Cavendish Laboratory, University of Cambridge, JJ Thompson Avenue, Cambridge CB3 0HE, U.K.
School of Biomedical Engineering, Faculty of Medicine, Dalian University of Technology, No. 2, Linggong Road, Dalian 116024, China.
J Am Chem Soc. 2023 Jun 7;145(22):12115-12123. doi: 10.1021/jacs.3c01649. Epub 2023 May 23.
Multiplexed nucleic acid sensing methods with high specificity are vital for clinical diagnostics and infectious disease control, especially in the postpandemic era. Nanopore sensing techniques have developed in the past two decades, offering versatile tools for biosensing while enabling highly sensitive analyte measurements at the single-molecule level. Here, we establish a nanopore sensor based on DNA dumbbell nanoswitches for multiplexed nucleic acid detection and bacterial identification. The DNA nanotechnology-based sensor switches from an "open" into a "closed" state when a target strand hybridizes to two sequence-specific sensing overhangs. The loop in the DNA pulls two groups of dumbbells together. The change in topology results in an easily recognized peak in the current trace. Simultaneous detection of four different sequences was achieved by assembling four DNA dumbbell nanoswitches on one carrier. The high specificity of the dumbbell nanoswitch was verified by distinguishing single base variants in DNA and RNA targets using four barcoded carriers in multiplexed measurements. By combining multiple dumbbell nanoswitches with barcoded DNA carriers, we identified different bacterial species even with high sequence similarity by detecting strain specific 16S ribosomal RNA (rRNA) fragments.
用于临床诊断和传染病控制的高特异性多重核酸传感方法至关重要，尤其是在后疫情时代。在过去的二十年中，纳米孔传感技术得到了发展，为生物传感提供了多功能工具，同时能够在单分子水平上实现高灵敏度的分析物测量。在这里，我们建立了一种基于 DNA 哑铃纳米开关的纳米孔传感器，用于多重核酸检测和细菌鉴定。当目标链与两个序列特异性传感突出端杂交时，基于 DNA 纳米技术的传感器会从“打开”状态切换为“关闭”状态。DNA 中的环将两组哑铃拉到一起。拓扑结构的变化导致电流迹中出现一个容易识别的峰。通过在一个载体上组装四个 DNA 哑铃纳米开关，实现了对四种不同序列的同时检测。通过使用四个带条形码的载体进行多重测量，对 DNA 和 RNA 靶标中的单个碱基变体进行区分，验证了哑铃纳米开关的高特异性。通过将多个哑铃纳米开关与带条形码的 DNA 载体相结合，我们甚至可以通过检测特定菌株的 16S 核糖体 RNA(rRNA) 片段来识别具有高序列相似性的不同细菌物种。
