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

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 sensing. 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_aqueous=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

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, 2026, 84(4): 459-464.

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, 2026, 84(4): 459-464.

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图/表 4

图1. 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. 通过将Aerolysin纳米孔道嵌入PEG-b-PPhMA两嵌段聚合物双层膜, 构建了可以在水-有机体系中维持稳定的单分子传感体系, 能够对疏水有机小分子进行灵敏检测

Figure 1. Schematic illustration of the single-molecule sensing platform based on a PEG-b-PPhMA polymer membrane. An Aerolysin nanopore embedded in the polymer membrane remains stable in organic-aqueous solvent mixtures, enabling highly sensitive detection of hydrophobic organic molecules

Molecule	Molecular weight/kDa	Solvent^a	Leakage current/pA ^b	Nanopore system ^c
DPhPC	0.8	decane	1.6±0.8	Y
PEG₄₄-b-PPhMA₅₂	8.7	toluene	29.5±1.9	N
PEG₄₄-b-PPhMA₇₉	13.8	toluene	9.5±0.5	N
PEG₄₄-b-PPhMA₁₃₅	20.3	toluene V_toluene/V_decane=4 V_toluene/V_decane=1 V_toluene/V_octane=1	16.2±0.1 10.4±2.7 2.5±0.4 1.9±1.0	Y Y Y Y
PEG₄₄-b-PPhMA₁₇₀	30.5	toluene	4.5±0.5	N

表1. PEG-b-PPhMA聚合物膜与传统磷脂膜的性能对比

Table 1. Performance comparison of PEG-b-PPhMA polymer versus traditional lipid membranes

图2. PEG44-b-PPhMA135聚合物膜支撑的WT Aerolysin纳米孔道单分子测量. (a) WT Aerolysin的I-V曲线(n=3); (b) 不同数量的纳米孔道蛋白依次嵌入聚合物膜的电流-时间轨迹图; (c)使用WT Aerolysin检测2 μmol/L Poly(dA)4分子的电流轨迹和特征信号(红色箭头); (d) Poly(dA)4分子的阻断时间与残余电流程度(I/I0)分布散点图(统计事件数1500). 实验条件: 缓冲液为200 μL的1 mol/L Tris-KCl, pH 8.0, 施加电压为＋100 mV, 采用20 mg/mL PEG44-b-PPhMA135/(V甲苯/V正辛烷=1)溶液成膜, 采样率为100 kHz, 低通滤波为5 kHz. 膜-孔体系基于MECA-16芯片构建, 电流信号通过Orbit 16仪器采集

Figure 2. Single-molecule sensing using Aerolysin nanopore based on PEG44-b-PPhMA135 polymer membrane. (a) Current-voltage (I-V) curves of WT Aerolysin (n=3). (b) Current traces showing stepwise insertion of different numbers of nanopores into the polymer membrane. (c) Representative current traces of WT Aerolysin detecting 2 μmol/L Poly(dA)4, with characteristic blockade signals indicated by red arrows. (d) Scatter plot of duration time versus residual current depth (I/I0) for Poly(dA)4 with 1500 events. All experiments were performed in 200 μL of 1 mol/L Tris-KCl, pH 8.0, at ＋100 mV with 20 mg/mL PEG44-b-PPhMA135/(Vtoluene/Voctane=1). Data were acquired using an Orbit 16 system with MECA-16 chips at 100 kHz sampling rate and at 5 kHz low-pass filter

图3. 聚合物膜支撑的Aerolysin纳米孔道在水-有机溶剂体系中对疏水有机小分子的检测. (a) 在不同丙酮含量下WT Aerolysin的开孔电流(I0)轨迹图. 上: PEG44-b-PPhMA135/(V甲苯/V正辛烷=1)聚合物膜. 下: DPhPC/正癸烷磷脂膜. V丙酮/V水分别为 0 (纯水相缓冲液)、0.01、0.05、0.1、0.2、0.3和0.4 (从左至右), 施加电压为＋100 mV; (b) WT Aerolysin检测10 mmol/L N,N'-间苯撑双马来酰亚胺有机小分子的电流轨迹图. 施加电压依次为＋100 mV, ＋120 mV, ＋140 mV和＋160 mV (从左至右); (c) WT Aerolysin检测N,N'-间苯撑双马来酰亚胺的阻断时间-残余电流程度分布散点图, 施加电压为＋100 mV, 统计事件数2000. 终体系V丙酮/V水=0.1. 实验条件: 缓冲液为1 mol/L Tris-KCl, pH 8.0, 采样率为100 kHz, 低通滤波为5 kHz, 膜-孔体系基于MECA-16芯片构建, 电流信号通过Orbit 16仪器采集

