Key Laboratory of Luminescence Analysis and Molecular Sensing (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, P. R. China.
Anal Chem. 2024 Sep 17;96(37):15066-15073. doi: 10.1021/acs.analchem.4c03766. Epub 2024 Sep 3.
In this work, by ingeniously integrating catalytic hairpin assembly (CHA), double-end Mg-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.
在这项工作中，通过巧妙地将催化发夹组装（CHA）、双端 Mg 依赖性 DNA 酶和杂交链式反应（HCR）集成作为三重级联信号放大器，构建了高效的串联 CHA-DNA 酶-HCR（CDH）系统，用于开发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）。在存在靶 miRNA-221 的情况下，通过与发夹 H1 和 H2 反应来启动 CHA 循环，形成 DNA 酶结构 H1-H2，该结构 H1-H2 催化底物发夹 H0 的切割以释放两个输出 DNA（输出 1 和输出 2）。随后，固定在电极板上的双链发夹 H 被输出 DNA 打开，在发夹 Ha 和 Hb 的辅助下触发 HCR。最后，亚甲基蓝插入 HCR 产物的长双链 DNA 聚合物中，导致电化学信号显著增强。令人惊讶的是，发夹 H 的双链结构可以显著降低背景信号，从而提高信号与噪声比（S/N）。作为概念验证，使用 CDH 系统开发了超灵敏电化学生物传感器，检测限低至 9.25 aM，可实现对各种癌细胞裂解物中 miRNA-221 的良好应用。得益于无酶、无标记、低背景和高灵敏度的特点，CDH 系统在分析各种临床研究中痕量生物标志物方面具有广泛的应用潜力。
