Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, College of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, P.R. China.
School of Water Conservancy and Environment, University of Jinan, Jinan 250022, P.R. China.
Analyst. 2019 May 7;144(9):3023-3029. doi: 10.1039/c9an00036d. Epub 2019 Mar 22.
A facile signal-on electrochemical DNA biosensor has been developed for ultrasensitive detection of pathogenic bacteria using an Exo III-assisted autonomous multiple-cycle amplification strategy. The strategy relies on pathogens and aptamer binding-initiated release of a trigger, which combines with the 3'-protruding terminus of the hairpin probe 1, leading to the formation of double-stranded DNA with a blunt 3' terminus which starts the Exo III-assisted multiple signal amplification reaction. In addition, hairpin probe 2 labeled with an electroactive reporter at the middle of the loop region is ingeniously designed to contain a short hairpin-embedded segment, which can fold into a hairpin structure via an Exo III-assisted cleavage reaction, thus bringing the redox molecule in proximity to the electrode surface for "signal-on" sensing. Under optimal conditions, this biosensor exhibits a very low detection limit as low as 8 cfu mL and a wide linear range from 1.0 × 10 to 1.0 × 10 cfu mL of target pathogenic bacteria. As far as we know, this is the first time that the Exo III-assisted autonomous multiple-cycle amplification strategy has been used for signal-on electrochemical sensing of pathogenic bacteria. In addition, the proposed sensor can also be used for highly sensitive detection of other targets by changing the aptamer sequence, such as nucleic acids, proteins and small molecules. Therefore, the proposed signal-on electrochemical sensing strategy might provide a simple and practical new platform for detection of pathogenic bacteria and related biological analysis, food safety inspection and environmental monitoring.
一种简便的电化学生物传感器已被开发出来，用于利用 Exo III 辅助自主多循环扩增策略对病原体进行超灵敏检测。该策略依赖于病原体与适体的结合引发触发物的释放，这与发夹探针 1 的 3'突出末端结合，导致形成具有钝 3'末端的双链 DNA，从而开始 Exo III 辅助的多信号放大反应。此外，发夹探针 2 在环区中部标记有电活性报告分子，巧妙地设计包含一个短发夹嵌入片段，通过 Exo III 辅助切割反应可以折叠成发夹结构，从而使氧化还原分子接近电极表面进行“信号开启”感应。在最佳条件下，该生物传感器的检测限低至 8 cfu mL，线性范围从 1.0 × 10 到 1.0 × 10 cfu mL 的目标病原体。据我们所知，这是首次将 Exo III 辅助自主多循环扩增策略用于病原体的电化学生物传感中的信号开启。此外，通过改变适体序列，该传感器还可用于对其他靶标进行高灵敏度检测，例如核酸、蛋白质和小分子。因此，所提出的信号开启电化学生物传感策略可能为病原体及相关生物分析、食品安全检测和环境监测提供一种简单实用的新平台。
