Circulating tumor DNA (ctDNA) is an ideal candidate for liquid biopsy biomarkers. Therefore, detecting a low abundance of ctDNA is essential for early cancer diagnosis. Here, we developed a novel triple circulation amplification system integrating entropy and enzyme cascade-driven three-dimensional (3D) DNA walker and branched hybridization strand reaction (B-HCR) for ultrasensitive detection of breast cancer-related ctDNA. In this study, the 3D DNA walker was constructed by inner track probes (NH) and complex S on a microsphere. Once the DNA walker was triggered by the target, the strand replacement reaction ran first and kept circulating to rapidly displace the DNA walker containing 8–17 DNAzyme. Secondly, the DNA walker could repeatedly cleave NH autonomously along the inner track, generating numerous initiators, and then promoting B-HCR to activate the third cycle. Subsequently, the split G-rich fragments were brought in close to form the G-quadruplex/hemin DNAzyme by adding hemin, with the addition of H₂O₂ and ABTS, the target could be observed. Benefiting from triplex cycles, the PIK3CA^E545K mutation detection possesses a good linear range from 1-10³ fM, and the limit of detection was 0.65 fM. Due to the low cost and high sensitivity, the proposed strategy has great potential in early diagnosis of breast cancer.

循环肿瘤 DNA (ctDNA) 是液体活检生物标志物的理想候选物。因此，检测低丰度的ctDNA对于早期癌症诊断至关重要。在这里，我们开发了一种新型三重循环扩增系统，集成了熵和酶级联驱动的三维 (3D) DNA 步行器和分支杂交链反应 (B-HCR)，用于超灵敏检测乳腺癌相关的 ctDNA。在这项研究中，3D DNA 步行器由内轨道探针 (NH) 和微球上的复合物 S 构建。一旦 DNA 助行器被目标触发，链置换反应首先运行并不断循环以快速取代含有 8-17 个 DNAzyme 的 DNA 助行器。其次，DNA walker 可以沿内部轨道自主重复切割 NH，产生大量启动子，然后促进B-HCR激活第三个循环。随后，通过添加血红素、添加 H₂ O ₂和ABTS，可以观察到目标。得益于三重循环，PIK3CA ^E545K突变检测具有良好的线性范围，范围为 1-10 ³ fM，检测限为 0.65 fM。由于成本低、灵敏度高，所提出的策略在乳腺癌的早期诊断中具有巨大的潜力。