一种用于核酸绝对定量检测的高鲁棒性液滴式数字PCR芯片

为满足液滴式数字聚合酶链式反应（PCR）技术对扩增反应过程中稳定保存液滴以及反应后高效检测的核心需求，构建了一种具有过滤气泡和增强荧光信号功能的液滴式数字聚合酶链式反应芯片.该芯片可在10 min内产生20多万个半径约为21 μm的液滴.利用“玻璃天花板”的方式构建了独立于芯片主体材料的液滴收集腔，为液滴提供稳定的保存与反应环境；还构建了过滤结构，可有效过滤混入液相中的空气，提高芯片鲁棒性.同时，在液滴收集腔中引入反射层，增强荧光信号，使单个视野荧光成像时间缩短约40%，提高了检测效率.利用该芯片定量检测EGFR基因第21号外显子，检测信号与DNA浓度在10¹~10⁵ copies/μL范围内呈现良好的线性关系（R²=0.998）.该方案在载玻片大小的芯片上实现了液滴产生、PCR扩增和荧光信号读取，并具有较高的鲁棒性与检测效率，在核酸检测等方面具有应用潜力.

Robust Droplet Digital PCR Chip for Absolute Quantitative Detection of Nucleic Acid

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.