Figure 3. Hydrophobic organic small molecule sensing using WT Aerolysin nanopore supported by polymer membranes in organic-aqueous solvent systems. (a) Open-pore current (I0) traces of WT Aerolysin inserted into the PEG44-b-PPhMA135/(Vtoluene/Voctane=1) membrane (top) and the DPhPC lipid membrane dissolved in decane (bottom). Measurements were performed in acetone-aqueous solvent systems, including pure aqueous buffer (Vacetone/Vaqueous=0, background) and mixtures with acetone-to-aqueous volume ratios of 0.01, 0.05, 0.1, 0.2, 0.3, and 0.4 (from left to right) at ＋100 mV; (b) Current traces of 10 mmol/L N,N'-1,3-phenylenedimaleimide detection with WT Aerolysin at ＋100 mV, ＋120 mV, ＋140 mV, and ＋160 mV, respectively; (c) Scatter plot of duration time versus I/I0 for N,N'-1,3-phenylenedimaleimide sensing. Statistics were collected in Vacetone/Vaqueous=0.1 system at ＋100 mV with 2000 events. All experiments were performed in 1 mol/L Tris-KCl, pH 8.0 as aqueous buffer solution with a sampling rate of 100 kHz and a 5 kHz low-pass filter using a MECA-16 chip and Orbit 16 instrument

