Gao Shujie, Huang Xiaowei, Zhang Xinai, Yuan Zhecong, Chen Haili, Li Zhihua, El-Mesery Hany S, Shi Jiyong, Zou Xiaobo
School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, P. R. China.
Faculty of Agricultural Engineering, Jiangsu University, Zhenjiang 212013, P. R. China.
Anal Methods. 2025 May 15;17(19):3902-3924. doi: 10.1039/d5ay00572h.
The investigation of proteins at the single-molecule level is urgent to reveal the relationship between their structure and function. Unlike traditional techniques for attaining the overall average effect of group systems, nanopore sensing mode can provide information on the characteristics of proteins at the single-molecule level. Assisting with the intensity, frequency, and period of current changes, nanopore sequencing technology is rapidly advancing due to its merits, including fast readout, high accuracy, low cost, and portability. In particular, the single-molecule nanopore sequencing mode enables in-depth studies of DNA-protein interactions, protein conformation, DNA sequencing, and microbial assay, including genome sequencing of new species. This review summarizes the sensing mechanisms of nanopore sequencing technology in DNA damage, DNA methylation, RNA sequencing, and protein post-translational modifications and unfolding, covering both biological and solid-state nanopores. Due to these significant advantages, nanopore sequencing provides new insights into complex biological processes and enables more precise real-time monitoring of molecular changes. Its applications extend to clinical diagnostics, environmental monitoring, food safety, and forensic analysis. Moreover, the review outlines the present challenges faced by nanopore sequencing patterns, such as the choice of raw reagents and the design of special construction, offering a deep understanding of nanoporous single-molecule sensing toward protein sequence information and structure prediction.
在单分子水平上研究蛋白质对于揭示其结构与功能之间的关系至关重要。与获取群体系统整体平均效应的传统技术不同，纳米孔传感模式能够提供单分子水平上蛋白质特性的信息。借助电流变化的强度、频率和周期，纳米孔测序技术因其快速读出、高精度、低成本和便携性等优点而迅速发展。特别是，单分子纳米孔测序模式能够深入研究DNA-蛋白质相互作用、蛋白质构象、DNA测序以及微生物检测，包括新物种的基因组测序。本综述总结了纳米孔测序技术在DNA损伤、DNA甲基化、RNA测序以及蛋白质翻译后修饰和去折叠方面的传感机制，涵盖了生物纳米孔和固态纳米孔。由于这些显著优势，纳米孔测序为复杂生物过程提供了新的见解，并能够更精确地实时监测分子变化。其应用扩展到临床诊断、环境监测、食品安全和法医分析。此外，本综述概述了纳米孔测序模式目前面临的挑战，如原始试剂的选择和特殊结构的设计，从而深入了解纳米多孔单分子传感对蛋白质序列信息和结构预测的作用。
