A highly sensitive fluorescence biosensor for detection of Staphylococcus aureus based on HCR-mediated three-way DNA junction nicking enzyme assisted signal amplification
A highly sensitive fluorescence biosensor for detection of Staphylococcus aureus based on HCR-mediated three-way DNA junction nicking enzyme assisted signal amplification
复制标题
基于HCR介导的三路DNA连接切口酶辅助信号放大的高灵敏金黄色葡萄球菌荧光生物传感器
作者：
Chuyan Zhang;Yixiang Duan;Mengfan Wu;Wei Ning;Ziyi Tian;Yixiang Duan;Yongxin Li
灵敏、高效地监测食源性细菌对食品安全控制具有重要意义。本文介绍了一种用于高灵敏度检测金黄色葡萄球菌（S.金黄色葡萄球菌）的表达载体。与基于上下游的电路不同，所提出的生物传感器集成了HCR电路和三向DNA连接切口酶辅助信号放大（3 WJ-NEASA），使其进入良性循环促进。在HCR介导的3 WJ-NEASA传感策略中，S. aureus引发HCR发夹（H1和H2）之间的自组装，暴露出差距以捕获分子信标（MB）并构建3 WJ结构。同时，MB增加了HCR纳米线的稳定性，提高了HCR电路的效率，从而在HCR纳米线中产生了更多的3 WJ-NEASA电路。利用HCR和3 WJ-NEASA的协同放大效应，该恒温生物传感器可以在30 min内一步检测低至6.7 pM的目标DNA。该方法的检出限（LOD）低至1.2 × 101 cfu mL-1，在牛奶中的检测中具有可靠的实用性。首次将HCR和3 WJ-NEASA技术结合起来，构建了一种双信号放大的DNA生物传感器，并应用于食源性细菌的灵敏度分析。此外，该方法还可以作为一个通用的平台，用于其他分析物的监测，因此在食品安全和环境监测中具有广阔的应用前景。
Sensitive and efficient monitoring of food-borne bacteria is of great importance for food safety control. Herein, a novel biosensor for highly sensitive detection of Staphylococcus aureus (S. aureus) was constructed by combining hybridization chain reaction (HCR) and nicking enzyme. Different from the upstream-downstream based circuit, the proposed biosensor integrated HCR circuit and three-way DNA junction nicking enzyme assisted signal amplification (3WJ-NEASA) into a virtuous circle of promotion. In the HCR-mediated 3WJ-NEASA sensing strategy, target DNA of S. aureus initiated the self-assembly between HCR hairpins (H1 and H2), which exposed the gap to capture molecular beacon (MB) and construct the 3WJ structure. Meanwhile, MB increased the stability of HCR nanowires and enhanced the efficiency of the HCR circuit, and thus more 3WJ-NEASA circuits were generated in HCR nanowires. Benefiting from the synergistic amplification coupling HCR and 3WJ-NEASA, this isothermal biosensor can detect as low as 6.7 pM of target DNA in one step within only 30 min. Furthermore, the HCR-mediated 3WJ-NEASA assay has been applied in the detection of S. aureus with a limit of detection (LOD) as low as 1.2 × 101 cfu mL-1, and has exhibited reliable practicability in spiked milk. It is the first time that a DNA biosensor combining HCR and 3WJ-NEASA for dual signal amplification was developed and has been adopted to the sensitive analysis of food-borne bacteria. Additionally, this strategy can serve as a universal platform for monitoring other analytes, and therefore possesses broad application prospects in food safety and environmental monitoring.