在这项工作中，通过巧妙地将催化发夹组装（CHA）、双端Mg2依赖的DNAzyme和杂交链式反应（HCR）作为三重级联信号放大器，构建了高效的串联CHA-DNAzyme-HCR（CDH）系统开发一种具有低背景信号的超灵敏电化学生物传感器，用于检测 microRNA-221 (miRNA-221)。在目标 miRNA-221 存在的情况下，CHA 循环通过与发夹 H1 和 H2 反应形成 DNAzyme 结构 H1-H2 来启动，催化底物发夹 H0 裂解，释放两个输出 DNA（输出 1 和输出 2） ）。随后，固定在电极板上的双环发夹H被输出的DNA打开，在发夹Ha和Hb的帮助下触发HCR。最后，亚甲基蓝嵌入到 HCR 产物的长 dsDNA 聚合物中，产生显着的电化学信号。令人惊讶的是，发夹H的双环结构可以显着降低背景信号，从而提高信噪比（S/N）。作为概念验证，使用 CDH 系统开发了一种超灵敏电化学生物传感器，检测限低至 9.25 aM，在检测各种癌细胞裂解物中的 miRNA-221 方面取得了良好的应用。得益于其无酶、无标记、低背景和高灵敏度的特点，CDH系统在各种临床研究中显示出分析微量生物标志物的广泛应用潜力。

In this work, by ingeniously integrating catalytic hairpin assembly (CHA), double-end Mg2+-dependent DNAzyme, and hybridization chain reaction (HCR) as a triple cascade signal amplifier, an efficient concatenated CHA-DNAzyme-HCR (CDH) system was constructed to develop an ultrasensitive electrochemical biosensor with a low-background signal for the detection of microRNA-221 (miRNA-221). In the presence of the target miRNA-221, the CHA cycle was initiated by reacting with hairpins H1 and H2 to form DNAzyme structure H1-H2, which catalyzed the cleavage of the substrate hairpin H0 to release two output DNAs (output 1 and output 2). Subsequently, the double-loop hairpin H fixed on the electrode plate was opened by the output DNAs, to trigger the HCR with the assistance of hairpins Ha and Hb. Finally, methylene blue was intercalated into the long dsDNA polymer of the HCR product, resulting in a significant electrochemical signal. Surprisingly, the double-loop structure of the hairpin H could prominently reduce the background signal for enhancing the signal-to-noise ratio (S/N). As a proof of concept, an ultrasensitive electrochemical biosensor was developed using the CDH system with a detection limit as low as 9.25 aM, achieving favorable application for the detection of miRNA-221 in various cancer cell lysates. Benefiting from its enzyme-free, label-free, low-background, and highly sensitive characteristics, the CDH system showed widespread application potential for analyzing trace amounts of biomarkers in various clinical research studies.