循环肿瘤DNA（ctDNA）被认为是非侵入性癌症诊断的理想候选生物标志物。然而，ctDNA缺乏方便且可靠的检测方法，限制了其在临床应用中的发展。因此，我们开发了一种双信号放大策略，基于杂交链反应（HCR）和邻近杂交调控CRISPR/Cas12a的敏感ctDNA检测。ctDNA通过两个发夹探针（H1和H2）的连续杂交启动HCR，产生由许多分裂片段组成的长轻微缺口的双链DNA纳米线，并依次连接以激活CRISPR/Cas12a的转切活性。在这种情况下，标记的单链DNA报告者会被切割产生强烈的荧光信号。由于HCR和CRISPR/Cas12a的双重放大，该策略对于ctDNA表现出高灵敏度，检测限制为5.43 fM。此外，所提出的方法在血清样本中成功应用于ctDNA检测，并取得了令人满意的结果，具有在临床癌症诊断中巨大的潜力。Copyright © 2023 Elsevier B.V. All rights reserved.

Circulating tumor DNA (ctDNA) is regarded as an ideal candidate biomarker for the non-invasive diagnosis of cancer. However, the lack of convenient and reliable detection methods for ctDNA restricts its clinical application. Herein, we developed a dual signal amplification strategy for sensitive detection of ctDNA based on hybridization chain reaction (HCR) and proximity hybridization-regulated CRISPR/Cas12a. The ctDNA initiates HCR through the continuous hybridization of two hairpin probes (H1 and H2), yielding long nicked double-stranded DNA nanowires composed of numerous split segments, which are successively connected to activate the trans-cleavage activity of CRISPR/Cas12a. In this case, the doubly labeled single-stranded DNA reporter can be cleaved to produce a strong fluorescent signal. Owing to the dual amplification of HCR and CRISPR/Cas12a, this strategy exhibits high sensitivity toward ctDNA with a low detection limit of 5.43 fM. Moreover, the proposed method was successfully applied for ctDNA detection in serum samples with satisfactory results, which has great potential in the clinical diagnosis of cancer.Copyright © 2023 Elsevier B.V. All rights reserved.