用于水-有机溶剂体系单分子测量的聚合物膜-蛋白质纳米孔道系统★

doi:10.6023/A25120423

摘要/Abstract

生物纳米孔道单分子电化学测量技术具有无标记、高灵敏和高分辨等优势, 已广泛应用于DNA、RNA、多肽及糖类等生物小分子的检测. 然而, 对于在水相中溶解度较低的疏水分子, 通常需引入有机溶剂以提高其溶解性, 而传统纳米孔道技术所采用的磷脂支撑膜对有机溶剂耐受性有限, 限制了该技术在疏水分子测量及相关研究中的应用. 为此, 本研究利用两亲性嵌段共聚物聚乙二醇-聚甲基丙烯酸苯酯(PEG-b-PPhMA), 构建了一种对有机溶剂具有良好耐受性的仿生双分子层聚合物膜. 通过调控聚合度和溶剂组成, 实现了膜体系的高稳定性和优良的隔绝离子传输性能, 可应用于气单胞菌溶素(Aerolysin)生物蛋白质纳米孔道的单分子测量研究. 结果表明, 在v_丙酮/v_水=0.3混合缓冲体系中, 该“膜-孔”系统仍可保持长期稳定测量并获得高信噪比离子电流信号, 从而实现了疏水有机小分子的单分子电化学测量. 本研究构建的聚合物膜有效解决了生物纳米孔道在水-有机体系中的稳定性难题, 拓展了纳米孔道技术在药物研发、生命分析、环境监测和单分子反应等研究中对疏水分子的测量潜力.

关键词: 聚合物膜, 纳米孔道, 单分子测量, 电化学, 膜-孔界面, 疏水小分子传感

Biological nanopore is a label-free and highly sensitive single-molecule electrochemical sensing technique that detect individual molecules by monitoring ionic current fluctuations through a pore-forming protein. In conventional nanopore measurements, transmembrane proteins such as Aerolysin are embedded in artificial phospholipid bilayers, enabling robust detection of small biomolecules including DNA, RNA, and peptides. However, extending biological nanopore sensing to poorly water-soluble organic molecules remains challenging, primarily due to the limited tolerance of phospholipid bilayers to organic solvents required for solubilizing hydrophobic analytes. To address this limitation, we developed an organic solvent tolerant biological nanopore sensing platform based on an amphiphilic block copolymer membrane composed of poly(ethylene glycol)-b-poly(phenyl methacrylate) (PEG-b-PPhMA). By systematically tuning the polymerization degree and solvent composition, the membrane thickness and mechanical stability were optimized to accommodate Aerolysin insertion and operation. The PEG-b-PPhMA polymer, dissolved in a v_toluene/v_octane=1 mixture, was spread across a microwell on a chip using an air-bubble method to form a uniform supporting membrane. Aerolysin nanopores were subsequently incorporated into the polymer membrane, yielding a low-leakage and electrically stable sensing interface. The resulting membrane-nanopore interface maintained stable ionic currents with high signal-to-noise ratios in acetone-aqueous systems containing organic solvent fractions up to v_acetone/v_buffer=0.3, under which conventional phospholipid bilayers typically lose structural integrity. Moreover, this platform enabled single-molecule electrochemical sensing of the hydrophobic organic molecule N,N’-1,3-phenylenedimaleimide in an organic-aqueous solvent environment. This work establishes a robust and versatile strategy for extending biological nanopore sensing beyond conventional aqueous environments, thereby expanding the applicability of nanopore technology to hydrophobic organic molecules. These advances hold promise for applications in small-molecule drug discovery, metabolism analysis, environmental monitoring, and single-molecule reaction studies.

Key words: polymer membrane, nanopore, single-molecule measurement, electrochemistry, membrane-nanopore interface, hydrophobic small molecule sensing

引用此文

张琳琳, 袁浩轩, 龙亿涛, 郎超, 应佚伦. 用于水-有机溶剂体系单分子测量的聚合物膜-蛋白质纳米孔道系统^★[J]. 化学学报, doi: 10.6023/A25120423.

Lin-Lin Zhang, Haoxuan Yuan, Yi-Tao Long, Chao Lang, Yi-Lun Ying. Organic Solvent Tolerant Polymer Membrane-Biological Nanopore Sensing Platform for Single-Molecule Measurement[J]. Acta Chimica Sinica, doi: 10.6023/A25120423.

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