A novel fuorescent biosensor based ondendritic DNA nanostructure incombination withligase reaction forultrasensitive detection ofDNA methylation
A novel fuorescent biosensor based ondendritic DNA nanostructure incombination withligase reaction forultrasensitive detection ofDNA methylation
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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
DOI：
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发表时间：
2019
影响因子：
10.2
通讯作者：
Junsong Zheng
中科院分区：
文献类型：
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作者：
Shu Zhang;Jian Huang;Jingrun Lu;Min Liu;Yan Li;Lichao Fang;Hui Huang;Jianjun Huang;Fei Mo;Junsong Zheng
背景：DNA甲基化检测对于包括恶性肿瘤在内的多种疾病的诊断和预后判断是必不可少的。因此，开发一种简单、灵敏、特异的检测策略至关重要。研究方法：利用功能性树状DNA纳米结构和信号富集聚苯乙烯微球结合连接酶检测反应（LDR）的简单双重信号放大策略，研制了一种新型荧光生物传感器。树状DNA是由Y-DNA和X-DNA在无酶DNA催化下自组装而成的发夹结构，通过加入peptide防止其在高温下解旋。然后将荧光染料Cy 5标记的树突状DNA聚合物与报告探针连接成偶联物。亲和素标记的聚苯乙烯微球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。通过添加Taq连接酶来触发LDR。当甲基化胞嘧啶存在时，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，产生荧光信号。然而，在亚硫酸氢盐处理后，未甲基化的胞嘧啶转化为尿嘧啶，导致单碱基错配。由于不存在双链体，未检测到荧光信号。结果：所制备的树枝状DNA聚合物体积较大。这种方法省时、成本低。在最佳实验条件下，亲和素标记的聚苯乙烯微球荧光信号得到明显放大，可超灵敏、高选择性地定量DNA甲基化。该传感器的探测范围为10−15至10−7。M，最低检测限可达0.4fM。所构建的生物传感器也成功地用于实际样品的分析。结论：该方法具有高灵敏度和高特异性，不需要PCR和酶切等复杂过程，在肿瘤诊断和生物医学研究中具有重要价值。
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 specifc detection strategy..Methods: A novel fuorescent biosensor was developed based on a simple dual signal amplifcation 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 fuorescent dye Cy5 was ligated with reporter probe into a conjugate. Avidin-labeled polystyrene microbeads were specifcally 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 fuorescent dye perfectly matched the target sequence, forming a stable duplex to .generate a fuorescence signal. However, after bisulfte treatment, unmethylated cytosine was converted into uracil, .resulting in a single base mismatch. No fuorescence 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 fuorescence signal was amplifed more obviously, and DNA methylation was quantifed 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 specifcity for DNA methylation quantifcation, without requiring complex processes such as PCR and enzymatic digestion, which is thus of great value in tumor diagnosis and .biomedical research.