Lucas Florian L R, Willems Kherim, Tadema Matthijs J, Tych Katarzyna M, Maglia Giovanni, Wloka Carsten
Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9712 CP Groningen, The Netherlands.
Lab for Nanobiology, Department of Chemistry, KU Leuven, 3001 Leuven, Belgium.
ACS Omega. 2022 Jul 19;7(30):26040-26046. doi: 10.1021/acsomega.2c00871. eCollection 2022 Aug 2.
Single-molecule nanopore electrophysiology is an emerging technique for the detection of analytes in aqueous solutions with high sensitivity. These detectors have proven applicable for the enzyme-assisted sequencing of oligonucleotides. There has recently been an increased interest in the use of nanopores for the fingerprinting of peptides and proteins, referred to as single-molecule nanopore spectrometry. However, the analysis of the resulting electrophysiology traces remains complicated due to the fast unassisted translocation of such analytes, usually in the order of micro- to milliseconds, and the small ion current signal produced (in the picoampere range). Here, we present the application of a generalized normal distribution function (gNDF) for the characterization of short-lived ion current signals (blockades). We show that the gNDF can be used to determine if the observed blockades have adequate time to reach their maximum current plateau while also providing a description of each blockade based on the open pore current ( ), the difference caused by the pore blockade (Δ ), the position in time (μ), the standard deviation (σ), and a shape parameter (β), leaving only the noise component. In addition, this method allows the estimation of an ideal range of low-pass filter frequencies that contains maximum information with minimal noise. In summary, we show a parameter-free and generalized method for the analysis of short-lived ion current blockades, which facilitates single-molecule nanopore spectrometry with minimal user bias.
单分子纳米孔电生理学是一种用于高灵敏度检测水溶液中分析物的新兴技术。这些检测器已被证明适用于寡核苷酸的酶促测序。最近，人们对使用纳米孔进行肽和蛋白质指纹识别（即单分子纳米孔光谱法）的兴趣日益增加。然而，由于此类分析物通常在微秒到毫秒量级的快速无辅助转运以及产生的小离子电流信号（皮安范围），对所得电生理迹线的分析仍然很复杂。在此，我们展示了广义正态分布函数（gNDF）在表征短暂离子电流信号（阻断）方面的应用。我们表明，gNDF可用于确定观察到的阻断是否有足够时间达到其最大电流平台，同时还能基于开孔电流（ ）、孔阻断引起的差异（Δ ）、时间位置（μ）、标准差（σ）和形状参数（β）对每个阻断进行描述，仅留下噪声成分。此外，该方法允许估计包含最大信息且噪声最小的低通滤波器频率的理想范围。总之，我们展示了一种用于分析短暂离子电流阻断的无参数广义方法，该方法以最小的用户偏差促进了单分子纳米孔光谱法。