参考文献 43

[1]	Ying, Y.-L.; Hu, Z.-L.; Zhang, S.; Qing, Y.; Fragasso, A.; Maglia, G.; Meller, A.; Bayley, H.; Dekker, C.; Long, Y.-T. Nat. Nanotechnol. 2022, 17, 1136. doi: 10.1038/s41565-022-01193-2 pmid: 36163504
[2]	Bai, H.; Tan, H.; Feng, J. Acta Chim. Sinica 2025, 83, 1089 (in Chinese). doi: 10.6023/A25050179
	(白宏震, 谭皓璟, 冯建东, 化学学报, 2025, 83, 1089.) doi: 10.6023/A25050179
[3]	Howorka, S. Nat. Nanotechnol. 2017, 12, 619. doi: 10.1038/nnano.2017.99 pmid: 28681859
[4]	Ren, M.; Liu, D.; Qin, F.; Chen, X.; Ma, W.; Tian, R.; Weng, T.; Wang, D.; Astruc, D.; Liang, L. Adv. Colloid. Interf. Sci. 2025, 338, 103417. doi: 10.1016/j.cis.2025.103417
[5]	Hu, Z.-L.; Ying, Y.-L.; Wang, J.; Zhong, C.-B.; Kong, X.; Yu, X.; Zhang, W.; Zhang, J.; Long, Y.-T. Univ. Chem. 2025, 40, 11 (in Chinese).
	(胡正利, 应佚伦, 王佳, 钟诚兵, 孔璇凤, 余晓冬, 章文伟, 张剑荣, 龙亿涛, 大学化学, 2025, 40, 11.)
[6]	Hu, Z.-L.; Xin, K.-L.; Liu, S.-C.; Zhong, C.-B.; Wu, X.-Y.; Ying, Y.-L.; Kong, X.; Yu, X.; Zhang, J.; Long, Y.-T. Univ. Chem. 2022, 37, 191 (in Chinese).
	(胡正利, 辛凯莉, 刘少创, 钟诚兵, 武雪原, 应佚伦, 孔璇凤, 余晓冬, 张剑荣, 龙亿涛, 大学化学, 2022, 37, 191.)
[7]	Ma, C.; Xu, W.; Liu, W.; Xu, C.; Sha, J. Acta Chim. Sinica 2023, 81, 857 (in Chinese). doi: 10.6023/A23040149
	(马超凡, 徐伟, 刘巍, 徐昌晖, 沙菁㛃, 化学学报, 2023, 81, 857.) doi: 10.6023/A23040149
[8]	Zhang, M.; Tang, C.; Wang, Z.; Chen, S.; Zhang, D.; Li, K.; Sun, K.; Zhao, C.; Wang, Y.; Xu, M.; Dai, L.; Lu, G.; Shi, H.; Ren, H.; Chen, L.; Geng, J. Nat. Methods 2024, 21, 609. doi: 10.1038/s41592-024-02208-7 pmid: 38443507
[9]	Wang, J.-H.; Ma, W.; Hu, Z.-L.; Gao, Z.; Long, Y.-T.; Li, T.; Ying, Y.-L. Research 2025, 8, 0850. doi: 10.34133/research.0850
[10]	Hao, W.-Y.; Wu, H.-C. J. Instrum. Anal. 2025, 44, 1723 (in Chinese).
	(郝文英, 吴海臣, 分析测试学报, 2025, 44, 1723.)
[11]	Harrington, L.; Alexander, L. T.; Knapp, S.; Bayley, H. Angew. Chem. Int. Ed. 2015, 54, 8154. doi: 10.1002/anie.201503141 pmid: 26058458
[12]	Qin, P.; Ma, H.; Zhang, F.-G.; Ma, J.-A. Acta Chim. Sinica 2023, 81, 697 (in Chinese). doi: 10.6023/A23040154
	(秦沛, 马海, 张发光, 马军安, 化学学报, 2023, 81, 697.) doi: 10.6023/A23040154
[13]	Ertugrul, S.; Yucel, C.; Sertoglu, E.; Ozkan, Y.; Ozgurtas, T. Chem. Phys. Lipids 2020, 230, 104932. doi: 10.1016/j.chemphyslip.2020.104932
[14]	Perera, R. T.; Fleming, A. M.; Johnson, R. P.; Burrows, C. J.; White, H. S. Nanotechnology 2015, 26, 074002. doi: 10.1088/0957-4484/26/7/074002
[15]	Gao, Z. F.; Wei, Q.; Xia, F. Sci. Bull. 2025, 70, 1186. doi: 10.1016/j.scib.2025.01.050
[16]	Liu, W.; Yang, Z.-L.; Yang, C.-N.; Ying, Y.-L.; Long, Y.-T. Chem. Sci. 2022, 13, 4109. doi: 10.1039/D1SC06837G
[17]	Ying, Y.-L.; Yang, C.-N.; Liu, W.; Wu, X.-Y.; Long, Y.-T. Nat. Chem. 2025, 17, 1450. doi: 10.1038/s41557-025-01905-w
[18]	Liu, W.; Yang, C.-N.; Yang, Z.-L.; Yu, R.-J.; Long, Y.-T.; Ying, Y.-L. Angew. Chem. Int. Ed. 2023, 62, e202304023. doi: 10.1002/anie.v62.27
[19]	Liu, W.; Zhu, Q.; Yang, C.-N.; Fu, Y.-H.; Zhang, J.-C.; Li, M.-Y.; Yang, Z.-L.; Xin, K.-L.; Ma, J.; Winterhalter, M.; Ying, Y.-L.; Long, Y.-T. Nat. Nanotechnol. 2024, 19, 1693. doi: 10.1038/s41565-024-01721-2
[20]	Zhou, S.; Tang, P.; Wang, Y.; Wang, L.; Wang, D.-Q. Chin. J. Anal. Chem. 2018, 46, 826 (in Chinese). doi: 10.1016/S1872-2040(18)61089-8
	(周硕, 唐鹏, 王赟姣, 王亮, 王德强, 分析化学, 2018, 46, 826.)
