Organic Solvent Tolerant Polymer Membrane-Biological Nanopore Sensing Platform for Single-Molecule Measurement

doi:10.6023/A25120423

Abstract

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

Cite this article

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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Fig. & Tab. 4

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

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

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

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

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