter the air mixed into the liquid phase, improving the robustness of the chip. The reflector, introduced into the collecting chamber, enhances the fluorescence signal, reducing exposure time and improving the detection efficiency. Using this chip to detect EGFR exon 21, a good linear correlation with the DNA concentration from 10¹ to 10⁵ copies/μL(R²=0.998) could be obtained. In general, the scheme, which achieved droplet generation, PCR amplification and fluorescence readout on the chip robustly and efficiently, has potential in nucleic acid detection and related applications.

Key words: Digital polymerase chain reaction(PCR), Droplet microfluidic chip, Nucleic acid detection, DNA