Nanopore analysis is a powerful technique for single molecule analysis by virtue of its electrochemically confined effects. As a single molecule translocates through the nanopore, the featured ionic current pattern on the time scale contains single molecule characteristics including volume, charge, and conformational properties. Although the characteristics of a single molecule in a nanopore have been written to the featured ionic current, extracting the dynamic information from a complex current trace is still a big challenge. Here, we present an applicable nanopore analysis method employing the Hilbert–Huang Transform (HHT) to study the vibrational features and interactions of a single molecule during the dynamic translocation process through the confined space of a nanopore. The HHT method is specially developed for analyzing nonlinear and non-stationary data that is highly compatible with nanopore data with a high frequency resolution. To provide proof-of-concept, we applied HHT to measure the frequency response for the wild-type (WT) aerolysin and mutant K238E aerolysin nanopores with and without the presence of poly(dA)₄, respectively. The energy–frequency–time distribution spectra demonstrate that the biological nanopore contributes greatly to the characteristics of the high frequency component (>2 kHz) in the current recording. Our results suggest that poly(dA)₄ undergoes relatively more consistent and confined interactions with K238E than WT, leading to a prolonging of the duration time. Therefore, the characteristics in frequency analysis could be regarded as an “single-molecule ionic spectrum” inside the nanopore, which encodes the detailed behaviours of single-molecule weak interactions.

中文翻译：

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测量与受限纳米孔的单分子相互作用的频谱

纳米孔分析凭借其电化学限制效应，是用于单分子分析的强大技术。当单个分子移位通过纳米孔时，时标上的离子电流特征图包含单个分子的特征，包括体积，电荷和构象性质。尽管已经将纳米孔中单个分子的特征写入了特征离子电流，但是从复杂的电流迹线中提取动态信息仍然是一个很大的挑战。在这里，我们介绍了一种适用的纳米孔分析方法，该方法采用希尔伯特-黄变换（HHT）来研究单个分子在纳米孔的受限空间内动态移位过程中的振动特征和相互作用。HHT方法是专门为分析非线性和非平稳数据而开发的，该数据与具有高分辨率的纳米孔数据高度兼容。为了提供概念验证，我们应用了HHT来测量存在和不存在poly（dA）的野生型（WT）溶血素和突变型K238E溶血素纳米孔的频率响应分别为₄。能量-频率-时间分布谱表明，生物纳米孔对当前记录中的高频分量（> 2 kHz）的特性有很大贡献。我们的研究结果表明，与WT相比，poly（dA） ₄与K238E经历了相对更一致和局限的相互作用，从而延长了持续时间。因此，频率分析的特征可以看作是纳米孔内部的“单分子离子光谱”，它编码了单分子弱相互作用的详细行为。