Andrés F Molina-Osorio

David Cheung

Colm O'dwyer

Andrew A Stewart

Manuel Dossot

Grégoire Herzog

Micheál D Scanlon

Self-assembly of Porphyrin Nanostructures at the Interface between Two Immiscible Liquids

One of the many evolved functions of photosynthetic organisms is to synthesize light harvesting nanostructures from photoactive molecules such as porphyrins. Engineering synthetic analogues with optimized molecular order necessary for the efficient capture and harvest of light energy remains challenging. Here, we address this challenge by reporting the self-assembly of zinc(II) meso-tetrakis(4-carboxyphenyl)porphyrins into films of highly ordered nanostructures.

The self-assembly process takes place selectively at the interface between two immiscible liquids (water|organic solvent), with kinetically stable interfacial nanostructures formed only at pH values close to the pKa of the carboxyphenyl groups. Molecular dynamics simulations suggest that the assembly process is driven by an interplay between the hydrophobicity gradient at the interface and hydrogen bonding in the formed nanostructure. Ex situ XRD analysis and in situ UV/vis and steady state fluorescence indicates the formation of chlathrate type nanostructures that retain the emission properties of their monomeric constituents. The self-assembly method presented here avoids the use of acidic conditions, additives such as surfactants and external stimuli, offering an alternative for the realization of light-harvesting antennas in artificial photosynthesis technologies.

1.

INTRODUCTION

Photosynthetic organisms universally exploit antenna systems to capture high energy photons and funnel this excitation energy toward a coupled reaction centre. [START_REF] Scholes | Lessons from Nature about Solar Light Harvesting[END_REF][START_REF] Mirkovic | Light Absorption and Energy Transfer in the Antenna Complexes of Photosynthetic Organisms[END_REF] Self-assembled molecular antennae consisting of multi-layers of chromophores, such as porphyrins, can potentially function as high efficiency light harvesters due to their exceptionally high molar absorption coefficients (10 5 cm -1 M -1 ). [START_REF] Otsuki | Supramolecular Approach towards Light-Harvesting Materials Based on Porphyrins and Chlorophylls[END_REF] However, to mimic the evolved efficiency of these antennas and avoid "concentration quenching" of the excited state at disordered trap sites, [START_REF] Otsuki | Supramolecular Approach towards Light-Harvesting Materials Based on Porphyrins and Chlorophylls[END_REF] the supramolecular packing of the individual chromophores must be precisely controlled and show long-range molecular order. [START_REF] Magna | The Assembly of Porphyrin Systems in Well-Defined Nanostructures: An Update[END_REF][START_REF] Medforth | Self-Assembled Porphyrin Nanostructures[END_REF] Molecular self-assemblies at "soft" liquid|air or immiscible liquid|liquid interfaces can exhibit the required macroscale long-range order. [START_REF] Bigioni | Kinetically Driven Self Assembly of Highly Ordered Nanoparticle Monolayers[END_REF] These soft interfaces are exceptionally smooth, and have no inherent defects leading to an unrivalled macroscale uniformity in molecule-interface interactions. [START_REF] Michael | Molecular Dynamics Simulation of the Water|nitrobenzene Interface[END_REF][START_REF] Strutwolf | Probing Liquid|liquid Interfaces Using Neutron Reflection Measurements and Scanning Electrochemical Microscopy[END_REF] By contrast, the grain boundaries, step defects and edge sites always present at solid|liquid interfaces can impede diffusion of adsorbed molecules, trapping them in local energy minima as molecules stick to defect sites. [START_REF] Mendes | Self-Assembly in Nature: Using the Principles of Nature to Create Complex Nanobiomaterials[END_REF][START_REF] Grzybowski | Self-Assembly: From Crystals to Cells[END_REF] The uniform templating of adsorbed molecules at liquid|air or immiscible liquid|liquid interfaces has been exploited to create a variety of porphyrin assemblies, [START_REF] Rong | Porphyrin Assemblies through the Air/water Interface: Effect of Hydrogen Bond, Thermal Annealing, and Amplification of Supramolecular Chirality[END_REF][START_REF] Ponce | Spectroscopic and Structural Studies of a Surface Active Porphyrin in Solution and in Langmuir-Blodgett Films[END_REF][START_REF] Babu | Self-Organization of Polar Porphyrinoids[END_REF][START_REF] Numata | Self-Assembly of Amphiphilic Molecules in Droplet Compartments: An Approach toward Discrete Submicrometer-Sized One-Dimensional Structures[END_REF][START_REF] Xie | Evolution of Nanoflowers and Nanospheres of Zinc Bisporphyrinate Tweezers at the Air/water Interface[END_REF][START_REF] Qiu | Interfacial Assemblies of Atypical Amphiphilic Porphyrins: Hydrophobicity/hydrophilicity of Substituents, Annealing Effects, and Supramolecular Chirality[END_REF] yet a facile and robust route to create highly-ordered porphyrin nanostructures remains challenging.

