One-Pot Species Release and Nanopore Detection in a Voltage-Stable Lipid Bilayer Platform
One-Pot Species Release and Nanopore Detection in a Voltage-Stable Lipid Bilayer Platform
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DOI：
10.1021/acs.nanolett.9b04446
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发表时间：
2019-12-01
期刊：
影响因子：
10.8
通讯作者：
Wanunu, Meni
中科院分区：
文献类型：
--
作者：
Kang, Xinqi;Alibakhshi, Mohammad Amin;Wanunu, Meni
生物纳米孔已被用作无标记检测和识别一系列生物分子的强大平台，用于生物传感应用和单分子生物物理学研究。尽管如此，由于低电压下生物分子捕获到生物纳米孔中的效率低下，分析物的检测限（LOD）较高，这对其生物传感功效造成了实际限制。已经提出了几种方法来提高膜的电压稳定性，包括聚合和水凝胶涂层，然而，这些方法会损害脂质的流动性。在这里，我们开发了一种基于芯片的平台，可以在晶圆级上大规模生产，能够承受 350 mV 的高电压，膜面积与传统系统相当。使用该平台，我们展示了在高电压下以纳摩尔状态检测 α-溶血素纳米孔中 DNA 发夹的传感。此外，我们还开发了一种工作流程，用于从磁性微珠中一锅酶释放具有不同茎长度的 DNA 发夹，然后在几分钟内对发夹进行多重纳米孔定量，为新型基于纳米孔的多重生物传感应用铺平道路。
Biological nanopores have been used as powerful platforms for label-free detection and identification of a range of biomolecules for biosensing applications and single molecule biophysics studies. Nonetheless, high limit of detection (LOD) of analytes due to inefficient biomolecular capture into biological nanopores at low voltage poses practical limits on their biosensing efficacy. Several approaches have been proposed to improve the voltage stability of the membrane, including polymerization and hydrogel coating, however, these compromise the lipid fluidity. Here, we developed a chip-based platform that can be massively produced on a wafer scale that is capable of sustaining high voltages of 350 mV with comparable membrane areas to traditional systems. Using this platform, we demonstrate sensing of DNA hairpins in alpha-hemolysin nanopores at the nanomolar regime under high voltage. Further, we have developed a workflow for one-pot enzymatic release of DNA hairpins with different stem lengths from magnetic microbeads, followed by multiplexed nanopore-based quantification of the hairpins within minutes, paving the way for novel nanopore-based multiplexed biosensing applications.