BACKGROUND DNA methylation detection is indispensable for the diagnosis and prognosis of various diseases including malignancies. Hence, it is crucial to develop a simple, sensitive, and specific detection strategy. METHODS A novel fluorescent biosensor was developed based on a simple dual signal amplification strategy using functional dendritic DNA nanostructure and signal-enriching polystyrene microbeads in combination with ligase detection reaction (LDR). Dendritic DNA self-assembled from Y-DNA and X-DNA through enzyme-free DNA catalysis of a hairpin structure, which was prevented from unwinding at high temperature by adding psoralen. Then dendritic DNA polymer labeled with fluorescent dye Cy5 was ligated with reporter probe into a conjugate. Avidin-labeled polystyrene microbeads were specifically bound to biotin-labeled capture probe, and hybridized with target sequence and dendritic DNA. LDR was triggered by adding Taq ligase. When methylated cytosine existed, the capture probe and reporter probe labeled with fluorescent dye perfectly matched the target sequence, forming a stable duplex to generate a fluorescence signal. However, after bisulfite treatment, unmethylated cytosine was converted into uracil, resulting in a single base mismatch. No fluorescence signal was detected due to the absence of duplex. RESULTS The obtained dendritic DNA polymer had a large volume. This method was time-saving and low-cost. Under the optimal experimental conditions using avidin-labeled polystyrene microbeads, the fluorescence signal was amplified more obviously, and DNA methylation was quantified ultrasensitively and selectively. The detection range of this sensor was 10-15 to 10-7 M, and the limit of detection reached as low as 0.4 fM. The constructed biosensor was also successfully used to analyze actual samples. CONCLUSION This strategy has ultrasensitivity and high specificity for DNA methylation quantification, without requiring complex processes such as PCR and enzymatic digestion, which is thus of great value in tumor diagnosis and biomedical research.

中文翻译：

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基于树突状DNA纳米结构结合连接酶反应的新型荧光生物传感器，用于DNA甲基化的超灵敏检测。

背景技术DNA甲基化检测对于包括恶性肿瘤在内的各种疾病的诊断和预后是必不可少的。因此，开发一种简单，灵敏和特定的检测策略至关重要。方法基于功能树突状DNA纳米结构和信号富集聚苯乙烯微珠结合连接酶检测反应（LDR）的简单双信号放大策略，开发了一种新型荧光生物传感器。通过无酶DNA催化的发夹结构从Y-DNA和X-DNA自组装树突状DNA，通过添加补骨脂素可防止其在高温下解开。然后，将用荧光染料Cy5标记的树突状DNA聚合物与报告探针连接成结合物。将抗生物素蛋白标记的聚苯乙烯微珠与生物素标记的捕获探针特异性结合，并与靶序列和树突状DNA杂交。LDR是通过添加Taq连接酶触发的。当存在甲基化的胞嘧啶时，用荧光染料标记的捕获探针和报告探针与靶序列完全匹配，形成稳定的双链体以产生荧光信号。但是，在亚硫酸氢盐处理后，未甲基化的胞嘧啶被转化为尿嘧啶，导致单个碱基错配。由于不存在双链体，未检测到荧光信号。结果获得的树突状DNA聚合物体积较大。这种方法既省时又低成本。在使用亲和素标记的聚苯乙烯微珠的最佳实验条件下，荧光信号被更明显地放大，并且DNA甲基化被超灵敏和选择性地定量。该传感器的检测范围是10-15至10-7 M，检测限低至0.4 fM。所构建的生物传感器也已成功用于分析实际样品。结论该策略对DNA甲基化定量具有超灵敏性和高特异性，不需要复杂的过程（例如PCR和酶消化），因此在肿瘤诊断和生物医学研究中具有重要的价值。