CY Cergy Paris Université, CNRS, LAMBE, Cergy, France; Université Paris-Saclay, Univ Evry, CNRS, LAMBE, Evry-Courcouronnes, France.
Laboratory for Membrane Physiology and Technology, Department of Physiology, Faculty of Medicine, University of Freiburg, Freiburg, Germany.
Methods Enzymol. 2021;649:587-634. doi: 10.1016/bs.mie.2021.01.017. Epub 2021 Feb 19.
We report here on the nanopore resistive pulse sensing (Np-RPS) method, involving pore-forming toxins as tools for polymer analytics at single molecule level. Np-RPS is an electrical method for the label-free detection of single molecules. A molecule interacting with the pore causes a change of the electrical resistance of the pore, called a resistive pulse, associated with a measurable transient current blockade. The features of the blockades, in particular their depth and duration, contain information on the molecular properties of the analyte. We first revisit the history of Np-RPS, then we discuss the effect of the configuration of the molecule/nanopore interaction on the molecular information that can be extracted from the signal, illustrated in two different regimes that either favor molecular sequencing or molecular sizing. Specifically, we focus on the sizing regime and on the use of two different pore-forming toxins, staphylococcal α-hemolysin (αHL) and aerolysin (AeL) nanopores, for the characterization of water-soluble polymers (poly-(ethylene glycol), (PEG)), homopeptides, and heteropeptides. We discuss how nanopore sizing of polymers could be envisioned as a new approach for peptide/protein sequencing.
我们在这里报告纳米孔电阻脉冲感应 (Np-RPS) 方法，该方法涉及孔形成毒素作为在单分子水平进行聚合物分析的工具。Np-RPS 是一种用于对单个分子进行无标记检测的电方法。与孔相互作用的分子会引起孔的电阻变化，称为电阻脉冲，与可测量的瞬态电流阻断相关。阻断的特征，特别是其深度和持续时间，包含有关分析物分子特性的信息。我们首先回顾 Np-RPS 的历史，然后讨论分子/纳米孔相互作用的配置对可以从信号中提取的分子信息的影响，通过两种不同的有利于分子测序或分子尺寸的模式进行说明。具体来说，我们专注于尺寸测定模式，并使用两种不同的孔形成毒素，即金黄色葡萄球菌α-溶血素 (αHL) 和 Aerolysin (AeL) 纳米孔，用于表征水溶性聚合物（聚乙二醇）(PEG)、同肽和异肽。我们讨论了如何将聚合物的纳米孔尺寸测定设想为一种新的肽/蛋白质测序方法。
