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Developing an Inositol Phosphate-Actuated Nanochannel System by Mimicking Biological Calcium Ion Channels Qi Lu, Qiu-Han Tang, Zhong-Hui Chen, Shi-Long Zhao, Guang-Yan Qing, and Tao-Lei Sun ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09992 • Publication Date (Web): 05 Sep 2017 Downloaded from http://pubs.acs.org on September 7, 2017

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ACS Applied Materials & Interfaces

Developing an Inositol Phosphate-Actuated Nanochannel System by Mimicking Biological Calcium Ion Channels Qi Lu,†,§ Qiuhan Tang,‡,§ Zhonghui Chen,† Shilong Zhao,⊥ Guangyan Qing,*,† and Taolei Sun*,†,‡ †

State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan

University of Technology, 122 Luoshi Road, Wuhan 430070, P. R. China ‡

School of Chemistry, Chemical Engineering and Life Science, Wuhan University of

Technology, 122 Luoshi Road, Wuhan 430070, P. R. China ⊥

Tsinghua―Berkeley Shenzhen Institute, Tsinghua University, 2279 Lishui Road, Shenzhen

518000, P. R. China KEYWORDS: smart polymer, bio-interfaces, calcium ion channel, inositol phosphate, gating behavior, porous alumina membrane,

ABSTRACT: In eukaryotic cells, ion channels—ubiquitously present as polypeptides or proteins—usually regulate the ion transport across biological membranes by conformational switching of the channel proteins in response to the binding of diverse signaling molecules (e.g.,

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inositol phosphate, abbreviated to InsP). To mimic the gating behaviors of natural Ca2+ channels manipulated by InsPs, a smart poly[(N-isopropylacrylamide-co-4-(3-acryloylthioureido) benzoic acid)0.2] (denoted as PNI-co-ATBA0.2) was integrated onto a porous anodic alumina (PAA) membrane, building an InsP–actuated nanochannel system. Driven by intensive hydrogen bonding complexation of ATBA monomer with InsP, the copolymer chains displayed a remarkable and reversible conformational transition from a contracted state to a swollen one, accompanying with significant changes in surface morphology, wettability and viscoelasticity. Benefiting from these features, dynamic gating behaviors of the nanochannels located on the copolymer–modified PAA membrane could be precisely manipulated by InsPs, reflecting as a satisfactory linear relationship between real-time variation in transmembrane ionic current and the InsP concentration over a wide range from 1 nmol·L–1 to 10 µmol·L–1, as well as a clear discrimination among InsP2, InsP3 and InsP6. This study indicates the great potential of biomolecule–responsive polymers in the fabrication of biomimetic ion nanochannels and other nanoscale bio-devices.

1. INTRODUCTON In living organisms, the fine organization of a biological system is constantly regulated by an intricate network of signaling pathways,1,2 among which ligand–gated ion channels3-5 play crucial roles in manipulating ion transport across diverse bio-interfaces through recognition and binding of specific signaling biomolecules. In particular, on endoplasmic reticulum (ER) membranes, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) receptor (a functional protein, denoted as InsP3R) constitutes a family of calcium ion (Ca2+) channels6 responsible for the release of intracellular Ca2+ stores, and subsequently


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affect the activation of Ca2+–dependent proteins.7 As illustrated in Scheme 1A, the gating of above Ca2+ channels has proved to be activated by the Ins(1,4,5)P3–binding–triggered conformational switching of the InsP3R,8 and returned to their original states by Ins(1,4,5)P3 metabolism toward a range of additional inositol phosphates (i.e., InsP2, InsP5, or InsP6), which are also widely acknowledged as essential signaling molecules.9 Hence, building biomimetic InsP–responsive nanochannels is of great significance for simulating the gating behavior of biological Ca2+ channels in vitro, which may help better understand of the Ca2+–related cellular signal pathways.10 In the past decade, smart polymer–based nanochannels or nanopores, capable of reversibly tuning the ionic transport properties according to external stimuli (e.g., pH, temperature, light, and special ions),11-15 have attracted increasing interest because of their wide applications in the interdisciplinary fields of chemistry, physics, life science16 and material science.17-20 However, compared with the highly sensitive and precisely controllable ligand (i.e., signaling biomolecules)–gated ion channels in vivo, there is a huge gap between the classical manufactured ion channels and natural biological systems,21-24 more intelligent ion channels manipulated by specific biomolecules are urgently needed. Nevertheless, relevant research is largely hampered by the lack of efficient synthetic receptors, which can specifically recognize and capture given biomolecules, and subsequently facilitate transforming this slight recognition signal into macroscopic property transition of material surface.25 Thus, the development of artificial biomolecule–gated ion channels still remains a great challenge for chemists and material scientists, and thus has been rarely reported before.26,27 Here, for the first time we report a biomimetic InsP–actuated nanochannel system constructed by immobilizing an InsP–responsive copolymer (i.e., PNI-co-ATBA0.2) onto multiple


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nanochannels of a PAA membrane (Scheme 1B). The copolymer comprises the core InsP recognition unit (ATBA) capable of specifically binding with phosphate groups in InsPs, and a flexible poly(N-Isopropyl acrylamide) (PNIPAAm) backbone with a smart hydrogen bonding network, which affords an ideal platform to intelligently modulate the movement of polymer chains in response to external stimuli.28-30 In addition, PAA membrane with high pore density and well-defined straight-through nanochannels (average diameter: 80―100 nm)31 was employed as the substrate material, allowing readily PNI-co-ATBA0.2 modification through surfaceinitiated atom transfer radical polymerization (SI-ATRP).32 Based on this design, the nanochannel system exhibited unique responsiveness and gating behaviors toward InsPs, particularly the InsP6 molecule, which is a typical Ca2+ channel–relevant signaling molecule, and substantial evidences have disclosed its striking anticancer potential both in prevention and therapy.33


