唾液中的miRNA-31是早期口腔鳞状细胞癌（OSCC）的可靠诊断标志物，然而由于其水平低且序列高度同源，对唾液样本中miRNA-31的准确检测是一项挑战。CRISPR/Cas12a系统具有实现简单核酸分析的卓越潜力，但其速度和灵敏度较低。为了通过CRISPR/Cas12a系统实现miRNA-31的快速和高灵敏度检测，我们开发了一种基于Cas12a的纳米收割机器人，利用聚合酶驱动的DNA行走机制激活，命名为双3D纳米机器人。目标迅速在DNA发夹修饰的磁性纳米颗粒表面上行走，由DNA聚合酶驱动产生了大量的双链DNA（dsDNA）。然后，Cas12a融合到生成的dsDNA上，激活其转底物酶活性，形成3D纳米收割机器人。随后，收割机器人切割并释放在感应界面上固定的含亚甲基蓝标记的DNA发夹，导致电化学信号的变化。我们发现，收割机器人的转底物酶活性高于传统的CRISPR/Cas12a系统。开发的双3D纳米机器人能够在唾液样本中快速（检测时间在60分钟内）、高灵敏度（检测限为飞摩尔级）和特异性地分析miRNA-31。因此，我们建立的电化学生物传感策略对于早期诊断OSCC具有巨大潜力。版权所有 © 2023 Elsevier B.V. 发布。

Salivary miRNA-31 is a reliable diagnostic marker for early-stage oral squamous cell carcinoma (OSCC), but accurate detection of miRNA-31 in saliva samples is a challenge because of its low level and high sequence homology. The CRISPR/Cas12a system has the exceptional potential to enable simple nucleic acid analysis but suffers from low speed and sensitivity. To achieve rapid and high-sensitive detection of miRNA-31 using the CRISPR/Cas12a system, a Cas12a-based nano-harvester activated by a polymerase-driven DNA walker, named as dual 3D nanorobots, was developed. The target walked rapidly on the surface of DNA hairpin-modified magnetic nanoparticles driven by DNA polymerase, generating numerous double-strand DNA (dsDNA). Then, the Cas12a bound to the generated dsDNA for activating its trans-cleavage activity, forming 3D nano-harvester. Subsequently, the harvester cut and released methylene blue-labeled DNA hairpins immobilized on the sensing interface, leading to the change in electrochemical signal. We found that the trans-cleavage activity of the harvester was higher than the conventional CRISPR/Cas12a system. The developed dual 3D nanorobots could enable rapid (detection time within 60 min), high-sensitive (detection limit of femtomolar), and specific analysis of miRNA-31 in saliva samples. Thus, our established electrochemical biosensing strategy has great potential for early diagnosis of OSCC.Copyright © 2023. Published by Elsevier B.V.