A novel fluorescent biosensor based on dendritic DNA nanostructure in combination with ligase reaction for ultrasensitive detection of DNA methylation

作者：Shu Zhang, Jian Huang, Jingrun Lu, Min Liu, Yan Li, Lichao Fang, Hui Huang, Jianjun Huang, Fei Mo, Junsong Zheng · 发表于：Journal of Nanobiotechnology · 年份：2019 · DOI：10.1186/s12951-019-0552-5 · 被引用次数：15 · 研究领域：Advanced biosensing and bioanalysis techniques、Epigenetics and DNA Methylation、DNA and Nucleic Acid Chemistry

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 ...