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Scheme 1. Illustration of InsP–responsive ion channels. (A) In living organisms, through multiple hydrogen bonding interactions among InsP3 and specific amino acid residues located on InsP3–receptor protein (InsP3R), InsP3 is bound to the receptor protein and triggers the conformational switching of InsP3R, which opens the Ca2+ channels on endoplasmic reticulum membrane, leading to a sharp increase in Ca2+ concentration in the cytoplasm. (B) Our biomimetic InsP–responsive ion channels are constructed by modifying InsP–responsive copolymer PNI-co-ATBA0.2 on a porous anodic alumina (PAA) membrane. The copolymer chains can undergo remarkable globule–to–coil transition upon exposure to InsPs in ultralow concentrations (at nanomolar level), which significantly decreases diameters of the nanochannels and obstructs ion transport across the nanochannels.

2. EXPERIMENTAL DETAILS 2.1. Materials. N-Isopropyl acrylamide (NIPAAm, 98%) that purchased from Sigma-Aldrich (China) was recrystallized in n-hexane three times before being used in copolymerization. Fluorescein–labeled ATBA was prepared through a coupling reaction of ATBA and fluorescein isothiocyanate, the synthesis route and characterization data are illustrated in Scheme S1 and in the corresponding text in Supporting Information (SI). Diverse inositol phosphates (InsP6, InsP3 and InsP2, purity: > 97%) used in the detection experiments were purchased from Sigma-Aldrich Corp. (China). Double distilled water (18.2 MΩ·cm, Milli-Q system) was used in the experiments. Other materials and instruments are described in the corresponding text in the SI. 2.2. Synthesis of ATBA Monomer. A two–step preparation method for the ATBA monomer is illustrated in Scheme S2 in the SI. Acryloyl chloride (0.906 g, 10 mmol) was added dropwise


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into a solution of anhydrous potassium thiocyanate (0.972 g, 10 mmol) in 20 mL dry acetone at ambient temperature. After being stirred for 10 h, the mixture was centrifuged and the supernatant was collected to concentrate using rotary evaporation. Then the intermediate product was added dropwise into a solution of 4-aminobenzoic acid (1.372 g, 10 mmol) in 20 mL acetone/H2O (1:1, v/v) mixture at ambient temperature, and continued to be stirred overnight. Finally, the suspension was centrifuged, and the precipitate was orderly washed twice with acetone and distilled water, respectively. Then the precipitate was dried under vacuum at ambient temperature to obtain the final product as yellow powder (1.950 g, yield: 78%, melting point: 163 ºC). Hydrogen nuclear magnetic resonance (1H NMR, 500 MHz, deuterated dimethylsulfoxide (d6-DMSO), δ (ppm)): 6.03 (d, J=10 Hz, 1H, C=CH), 6.48 (d, J=17 Hz, 1H, C=CH), 6.61–6.67 (m, 1H, C=CH), 7.87 (d, J=8.5 Hz, 2H, Ph-H), 7.97 (d, J=8.5 Hz, 2H, Ph-H), 11.73 (s, 1H, CNHCS), 12.79 (s, 1H, CNHCS), 12.90 (s, 1H, COOH); Carbon nuclear magnetic resonance (13C NMR, 500 MHz, d6-DMSO, δ (ppm)): 123.8, 128.6, 130.2, 130.4, 132.5, 142.2, 167.5, 171.1, 175.7; Infrared spectra (IR): 3183, 2972, 2851, 1673, 1599, 1570, 1410, 1252, 1165, 978, 875 cm−1; Matrix–assisted laser desorption ionization mass spectrometry (MADLIMS): m/z calcd. for C11H10N2O3S: 250.04; found: 251.0311 (M+H). Elemental analysis calcd. (%) for C11H10N2O3S: C, 52.79; H, 4.03; N, 11.19; S, 12.81. Found: C, 52.81; H, 4.01; N, 11.39; S, 12.69. 2.3. Fluorescent Titration Experiment. To investigate binding affinity of ATBA toward InsP6, a fluorescent titration experiment was conducted, which is a typical and widely adopted method for calculating association constant (Ka) in host–guest chemistry.34 The host fluorescein– labeled ATBA was prepared as stock solution (5.0×10‒6 mol·L‒1) in tris(hydroxymethyl)aminomethane and hydrochloric acid (Tris–HCl) buffer solution (1.0 mmol·L−1, pH 7.4).


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Guest InsP6 salt was synthesized by a reaction of InsP6 and six equivalents of tetrabutylammonium hydroxide in methanol,35 and was then prepared to two stock solutions in H2O (0.025 and 0.25 mol·L‒1, respectively). The work solutions were prepared by adding different volumes of guest solution to a series of test tubes, and then the same amount of stock solution of the fluorescein-labeled ATBA host was added into each test tube, followed by dilution to 3.00 mL by Tris-HCl buffer solution. After being shaken for one min, the work solutions were measured immediately at 20 °C using a Perkin-Elmer LS-55 spectrometry (excitation wavelength: 470 nm). The Ka value between ATBA and InsP6 salt was obtained through non-linear fitting calculation according to the fluorescence intensity changes of in the maximum emission peak (514 nm), and the corresponding calculation formula is illustrated in the SI. 2.4. Bio-Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (BioATR-FT-IR) Titration Experiment. The infrared spectra were recorded on a Bruker Vertex 80v FT–IR spectrometer in Bio-ATR cell II accessory [the accessory is based on dual crystal technology: the top crystal is made of silicon, and the second crystal is made of zincselenide (ZnSe) and has a hemispherical design].36,37 All samples were dissolved in 16 µL d6-DMSO. For each sample, the concentrations (ATBA: 40 mmol·L‒1, InsP6 salt: 40 mmol·L‒1) and total volume (16 µL) were strictly controlled. For each measurement, the equipment remained in standby mode for 15 min to ensure the equilibrium of temperature (20 °C) prior to the test, and all the spectra of samples were obtained through 1200 scans subtracting the d6-DMSO background at a 4 cm−1 resolution. Before each measurement, the Bio-ATR cell was orderly cleaned with distilled water and ethanol, respectively. Then it was sufficiently dried under a nitrogen gas flow.


