Novel force spectroscopy with nanopores

纳米孔不仅是每个生物体的基本组成部分，而且是表征水溶液中单个分子的新兴工具。用于检测纳米孔中的分子的主要探针是由外部电压驱动的离子电流。电导率的短暂变化表明单个分子通过含有孔的膜。到目前为止，纳米孔已成功用于检测DNA、RNA、蛋白质甚至单个离子的存在。纳米孔有两个主要来源。自上而下的方法采用纳米技术和高度聚焦的电子束在绝缘膜上制造单个孔。自下而上的可能性是从细菌中提取生物通道，并将其用作嵌入天然脂质膜中的传感器。本研究的目的是利用生物纳米孔的固态界面，在单分子水平上开发一种新的力谱。纳米孔提供了一个独特的可能性，分析分子在一分钟的体积和研究的单分子在强约束。电场允许将带电的大分子拉入并穿过孔，而离子电流包含关于分子电荷、直径、长度及其与通道表面的相互作用的信息。在这里，我们使用带有离子电流检测的光镊来施加力并精确地将分子定位在孔中。我们将对生物纳米孔进行遗传修饰，以研究相互作用对易位时间和力的影响。这将导致一种新的，基于纳米孔的力谱，使皮牛顿力检测，同时控制与纳米分辨率沿着在生物纳米孔中的分子的距离。我们的工具将被用来研究孔隙中大分子的相互作用，易位或展开的基本物理学。目前的联合体将在这项技术上占据世界领先地位。我们提出的技术可能导致生物物理学，生物化学和生物学领域的突破性实验。

Nanopores are not only basic building blocks in every living organism but emerging tools for the characterisation of single molecules in aqueous solution. The main probe for detection of a molecule in a nanopore is the ionic current driven by an external voltage. A brief change in conductance indicates the passage of a single molecule through the membrane containing the pore. Until now, nanopores were successfully used to detect the presence DNA, RNA, proteins and even single ions. There are two main sources for nanopores. The top-down approach employs nanotechnology and highly focussed electron beams to fabricate single pores in insulating membranes. The bottom-up possibility is to extract biological channels from bacteria and use them as sensors embedded in a native lipid membrane. The aim of this proposal is to use solid-state interfaced with biological nanopores to develop a novel force spectroscopy on the single molecule level. Nanopores offer a unique possibility for analysis of molecules in a minute volume and studies of single molecules in strong confinement. Electric fields allow to pull charged macromolecules into and through the pore, while the ion current contains information about the molecule charge, diameter, length and its interaction with the channel surface. Here we use optical tweezers with ion current detection to apply forces and accurately position a molecule in a pore. We will genetically modify the biological nanopores to study the influence of interactions on the translocation time and forces. This will lead to a novel, nanopore-based force spectroscopy enabling pico-Newton force detection while controlling the distance with nanometer resolution along a molecule in a biological nanopore. Our tool will be used to investigate the underlying physics of interaction, translocation or unfolding of macromolecules in pores. The current consortium will claim a worldwide lead position with this technique. Our proposed technology could lead to groundbreaking experiments in the areas of biological physics, biochemistry and biology.