微小RNA（miRNA）的检测方法具有鲁棒性和敏感性对于癌症临床诊断至关重要。在本研究中，我们开发了一种新型的高灵敏度miRNA-21检测电化学生物传感器，该传感器依赖于肽核酸（PNA）-DNA异种三路结合（H3WJ）和目标回收催化发夹装配（CHA）扩增的形成。首先，在金电极上固定了电荷中性的PNA探针以构建传感器。引入miRNA-21后，启动目标回收CHA，产生丰富的双链CHA产物。随后，PNA探针与这些产物结合形成PNA-DNA H3WJ。因此，电极表面密布有众多电活性的二茂铁（Fc）基团，从而实现对浓度低至0.15 fM的miRNA-21的显著增强电流响应的高灵敏度检测。该方法在目标miRNA上表现出显著的特异性，并可用于定量监测人类癌细胞中miRNA-21的表达。更重要的是，该传感器表现出卓越的稳定性，并在miRNA检测过程中降低了背景噪音，使该方法成为监测各种miRNA生物标志物以促进不同类型癌症诊断的高度有前途的传感平台。版权所有©2023 Elsevier B.V.保留所有权利。

Robust and sensitive methods for the detection of microRNAs (miRNAs) are crucial in the clinical diagnosis of cancers. In this study, a novel electrochemical biosensor with high sensitivity for miRNA-21 detection is developed, which relies on the formation of a peptide nucleic acid (PNA)-DNA hetero-three-way junction (H3WJ) and target-recycling catalytic hairpin assembly (CHA) amplification. The electroneutral PNA probes are initially immobilized onto a gold electrode to construct the sensor. Upon introduction of miRNA-21, target-recycling CHA is initiated, resulting in abundant double-stranded CHA products. Subsequently, association between the PNA probes and these products leads to the formation of PNA-DNA H3WJs. Consequently, the electrode surface is densely populated with numerous electroactive Ferrocene (Fc) groups, resulting in a significantly amplified current response for highly sensitive detection of miRNA-21 at concentrations as low as 0.15 fM. This approach demonstrates remarkable specificity towards target miRNAs and can be utilized for quantitative monitoring of miRNA-21 expression in human cancer cells. More importantly, the sensor exhibits exceptional stability and shows a significant reduction in background noise during miRNA detection, making this method a highly promising sensing platform for monitoring various miRNA biomarkers to facilitate the diagnosis of diverse cancers.Copyright © 2023 Elsevier B.V. All rights reserved.