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2.5. 1H NMR and 1H–13C Correlated NMR Titration Experiments. In order to investigate the complexation of ATBA with InsP6, 1H and 1H‒13C NMR titration experiments were employed to explore the binding details.38 To avoid the interference of deuterated water (D2O) with strong suppression effect on hydrogen bonding, d6-DMSO was chosen as the solvent because both ATBA and InsP6 salt were well soluble in it. The host solution of ATBA monomer (40 mmol·L‒1), gest solution of InsP6 salt (40 mmol·L‒1), and their mixture at a molar ratio of 1:0.2 were prepared, then the chemical shift changes of active hydrogen protons were recorded on a Varian INOVA-400 MHz and analyzed using MestReNova software. In addition, twodimensional 1H‒13C correlation spectroscopy was used to verify the interaction of ATBA with InsP6 salt in d6-DMSO by investigating the sample of ATBA monomer and the mixture of ATBA with InsP6 salt at a molar ratio of 1:0.2. 2.6. Quartz Crystal Microbalance–Dissipation (QCM-D) Adsorption Experiment 2.6.1. Synthesis of ATBA monolayer, PNI-co-ATBA0.2 and PNIPAAm modified quartz– crystal (QC) resonators. First, Au-coated QC resonator with an intrinsic frequency (F0) of 5 MHz [purchased from QSense Corp. (Sweden)] was orderly washed with distilled water and ethanol three times, respectively. Then a monolayer of 2-mercaptoethylamine was grafted onto the gold surface after the Au-coated QC resonator was immersed in a solution of 2-mercaptoethylamine (0.01 mol·L‒1) in ethanol for 24 h at ambient temperature. To remove the physically adsorbed 2mercaptoethylamine, the QC resonator was rinsed with ethanol three times and dried under a flow of nitrogen gas. The ATBA monolayer was obtained by immersing this 2-


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mercaptoethylamine–grafted QC resonator in a solution of 4-carboxyphenyl isothiocyanate (0.05 mol·L‒1) for 12 h at ambient temperature. For the modifications of PNI-co-ATBA0.2 and PNIPAAm, the 2-mercaptoethylamine–grafted QC resonator was immersed in 50 mL dry dichloromethane (CH2Cl2) solution containing pyridine (0.8%, v/v). The polymerization initiator 2-bromoisobutyryl bromide (0.4 mL) was added dropwise into the solvent containing the QC resonator at 0 ºC, and the mixture was left at this temperature for 1 h then at ambient temperature for 12 h. Subsequently, the QC resonator was rinsed with CH2Cl2, and dried under a nitrogen flow, receiving a bromine-modified QC resonator for polymerization. Then PNI-co-ATBA0.2 was grafted from the surface of brominemodified QC resonator through SI-ATRP according to the literature.39 The polymeric film was achieved by immersing the bromine-modified QC resonator in a degassed solution of NIPAAm (0.1828 g, 1.6 mmol) and ATBA (0.1016 g, 0.4 mmol) in 10 mL dimethylfumarate (DMF) containing cuprous bromide (CuBr, 0.0143 g, 0.1 mmol) and N,N,N',N'',N''-pentamethyldiethylenetriamine (PMDETA, 0.1 mL, 0.47 mmol). The reaction was carried out in nitrogen atmosphere for 15 h at 60 °C. After that, the polymerization was stopped by removing the QC resonator from the reaction bath, and then the copolymer–modified QC resonator was orderly cleaned with 20 mL DMF, 10 mL water and 10 mL ethanol, respectively, and subsequently dried under a nitrogen flow. The PNIPAAm-modified QC resonator was prepared through the similar method as described above. 2.6.2. QCM-D adsorption experiments. All QCM-D measurements were performed at 20 °C on a Q-Sense E4 system (Sweden). Prior to binding assays between InsP6 and QC resonator that modified with ATBA monolayer, PNI-co-


