基于催化发夹自组装技术构建信号放大的电化学生物传感器检测miRNA

Development of a Catalytic Hairpin Self-Assembly-Based for Electrochemical Biosensors with Signal Amplification for miRNA Detection

摘要: 本研究旨在开发一种基于等温无酶式的催化发夹自组装技术(catalytic hairpin assembly, CHA)构建新型信号放大的电化学生物传感器，以DNA为模版制备的银纳米簇(DNA-AgNCs)作为信号探针，特异性的检测miRNA。首先研究选择以let-7a miRNA为分析对象，设计一对与目标物let-7a miRNA部分碱基互补的发夹链H1和H2，其中H1链富含胞嘧啶，可作为模板合成具有良好导电性的DNA-AgNCs，而H2则被设计为端基带有氨基的发夹结构，作为捕获探针H2能够借助Au-NH键，修饰在经过纳米金修饰的玻碳电极(dpAu/GCE)表面，实现对目标物let-7a miRNA的特异性识别与捕获。当目标物let-7a miRNA和DNA-AgNCs滴涂在修饰电极表面时，H1和H2发夹打开，引发CHA反应，将导电性能好的DNA-AgNCs (荧光基团)修饰在电极表面，可显著增强的电流信号。实验结果表明，该生物传感器在50 pM~1 μM浓度范围内，对let-7a miRNA具有良好的线性响应，测限低至16.67 pM，且具有优异的特异性，为电化学生物传感器在miRNA检测领域的应用提供了新的策略。

Abstract: This study aims to develop a novel electrochemical biosensor with signal amplification based on isothermal enzyme-free catalytic hairpin assembly (CHA) technology. Silver nanoclusters prepared using DNA as a template (DNA-AgNCs) are used as signal probes to specifically detect miRNA. Firstly, let-7a miRNA is selected as the analysis object. A pair of hairpin strands H1 and H2 that are partially complementary to the target let-7a miRNA in terms of base sequence are designed. Among them, strand H1 is rich in cytosine and can serve as a template for synthesizing DNA-AgNCs with good electrical conductivity. Hairpin strand H2 is designed to have an amino group at the end. As a capture probe, H2 can be modified on the surface of a glassy carbon electrode modified with gold nanoparticles (dpAu/GCE) through the Au-NH bond, enabling specific recognition and capture of the target let-7a miRNA. When the target let-7a miRNA and DNA-AgNCs are drop-coated on the surface of the modified electrode, hairpins H1 and H2 open, triggering the CHA reaction. DNA-AgNCs (fluorescent groups) with good electrical conductivity are modified on the electrode surface, which can significantly enhance the current signal. The experimental results show that this biosensor exhibits a good linear response to let-7a miRNA within the concentration range of 50 pM to 1000 nM, with a detection limit as low as 16.67 pM, and has excellent specificity. It provides a new strategy for the application of electrochemical biosensors in the field of miRNA detection.