[21]	Wang, J.; Li, S.; Li, K.; Wang, Y.; Li, W. J. Mol. Liq. 2022, 362, 119697. doi: 10.1016/j.molliq.2022.119697
[22]	Posokhov, Y. O.; Kyrychenko, A. Comput. Biol. Chem. 2013, 46, 23. doi: 10.1016/j.compbiolchem.2013.04.005 pmid: 23764528
[23]	Rai, R.; Kumar, D.; Dhule, A. A.; Rudani, B. A.; Tiwari, S. Langmuir 2024, 40, 14057. doi: 10.1021/acs.langmuir.4c01499
[24]	Nardin, C.; Winterhalter, M.; Meier, W. Langmuir 2000, 16, 7708. doi: 10.1021/la000204t
[25]	Martin, M.; Dubbs, T.; Fried, J. R. Langmuir 2017, 33, 1171. doi: 10.1021/acs.langmuir.6b03309
[26]	Vreeker, E.; Grünewald, F.; Heide, N. J.; Bonini, A.; Marrink, S. J.; Tych, K.; Maglia, G. Adv. Mater. 2025, 37, 2418462. doi: 10.1002/adma.v37.15
[27]	Yu, L.; Kang, X.; Alibakhshi, M. A.; Pavlenok, M.; Niederweis, M.; Wanunu, M. Biophys. J. 2021, 120, 1537. doi: 10.1016/j.bpj.2021.02.019
[28]	Kihara, H.; Sato, M.; Kawano, R. Phys. Chem. Chem. Phys. 2025, 27, 12483. doi: 10.1039/D4CP04782F
[29]	Heitz, B. A.; Jones, I. W.; Hall, H. K.; Aspinwall, C. A.; Saavedra, S. S. J. Am. Chem. Soc. 2010, 132, 7086. doi: 10.1021/ja100245d
[30]	Cressiot, B.; Ouldali, H.; Pastoriza-Gallego, M.; Bacri, L.; Van der Goot, F. G.; Pelta, J. ACS Sensors 2019, 4, 530. doi: 10.1021/acssensors.8b01636
[31]	Li, M.-Y.; Ying, Y.-L.; Long, Y.-T. Acta Chim. Sinica 2019, 77, 984 (in Chinese). doi: 10.6023/A19060202
	(李孟寅, 应佚伦, 龙亿涛, 化学学报, 2019, 77, 984.) doi: 10.6023/A19060202
[32]	Niu, H.-Y.; Hu, Z.; Ying, Y.; Long, Y.-T. Acta Chim. Sinica 2019, 77, 989 (in Chinese). doi: 10.6023/A19060230
	(牛红艳, 胡正利, 应佚伦, 龙亿涛, 化学学报, 2019, 77, 989.) doi: 10.6023/A19060230
[33]	Li, M.-Y.; Jiang, J.; Niu, H.-Y.; Ying, Y.-L.; Long, Y.-T. Chin. Sci. Bull. 2023, 68, 2148 (in Chinese).
	(李孟寅, 蒋杰, 牛红艳, 应佚伦, 龙亿涛, 科学通报, 2023, 68, 2148.)
[34]	Zhang, X.-J.; Qi, G.-D.; Yang, M.-S.; Weng, T.; Liang, L.-Y.; Wang, D.-Q. Chin. J. Anal. Chem. 2023, 51, 1867 (in Chinese).
	(张晓君, 奇国栋, 杨淼森, 翁婷, 梁丽媛, 王德强, 分析化学, 2023, 51, 1867.)
[35]	Rideau, E.; Dimova, R.; Schwille, P.; Wurm, F. R.; Landfester, K. Chem. Soc. Rev. 2018, 47, 8572. doi: 10.1039/C8CS00162F
[36]	Antonov, V. F.; Smirnova, E. Y.; Anosov, A. A.; Norik, V. P.; Nemchenko, O. Y. Biophysics 2008, 53, 390. doi: 10.1134/S0006350908050126
[37]	Wang, S.; Li, L.; Liu, Q.; Urban, M. W. Macromolecules 2022, 55, 4703. doi: 10.1021/acs.macromol.2c00739
[38]	Maglia, G.; Heron, A. J.; Stoddart, D.; Japrung, D.; In Methods in Enzymology, Vol. 475, Ed.: Walter, N. G., Elsevier Inc., San Diego, CA, USA, 2010, pp. 591-623.
[39]	Wu, X.-Y.; Ying, Y.-L.; Long, Y.-T. Chem. J. Chin. U. 2019, 40, 1825 (in Chinese).
	(武雪原, 应佚伦, 龙亿涛, 高等学校化学学报, 2019, 40, 1825.) doi: 10.7503/cjcu20190364
[40]	Zhang, L.-L.; Zhong, C.-B.; Li, J.-G.; Niu, H.-Y.; Ying, Y.-L.; Long, Y.-T. J. Electroanal. Chem. 2022, 915, 116266. doi: 10.1016/j.jelechem.2022.116266
[41]	Zhang, L.-L.; Zhong, C.-B.; Huang, T.-J.; Zhang, L.-M.; Yan, F.; Ying, Y.-L. Chem. Sci. 2024, 15, 8355. doi: 10.1039/D3SC06795E
[42]	Zhu, T.; Yu, Q.; Ding, L.; Di, T.; Zhao, T.; Li, T.; Li, L. Green Chem. 2019, 21, 2326. doi: 10.1039/C8GC03891K
[43]	Wu, L.; Deng, Y.; Guo, D.; Zhang, G.; Yu, T.; Chen, X.; Li, N. J. Membr. Sci. 2026, 745, 125245. doi: 10.1016/j.memsci.2026.125245