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ATBA0.2 or PNIPAAm homopolymer, respectively, QCM channels and tubes were washed with distilled water carefully and dried under a flow of nitrogen gas, followed by installing the functionalized QC resonator into a flow-cell for frequency and dissipation measurement. After stabilization of the fundamental resonance frequency with pure water, InsP6 solution (10 µmol·L‒1) was pumped into the flow-cell by a peristaltic pump at a constant speed of 100 µL·min‒1. All the time–dependent frequency and dissipation curves were recorded by Q-Sense software and analysed by Q-Tools.40 2.7. Atomic Force Microscopy (AFM) Experiment. AFM measurements were performed using a Multimode 8 AFM (Bruker, USA). To determine the thickness of PNI-co-ATBA0.2 film, the surfaces of Au-coated QC resonators before and after copolymer modification were scratched by a syringe needle,41 and corresponding AFM images were subsequently acquired in a tapping mode under ambient condition, as shown in Figure S1 in SI. In order to discern the changes in surface morphology and roughness of the copolymer film before and after InsP6 adsorption, PNIco-ATBA0.2–modified QC resonator was treated by a solution of InsP6 (10 µmol·L‒1) for 10 min at 20 oC, and followed by AFM measurements in the tapping mode under ambient condition. The comparative AFM analysis of PNI-co-ATBA0.2–modified PAA membrane before and after InsP6 adsorption was conducted through a similar method. 2.8. Surface Contact Angle (CA) Measurement. Static CA was measured at ambient atmosphere and a constant temperature of 20 °C.42 In the CA cycling experiment, a PNI-coATBA0.2 modified QC resonator was alternately treated with InsP6 solution (10 µmol·L−1) for 10 min or with pure water for 10 min at 20 oC, respectively, followed by drying with a flow of nitrogen gas before each measurement. Then the static CA was recorded for each substrate using


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the sessile drop method and pure water as a solvent. Each measurement was repeated in triplicate to ensure the reliability of data. 2.9. Synthesis of PNI-co-ATBA0.2 Modified PAA Membrane. 2.9.1. Synthesis of PAA membrane The PAA membrane with well-defined straight through-holes was synthesized according to the literature.43 First, to enhance the grain size in the metal and to obtain homogeneous condition for pore growth over large areas, purity aluminum foils (99.999%, 20 mm × 30 mm × 0.2 mm) were annealed under nitrogen atmosphere at 500 °C for 3 h. Then the annealed foils were immersed in a sodium hydroxide aqueous solution (50 mL, 5 %wt) for 2 min, rinsed with distilled water and then electropolished in a mixture of perchloric acid and ethanol (1:5, v/v). Subsequently, anodization was carried out under a constant potential of 50 V in an oxalic acid solution (0.4 mol·L−1) for 3 h at 0 °C. The alumina formed was then removed with a mixture of phosphoric acid (6%wt) and chromic acid (1.5%wt), and the foils were anodized again under the same condition as aforementioned for 12 h. After that, the foils were immersed in a phosphoric acid solution (5%wt) at 60 °C for 1.5 h for pore-widening treatment. Then the foils were immersed in a copper chloride (0.3 mol·L−1) and hydrochloric acid (HCl, 10%wt) solution to remove the aluminum substrate, a PAA membrane with straight through-holes (average pore size: 80―100 nm) was ultimately obtained. 2.9.2. Modification of PAA membrane with PNI-co-ATBA0.2 polymeric film The prepared PAA membrane was orderly immersed in distilled water for 10 min, in ethanol for 10 min, in a hydrochloric acid aqueous solution (5%, v/v) for 35–50 s, and in a heated


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hydrogen peroxide at 100 °C for 1 h to generate surface hydroxyl groups. After that the membrane was orderly washed with distilled water and ethanol, respectively, followed by drying under a nitrogen flow. Then the membrane was immersed in a solution of 40 mL toluene containing 1.0 mL 3-aminopropyl triethoxysilane (APTES), the mixture was left at 65 °C for 3 h. The obtained amino–modified membrane was rinsed with ethanol to remove the remaining APTES, and dried under a nitrogen flow. Then the same method described in Part 2.6.1 was adopted to graft polymerization initiator 2-bromoisobutyryl bromide and subsequently the PNIco-ATBA0.2 film onto the inner surface of nanochannels located on the PAA membrane. The whole modification process is illustrated in Scheme S3 in the SI. 2.10. Electrical measurements. A piece of PAA membrane (before or after modification) was mounted in between a two-compartment electrochemical cell according to the literature.44 Then, approximately 0.6 mL work solutions containing electrolyte [sodium chloride (NaCl, 0.1 mol·L−1) or calcium chloride (CaCl2, 0.1 mol·L−1)] and InsP6 (concentrations range from 1 nmol·L−1 to 10 µmol·L−1) were injected into the electrochemical cell, respectively. The contact area between PAA membrane and these work solutions was approximately 20 mm2. After the PAA membrane was exposed to the work solutions for 10 min at 20 oC, Ag/AgCl electrodes with a voltage source were inserted to apply a transmembrane potential across the membrane. Then the transmembrane ionic current was measured using a Keithley 6487 picoammeter (Keithley Instruments) at 20 oC.

3. RESULTS AND DISCUSSIONS 3.1. Complexation of ATBA with InsP6. As the first step toward the design of InsP6–responsive polymer, binding affinity of ABTA monomer with InsP6 was investigated by fluorescent titration,34 Bio-ATR-FT-IR titration


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experiment,37 and 1H and 1H–13C NMR experiment.38 As shown in Figure 1A, an evidential increase in fluorescence intensity (at 514 nm) of fluorescein-labeled ATBA was observed upon the additions of various equivalents of InsP6 salt (six tetrabutylammonium worked as cations45). Through non-linear fitting calculation according to the fluorescence intensity change (Inset in Figure 1A), an association constant of 2786 ± 370 L·mol–1 was obtained, indicating the strong binding capacity of ATBA toward InsP6.


