An enzyme-mediated universal fluorescent biosensor template for pathogen detection based on a three-dimensional DNA walker and catalyzed hairpin assembly
An enzyme-mediated universal fluorescent biosensor template for pathogen detection based on a three-dimensional DNA walker and catalyzed hairpin assembly
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基于三维 DNA 步行器和催化发夹组装的酶介导通用荧光生物传感器模板，用于病原体检测
DOI：
10.1039/d0nr07593k
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发表时间：
2021-01-28
期刊：
影响因子：
6.7
通讯作者：
Duan, Yixiang
中科院分区：
文献类型：
--
作者：
Li, Dan;Yang, Enlai;Duan, Yixiang
基于三维DNA步行者和催化发夹组装（CHA）反应的策略，构建了一种通用的酶介导荧光生物传感器模板，用于病原体的快速检测。在细菌识别步骤中，细菌和双链复合物之间的链置换反应导致步行者链的释放。步行者链触发DNA步行者产生酶片段，DNA步行者使用金纳米颗粒（AuNP）作为轨道以提供优异的DNA配体锚定区域。在CHA步骤中，酶片段诱导CHA循环产生荧光信号，这大大提高了触发DNA的转化率和荧光生物传感器的灵敏度。通过调节多聚腺苷酸（PolyA）的长度，研究了DNA配体的距离和密度对反应的影响，并进一步探讨了其反应动力学。通过比较最大反应速率（V-max）、米氏常数（K-m）和转换数（K-cat），对优化后的PolyA探针进行评价和鉴定。在这项工作中，优化的PolyA-DNA探针在鼠伤寒沙门氏菌（S。ty）检测，分别是SH-DNA和MCH处理的SH-DNA的11.9倍和4.6倍。对大肠杆菌的检出限为28.1CFU/mL。coli）检测。该传感器对真实的样品检测的回收率在91%~ 115%之间，具有良好的选择性和重复性。考虑到这些优点，该模板作为病原体检测的常规工具具有很大的潜力，并在全球公共卫生和食品安全领域具有广泛的应用。
An enzyme-mediated universal fluorescent biosensor template for rapid detection of pathogens was developed based on the strategy of a three-dimensional (3D) DNA walker and catalyzed hairpin assembly (CHA) reaction. In the bacterial recognition step, a strand displacement reaction between bacteria and the double-stranded complex caused the release of the walker strand. The walker strand triggered the DNA walker to produce an enzyme fragment, and the DNA walker used gold nanoparticles (AuNPs) as the track to provide an excellent DNA ligand anchoring area. In the CHA step, the enzyme fragment induced the CHA cycle to yield fluorescence signals, which greatly enhanced the conversion ratio of trigger DNA and the sensitivity of the fluorescent biosensor. The effect of the distance and density of the DNA ligand was studied by adjusting the length of poly-adenine (PolyA), and was further explored by its reaction kinetics. By comparing the maximum reaction rate (V-max), Michaelis constant (K-m) and turnover number (K-cat), the optimized PolyA probe was assessed and identified. In this work, the optimized PolyA-DNA probe exhibited an outstanding sensitivity in Salmonella typhimurium (S. ty) detection, which is 11.9 times and 4.6 times higher than those of the SH-DNA and the MCH treated SH-DNA. Meanwhile, a detection limit of 28.1 CFU mL(-1) was achieved in Escherichia coli (E. coli) detection. Furthermore, the biosensor achieved good selectivity and high repeatability with recoveries of 91%-115% for real sample detection. Considering these advantages, this template has great potential as a routine tool for pathogen detection and has wide applications in the field of global public health and food safety.