Methylated DNA is a promising epigenetic biomarker for diseases diagnosis, therapy monitoring, and prognosis. Here, we developed a sensitive ratiometric electrochemical biosensor for the detection of methylated DNA based on the design of multistep DNA amplification circuits. The methylated target DNA firstly initiates the DNAzyme-assisted amplification circuit and produces a number of single-stranded triggers that catalyze the subsequent double-step catalytic hairpin assembly circuits to release numerous four-way DNA junctions. The four-way DNA junctions displace the methylene blue (MB)-modified signal probes for intercalating doxorubicin (DOX) molecules on the sensing interface, resulting in a significantly increased value of i_DOX/i_MB to achieve sensitive monitoring of methylated DNA. Taking advantage of the significant signal amplification of multistep DNA amplification circuits, the proposed method exhibited high sensitivity for target analysis with a detection limit of 4 aM and a dynamic linear range of 10 aM to 20 pM. Moreover, the proof-of-concept application of the sensing platform was investigated, suggesting that the system is promising for potential applications in early cancer diagnosis and the fundamental research of epigenetics.

甲基化 DNA 是一种很有前途的表观遗传生物标志物，可用于疾病诊断、治疗监测和预后。在这里，我们基于多步 DNA 扩增电路的设计开发了一种灵敏的比率电化学生物传感器，用于检测甲基化 DNA。甲基化的目标 DNA 首先启动 DNAzyme 辅助的放大电路，并产生许多单链触发器，这些触发器催化随后的双步催化发夹组装电路，以释放大量的四向 DNA 连接。四通 DNA 连接取代了亚甲基蓝 (MB) 修饰的信号探针，用于在传感界面上嵌入阿霉素 (DOX) 分子，导致i _DOX / i _{MB值显着增加}实现对甲基化 DNA 的灵敏监测。利用多步 DNA 放大电路的显着信号放大优势，该方法对目标分析表现出高灵敏度，检测限为 4 aM，动态线性范围为 10 aM 至 20 pM。此外，还研究了传感平台的概念验证应用，表明该系统在早期癌症诊断和表观遗传学基础研究中具有潜在应用前景。