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Figure 1. (A) Fluorescence spectra of fluorescein–labeled ATBA (5.0 × 10−6 mol·L−1) upon the addition of various equivalents of InsP6 salt (six tetrabutylammonium worked as cations) in Tris-HCl buffer solution (1.0 mmol·L−1, pH 7.4) at 20 °C. The inset shows the relationship between the fluorescence intensity at 514 nm and the molar ratios of InsP6 to


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fluorescein-labeled ATBA, the red line is the non-linear fitting curve. (B) Representative FT–IR spectra of ATBA (balck), InsP6 salt (red), and their mixtures at a molar ratio of 1:1 (blue dots) in d6-DMSO at 20 oC. Color ribbons illustrate the characteristic peaks of InsP6 salt (green ribbon: C–H deformation vibration), and two characteristic peaks of ATBA, corresponding to the C=S stretching vibration (blue ribbon) and N–H deformation vibration (red ribbon). (C) Partial 1H NMR spectra of InsP6 salt (40 mmol·L−1), ATBA (40 mmol·L−1) and their mixture at a molar ratio of 1:5 in d6-DMSO at 20 oC. Chemical shift changes of the ATBA protons are indicated by red box and arrows. The InsP6 salt has no characteristic proton signals in this region. (D, E) Corresponding 1H‒13C correlation spectroscopy of ATBA monomer before (D) and after (E) interaction with 0.2 equivalents of InsP6 salt in d6-DMSO at 20 oC.

This strong binding was further verified by Bio-ATR-FT-IR measurement, in which a sharp decrease was found in the intensity of characteristic peaks corresponding to the C=S stretching vibration (at 1412 cm−1) and N–H deformation vibration (at 1412 cm−1) in ATBA monomer (Figure 1B). Meanwhile, evidential intensity decrease at 1491 cm−1 and redshift change from 1468 cm−1 to 1462 cm−1 were observed in the characteristic band of C–H deformation variation, which demonstrated that the C–H bonds in the carbocyclic ring skeleton of InsP6 salt were also influenced by the intensive complexation between ATBA and InsP6.To gain more detailed information of this evidential binding, 1H NMR titration experiment was performed (Figure 1C). Only with the addition of 0.2 equivalent of InsP6 salt, the amide and carboxyl proton signals of ATBA monomer disappeared, accompanying with remarkable chemical shift changes of alkene proton signals, which were further confirmed by 1H−13C correlation NMR spectroscopy (Figure


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1D and 1E). These data indicated that the binding of ATBA with InsP6 was mainly driven by multiple hydrogen bonding interactions between the thiourea or carboxyl groups in ATBA and phosphate group in InsP6 salt. 3.2. Conformational Transition of PNI-co-ATBA0.2 Polymer Chains. To improve the binding affinity of ATBA toward InsP6 and further amplify this recognition signal, ATBA was copolymerized with PNIPAAm through the SI–ATRP method,46,47 generating a PNI-co-ATBA0.2 thin film (film thickness: approximately 18 nm, Figure S1 in the SI) on the surface of Au–coated quartz–crystal (QC) resonator. Subsequently, dynamic adsorption behaviors of InsP6 on the PNI-co-ATBA0.2 surface was monitored using a quartz crystal microbalance with dissipation (QCM-D), which simultaneously measured the real-time variation in resonance frequency (∆ƒ) and energy dissipation (∆D) when the mass adsorbed on an oscillated piezoelectric crystal changes.40 As shown in Figure 2A, InsP6 displayed a rapid dynamic adsorption on the PNI-co-ATBA0.2 surface with a maximal ∆ƒ of –27.5 Hz after 2 min, corresponding to an adsorption quantity of 162 ng·cm−2 according to the Sauerbrey equation.48 By comparison, when the PNIPAAm homopolymer or ATBA monolayer modified QC resonator was introduced in the control experiment, the frequency change caused by InsP6 adsorption (∆ƒ: 0.6 Hz or 3.9 Hz, respectively) was substantially lower than that on the PNI-co-ATBA0.2 surface. The negligible ∆ƒ obtained on the PNIPAAm film indicated that the InsP6-adsorption capacity of copolymer film mainly originated from the aforementioned complexation of ATBA with InsP6, rather than physical or electrostatic InsP6 adsorption.40 On the other hand, substantially higher ∆ƒ obtained on PNI-co-ATBA0.2 than that on ATBA monolayer validated our design strategy, the introduction of polymeric matrix provided sufficient binding sites for continuous adsorption of InsP6.


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In addition to the time dependence frequency curve, QCM-D also provides energy dissipation curve, which records real-time information about the variation in viscoelasticity and thickness of the copolymer film.49 As illustrated by the dashed lines in Figure 2A, remarkable dissipation increase (∆D: 5.9 × 10−6) was observed for InsP6 adsorption on the PNI-co-ATBA0.2 film only. According to the classical QCM adsorption theory,40 this data demonstrated an evidential increase in viscoelasticity and thickness of the PNI-co-ATBA0.2 film after interaction with InsP6, suggesting that the copolymer chains stretched into relaxed and swollen states. This result was further verified by morphological changes of the copolymer film discerned using atomic force microscopy (AFM).50 As shown in Figure 2C and 2D, the copolymer film expanded considerably after being immersed in an InsP6 solution (10 µmol·L−1) for 10 min, displaying a clear surface morphology transition from a smooth state (Rq: 2.35 nm) to a notably rough state (Rq: 5.36 nm). Meanwhile, through alternate treatment with InsP6 solution (10 µmol·L−1) and pure water, the copolymer thin film exhibited reversible surface wettability switching between hydrophobic state (CA: 82°) and relatively hydrophilic state (CA: 54°),42 as shown in Figure 2B. This revealed another attractive feature of this copolymer film—excellent reversibility in response to the InsP6 adsorption, which would largely facilitate the development of relevant bio-device.


