Circulating tumor DNA (ctDNA) is a promising biomarker for tumor genotyping and therapy monitoring. Herein, we developed a digital PCR chip with embedded microwell and bidirectional partition network for highly sensitive ctDNA analysis. The embedded microwell contributes to increasing microreaction density (up to 7000 microwells/cm²) and reducing evaporation during amplification. The bidirectional partition network can achieve fast and random distribution of targets, ensuring the precise quantification of nucleic acid. We used plasmids, artificial samples and 32 clinical blood samples from non-small cell lung cancer patients to test the performance of this platform. The results demonstrated that our chip has not only comparable quantification performance to commercial counterpart but also the ability to detect EGFR mutations with as low as 0.01% mutation rate and 20 alter molecules in 27 ng genomic DNA. The identification of EGFR mutations in plasma using developed chip exhibited 85.71% sensitivity and 94.44% specificity for L858R mutation and 100% sensitivity and 86.96% specificity for T790 M mutation. Moreover, the monitoring of mutant allele in plasma was accomplished in this work. In conclusion, the developed chip has a potential in lung tumor genotyping and therapy monitoring for precision medicine, even other tumors.

循环肿瘤DNA（ctDNA）是用于肿瘤基因分型和治疗监测的有前途的生物标志物。本文中，我们开发了一种具有嵌入式微孔和双向分区网络的数字PCR芯片，用于高度敏感的ctDNA分析。嵌入式微孔有助于提高微反应密度（高达7000微孔/ cm ²）并减少扩增过程中的蒸发。双向分配网络可实现目标的快速随机分布，从而确保核酸的精确定量。我们使用了来自非小细胞肺癌患者的质粒，人工样本和32种临床血液样本来测试该平台的性能。结果表明，我们的芯片不仅具有与商业同类产品相当的定量性能，而且还具有检测低至0.01％突变率和27 ng基因组DNA中20个可变分子的EGFR突变的能力。使用开发的芯片在血浆中鉴定EGFR突变对L858R突变的敏感性为85.71％，特异性为94.44％，对T790 M突变的敏感性为100％，特异性为86.96％。此外，这项工作完成了对血浆中突变等位基因的监测。总之，开发的芯片在肺肿瘤基因分型和精确医学甚至其他肿瘤的治疗监测中具有潜力。