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Figure 2. (A) Time dependence of frequency (solid) and dissipation (dash) changes during InsP6 (10 µmol·L−1 in pure water) adsorption on the ATBA monolayer (black), PNIPAAm homopolymer (red), or PNI-co-ATBA0.2 copolymer (blue) modified QC resonator surfaces at 20 °C. (B) Contact angle (CA) cycling experiment, in which the copolymer film was alternately treated with InsP6 solution (10 µmol·L−1) for 10 min or with pure water for 10 min at 20 oC, respectively, followed by drying with nitrogen gas before each measurement. All data are shown as mean ± standard error (n = 3). (C, D)


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AFM images of PNI-co-ATBA0.2 polymer film before (C) and after (D) treatment with the InsP6 solution (10 µmol·L−1) at 20 °C and the corresponding section profiles along the green lines. Rq is an abbreviation of root mean square roughness. Insets: water droplet profiles before and after immersion of the PNI-co-ATBA0.2 film in InsP6 solution (10 µmol·L−1) for 10 min at 20 oC.

Based on the above analysis, a possible model was proposed for the globule–to–coil conformational transition of the copolymer chains in response to InsP6 adsorption, as shown in Figure 3A. Initially, owing to multiple hydrogen bonding interactions between thiourea groups in ATBA and carbonyl groups in NIPAAm as described by their binding model in Figure 3B (result of quantum chemistry calculation51), the copolymer chains maintained a globule state, contributing to a contracted copolymer film. When the copolymer chains were exposed to the InsP6 solution, intensive and multisite complexation among several ATBA residues and an InsP6 would happen sequentially (Figure 3C). This presumption was confirmed by the results of isothermal titration calorimetric (ITC) experiment (Figure 3D), in which the sequential binding sites model with four identical sites yielded the best ﬁtting of ITC curves (Figure 3E), accompanying with a remarkable exothermic process.52 This competitive binding of ATBA with InsP6 destroyed the initial hydrogen bonding network constructed by ATBA and NIPAAm, which triggered a rapid polymer conformation transition from globule to coil state, resulting in evidential changes in the surface morphology, wettability and viscoelasticity of the copolymer film. These results illustrated the significant advantages of our copolymer design, which affords an ideal platform for the sensitive recognition of InsP6


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and the efficient amplification of this slight signal into the macroscopic effects on the material surface.53


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Figure 3. (A) Graphic illustration of potential reversible globule–to–coil conformational transition of PNI-co-ATBA0.2 copolymer chains triggered by InsP6 adsorption and desorption. (B, C) Optimized interaction model of N-isopropylacrylamide (NIPAAm) with ATBA (B), and model of InsP6 with ATBA (C), obtained from quantum chemistry calculation [Gaussian, density function theory (DFT), at 6-311g level of theory]. Calculated hydrogen bonds are indicated by green dashed lines with different bond lengths. (D, E) Isothermal titration calorimetric data for the titration of PNI-co-ATBA0.2 solution (1.0 mmol·L−1) with the additions of various equivalents of InsP6 (20 mmol·L−1) in DMSO. In E, the red line denotes a non–linear fitting curve using a sequential binding sites model (N = 4).

3.3. Construction and characterization of PNI-co-ATBA0.2 modified nanochannels. Furthermore, to construct biomimetic ion channel, the developed InsP-responsive copolymer was grafted onto the PAA membrane with multiple nanochannels54 (average pore size: 80–100 nm) through SI–ATRP. The copolymer–modified PAA membrane was characterized through scanning electron microscopy (SEM), thermal gravimetric analysis (TGA), electrical measurement, X-ray photoelectron spectrometry (XPS), and infrared (IR) spectroscopy. The SEM images (Figure 4A and 4B) demonstrated that after being modified with the copolymer brushes,55 the porosity of the PAA membrane decreased considerably with average diameter of nanopores changing from 80–100 nm to 55–70 nm. In comparison with the bare membrane, signals of N1s and S2p with binding energies of 399.5 eV and 162.4 eV were only observed in the XPS spectrum of the copolymer–modified PAA membrane (Figure 4C and 4D, and Figure S2 in the SI), while the characteristic vibration peaks of C=O (1742, 1690, and 1645 cm−1) and –


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CS–NH– (1550, 1514, and 1464 cm−1) were only present in the FT–IR spectrum of the copolymer–modified membrane (Figure S3 in the SI). The appearance of these characteristic signals indicated that the PNI-co-ATBA0.2–modified PAA membrane had been successfully prepared. Moreover, a grafting rate of approximately 16% was determined by the TGA data56 (Figure 4E).To test the ion transport properties of the prepared nanochannels, PAA membrane (before or after copolymer modification) was mounted in between a two–compartment electrochemical cell (Figure S4 in the SI),44 and ionic current across the nanochannels was measured with NaCl solution (0.1 mol·L−1) serving as the electrolyte.57 As shown in Figure 4F, the immobilization of the copolymer with an average thickness of 18 nm remarkably reduced the average diameter of the PAA nanochannels, resulting in an obvious decrease in the transmembrane ionic current. Under this condition, the prepared PAA membrane maintained good permeability for ion transport with an ionic current of 26.4 µA measured by a picoammeter.


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Figure 4. (A, B) SEM images of PAA membrane before (A) and after (B) PNI-coATBA0.2 modification. Insets: high-magnification SEM images of a single nanochannel. (C, D) Narrow scan XPS spectra of PAA membrane before (black) and after (red) copolymer modification: (C) N1s; (D) S2p. (E) TGA curves of the PAA membranes before (black) and after (red) copolymer modification. (F) Voltage-dependent


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transmembrane ion current curves of the PAA membranes in 0.1 mol·L−1 NaCl aqueous solution before (black) and after (red) copolymer modification at 20 oC.

To evaluate the responsiveness of this multi-nanochannel system toward InsP6, the transmembrane ionic currents were measured after the bare, PNIPAAm or PNI-coATBA0.2 modified PAA membrane was exposed to the NaCl solution (0.1 mol·L−1) containing diverse concentrations of InsP6 for 10 min. Figure 5A illustrates the InsP6 concentration dependence of the ionic current (at +0.2 V) change ratio [defined as (I − I0)/I0, where I0 is the initial current] for the bare, PNIPAAm or PNI-co-ATBA0.2 modified PAA membrane. The ionic current sharply decreased by 27% after the copolymer– modified membrane was immersed in the electrolyte containing only 10 µmol·L−1 InsP6. Under the same condition, the current change ratio was negligible for the bare or PNIPAAm–modified PAA membrane. Notably, even when the concentration of InsP6 was only 1 nmol·L−1, the decrease of ionic current (15%) was still evidential and readily detected. Additional electrical tests contributed to a satisfactory linear relationship between the ionic current change ratio and the concentration of InsP6 over a wide range from 1 nmol·L−1 to 10 µmol·L−1, which revealed the excellent responsiveness of our nanochannel system toward InsP6, as well as its good potential in InsP6 quantitative analysis. When CaCl2 solution (0.1 mol·L−1) was employed as the electrolyte in the control experiments, similar linear relationship between the ionic current change ratio and the concentration of InsP6 was observed (Figure S5 in the SI), indicating that our nanochannels displayed no evidential selectivity toward different ionic species. Moreover, we repeatedly measured the transmembrane ionic current at +0.2 V after alternately


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immersing the copolymer–modified membrane in a 0.1 mol·L−1 NaCl solution containing InsP6 (10 µmol·L−1) for 10 min or in the NaCl solution (0.1 mol·L−1) for 10 min. As shown in Figure 5B, when the InsP6 molecules were removed, the ionic current could still recover to the original value even after six cycles, demonstrating the good reversibility of the ionic gating behaviors of the copolymer–modified nanochannels.

Figure 5. (A) InsP–concentration dependence of ionic current (at +0.2 V) change ratio of bare nanochannels (black), PNIPAAm (blue) or PNI-co-ATBA0.2 (red) modified


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nanochannels in 0.1 mol·L−1 NaCl solutions containing InsP6 (concentrations range from 1 nmol·L−1 to 10 µmol·L−1) at 20 oC. (B) Ionic current cycling measurement, in which the PNI-co-ATBA0.2–modified membrane was alternately immersed in a 0.1 mol·L−1 NaCl solution containing InsP6 (10 µmol·L−1) for 10 min or immersed in the NaCl solution (0.1 mol·L−1) for 10 min at 20 oC, respectively, before each electrical measurement. All above data are shown as mean ± standard error (n = 3). (C, D) AFM images of PNI-co-ATBA0.2 modified PAA membrane surface before (C) and after (D) treatment with the InsP6 solution (10 µmol·L−1) for 10 min at 20 oC, and the corresponding section profiles along the green lines.

In combination of the mechanism analysis of the copolymer conformational transition (Figure 3A–C), we reasonably presumed that the PNI-co-ATBA0.2 copolymer immobilized on the inner surface of PAA nanochannels occurred remarkable expansion after complexation with InsP6, leading to a sharp decrease in the size of nanochannels,58 which further decreased the ionic flux through the nanochannels. This presumption was verified by AFM observation of the PNI-co-ATBA0.2 modified PAA membrane surface before and after being immersed in an InsP6 solution (10 µmol·L−1). As shown in Figure 5C, clear honeycombed nanopores were observed with numerous polymeric beads immobilized both on the surface of PAA membrane and on the inner surface of these nanopores. After treatment with InsP6 solution, the initially well-defined nanostructures became substantially indistinct (Figure 5D), which could be attributed to the obvious expansion of the polymeric beads. According to the large-scale statistical analysis, the average PAA pore size significantly decreased from 65 nm to less than 25 nm, which


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resulted in a PAA membrane with lower permeability, and would induce a decrease in transmembrane ionic current. It is worth mentioning that InsP6 adsorption–triggered wettability change of the polymeric film might improve the hydrophilicity of inner surface of the nanochannel, which would increase the ionic flux through the channel,55 and lead to an increased ionic current. Considering the ionic current was finally decreased upon the addition of InsP6, the conformational change of polymer chains should be the dominant factor that resulted in ionic current changes. In addition to the sensitive response to the focused InsP6 molecule, the copolymer– modified PAA membrane displayed satisfactory discrimination capacity among Ins(1,3)P2, Ins(1,3,5)P3 and InsP6. Even when the concentrations of diverse InsPs were as low as 1 n mol·L−1, the modified nanochannels still displayed evidentially distinct ionic current decreases toward InsP2, InsP3, and InsP6 adsorption, the corresponding change ratios were 5.6%, 10.1% and 14.0%, respectively (Figure 6C). Distinct ionic current changing curves induced by the adsorption of different InsPs (Figure 6A and 6B) also proved this discrimination capacity, which could be attributed to the gradually increased binding affinity of ATBA units toward InsPs with more phosphate groups. It is worth mentioning that in the control experiment, the bare or PNIPAAm―modified nanochannels had negligible and unbiased responses to all these InsPs, which further validated our design strategy for InsP–responsive copolymer. Moreover, to evaluate the selectivity of this nanochannel system toward InsPs, other four molecules containing phosphate groups [i.e. phenylphosphonic acid (PPA), phosphoric acid (PA), adenosine 5’-diphosphate (ADP) and adenosine 5’-triphosphate (ATP)] were used to perform the control experiments. As shown in Figure 6D, even the most marked current decrease ratio induced by ATP


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addition (1 nmol·L−1) was only 2.3%, which was less than half of that induced by the addition of 1 nmol·L−1 InsP2 (5.6%). This data indicated that among these model molecules containing phosphate groups, our nanochannel system displayed a preferential response toward InsPs, which may facilitate its application in InsP detection.

Figure 6. (A, B) InsP–concentration dependence of ionic current (at +0.2 V) change ratio of bare nanochannels (black), PNIPAAm (blue) or PNI-co-ATBA0.2 (red) modified nanochannels in a NaCl solutions (0.1 mol·L−1) containing InsP3 (A) or InsP2 (B) (concentrations range from 1 nmol·L−1 to 10 µmol·L−1) at 20 oC. (C) Comparative analysis of current change ratios of the bare nanochannels (black), PNIPAAm (blue) or PNI-co-ATBA0.2 (red) modified nanochannels in a 0.1 mol·L−1 NaCl solution at 20 oC upon the addition of 1 nmol·L−1 InsP2, InsP3, or InsP6, respectively. (D) Comparative


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analysis of current change ratios of the PNI-co-ATBA0.2 modified nanochannels in a 0.1 mol·L−1 NaCl solution at 20 oC upon the addition of 1 nmol·L−1 InsP6 (red), ATP (orange), ADP (yellow), PPA (olive) or PA (blue), respectively. All data are shown as mean ± standard error (n = 3).

4. CONCLUSIONS By mimicking biological Ca2+ channels, we successfully developed an InsP–actuated nanochannel system, the gating behaviors of which could be precisely modulated by structurally dynamic copolymers that show macroscopic responses on exposure to the InsP solutions. Trace amount of InsP molecules across the nanochannel system could be sensitively recognized and captured, accompanying with remarkable variation in the transmembrane ionic current and clear discrimination among InsP2, InsP3 and InsP6. Compared with the mainstreamed isotope labeling and mass spectrometry–based strategies for InsPs analysis that usually involve in complicated pretreatment and expensive instruments,59,60 this method displays its unique advantages in more rapid, high-efficiency and dynamic InsP detection, and in fast discrimination of different InsP species.61 Moreover, this work well conforms to the burgeoning trend in biomimetic materials design that exploits the dynamic bonds (e.g., multiple hydrogen bonds) to construct smart bio-devices with high selectivity, controllability and reproducibility.62-64 In particular, this study demonstrates the feasibility of utilizing biomolecule–responsive polymers65, 66 in building artificial signaling biomolecule–gated ion channels, whose gating behavior is more like that of biological ion channels. It is mentionable that a big gap between synthetic responsive polymers and natural protein/peptide receptors still exists in sensing specificity, response speed, and


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accuracy of conformational transitions that may greatly influence the gating of ion channels. Therefore, it’s just a beginning for the construction of artificial biomolecule–gated ion channels, and substantial efforts should be spent in designing more specific biomolecule receptors and highly reversible polymer platform,67 as well as the converge of these substrate materials with elaborate nanochannels or nanopores.68-70 ASSOCIATED CONTENT Supporting Information. Materials and instruments, details for the synthesis and characterization of ATBA and fluorescein-labeled ATBA, 1H NMR titration tests, ITC experiments, AFM measurements, illustration of the electrical measurement and reference materials. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. (G.Q.) *E-mail: [email protected]. (T.S.) Author Contributions §

Q.L. and Q.T. contributed equally to this work. Q.L. designed and synthesized the copolymers

and performed the QCM adsorption experiment; Q.T. prepared the copolymer–modified PAA membrane and performed the electrochemical tests. Z. C. and S. Z. helped the PAA membrane preparation and electrochemical analysis. G.Q., T. S. and Q.L. analyzed the experimental data and wrote this paper. All authors commented on the final draft of the manuscript and contributed to the analysis and interpretation of the data.


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Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We thank the Major State Basic Research Development Program of China (973 Program, Grant 2013CB933002), the National Natural Science Foundation of China (51473131, 21275114, 51533007, 51521001 and 21775116), China National Funds for Distinguished Young Scientists (51325302) and Program for Changjiang Scholars and Innovative Research Team in University (IRT_15R52). G.Q. acknowledges Hubei Provincial Department of Education for financial assistance through the “Chutian Scholar” Program and Hubei Provincial Natural Science Foundation of China (2014CFA039). REFERENCES (1) Weng, G. Z.; Bhalla, U. S.; Iyengar, R. Complexity in Biological Signaling Systems. Science 1999, 284, 92–96. (2) Kiel, C.; Yus, E.; Serrano, L. Engineering Signal Transduction Pathways. Cell 2010, 140, 33–47. (3) Delmas, P.; Coste, B. Mechano-Gated Ion Channels in Sensory Systems. Cell 2013, 155, 278–284. (4) James, Z. M.; Borst, A. J.; Haitin, Y.; Frenz, B.; DiMaio, F.; Zagotta, W. N.; Veesler, D. CryoEM Structure of a Prokaryotic Cyclic Nucleotide-Gated Ion Channel. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 4430–4435. (5) Lev, B.; Murail, S.; Poitevin, F.; Cromer, B. A.; Baaden, M.; Delarue, M.; Allen, T. W. String Method Solution of the Gating Pathways for a Pentameric Ligand-Gated Ion Channel. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, E4158–E4167.


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