Here we describe the self-assembly of light harvesting nanostructures from readily available, water-soluble and symmetrically substituted porphyrins at mild pH conditions. Thus, we avoid acidic conditions (that would lead to the expulsion of the central metal ion), [START_REF] Nagatani | Direct Spectrophotometric Measurement of Demetalation Kinetics of 5,10,15,20-tetraphenylporphyrinatozinc(II) at the Liquid-liquid Interface by a Centrifugal Liquid Membrane Method[END_REF] as well as the use of synthetically challenging amphiphilic porphyrin molecules, [START_REF] Lin | In Situ Measurement of the Supramolecular Chirality in the Langmuir Monolayers of Achiral Porphyrins at the Air/aqueous Interface by Second Harmonic Generation Linear Dichroism[END_REF] and more complicated routes using additives (e.g., divalent cations or surfactants) [START_REF] Qian | Multiporphyrin Array from Interfacial Metal-Mediated Assembly and Its Langmuir-Blodgett Films[END_REF][START_REF] Cheung | How Stable Are Amphiphilic Dendrimers at the Liquid-liquid Interface?[END_REF][START_REF] Guo | Porphyrin Nanoassemblies via Surfactant-Assisted Assembly and Single Nanofiber Nanoelectronic Sensors for High-Performance H2O2 Vapor Sensing[END_REF] or external stimuli (e.g., electric fields). [START_REF] Nagatani | Adsorption and Aggregation of Meso-tetrakis(4-Carboxyphenyl)porphyrinato Zinc(II) at the Polarized water|1,2-Dichloroethane Interface[END_REF][START_REF] Yamamoto | Potential-Induced Aggregation of Anionic Porphyrins at Liquid|liquid Interfaces[END_REF][START_REF] Yamamoto | Potential-Dependent Adsorption and Orientation of Meso-Substituted Porphyrins at Liquid|liquid Interfaces Studied by Polarization-Modulation Total Internal Reflection Fluorescence Spectroscopy[END_REF] We chose zinc(II) meso-tetrakis(4-carboxyphenyl)porphyrin (ZnTPPc) as a prototypical model system to demonstrate this new means of self-assembly at an immiscible aqueous|organic interface.

Simply contacting aqueous ZnTPPc solutions prepared in citrate buffer at pH values between 5.1 and 6.0 with a neat, immiscible organic phase of -trifluorotoluene (TFT) lead to the immediate formation of films of porphyrin nanostructures. We rationalise our findings using molecular dynamics (MD) simulations that highlights the key role of the hydrophobicity gradient at the immiscible aqueous|organic interface, and carboxylic acid-carboxylate hydrogen bonding interactions in the formed nanostructure. The latter were found to be maximised at pH values close to the pKa of the carboxylic groups within the nanostructure (pH 5.8), [START_REF] Maiti | J-and H-Aggregates of Porphyrin-Surfactant Complexes: Time-Resolved Fluorescence and Other Spectroscopic Studies †[END_REF] leading to kinetically stable interfacial nanostructures. The presence of multi-layers with strong visible light absorption, and the crystalline macroscale long-range molecular order in the porphyrin nanostructure, suggests these films as promising light-harvesting antennae in artificial photosynthetic technologies.

2.

EXPERIMENTAL SECTION 2.1. Chemicals. All reagents were used as received without further purification. Mesotetrakis(4-carboxyphenyl)porphyrin (H2TPPc, ≥98%) and its zinc(II) derivative (ZnTPPc, ≥98%)

were obtained from Porphychem. Lithium hydroxide (LiOH, ≥98%), citric acid (H3Cit, ≥99.5%), decamethylferrocene (97%), and 1,2-dichloroethane (DCE, ≥99.0%) were purchased from Sigma-Aldrich, and α,α,α-trifluorotoluene (TFT, ≥99%) from Acros Organics. All aqueous solutions were prepared using Milli-Q® deionized water (18.2 MΩ•cm). Aqueous solutions of ZnTPPc were prepared by directly dissolving the solid in the lithium citrate buffer pre-adjusted to the desired pH, followed by sonication of the solution for three minutes. Initially, H2TPPc was insoluble in the buffer at neutral pH. Therefore, the solid was dissolved first in LiOH and the pH subsequently adjusted with H3Cit.

RESULTS AND DISCUSSION

Triggering the formation of interfacial nanostructures. The selective formation of

ZnTPPc nanostructures at the interface between water and TFT was observed upon contacting a ZnTPPc aqueous solution with neat TFT. A yellow/green colour was observed at the water|TFT interface within minutes, easily distinguishable from the purple colour of the bulk ZnTPPc aqueous solution, and associated with the formation of porphyrin interfacial nanostructures (Por-INs) (Figure 1a). Self-assembly was observed only at pH values where the ratio between neutral, protonated (H4[ZnTPPc]) and tetra-anionic, deprotonated ([ZnTPPc] 4-) species was close to 1 (pH= pKa COOH = 5.8), see Figure S5. Mono-, di-and triprotonated ZnTPPc species may also be present. However, only one pKa was observed for the potentiometric titration of ZnTPPc with hydrochloric acid (Figure S6) suggesting that the different pKa values of the carboxylic groups fall within a very narrow interval of pH. Nevertheless, it must be noted that the interfacial protonation equilibria is complex. Despite using a buffered aqueous electrolyte, an implicit assumption cannot be made that the pH at the liquid|liquid boundary, where nanostructure formation is initiated, is the same as in the bulk. Numerous studies have shown that local interfacial pH values can be several units different from the bulk aqueous phase. [START_REF] Zhao | New Method for Determination of Surface pKa Using Second Harmonic Generation[END_REF][START_REF] Xiao | Probing the Proton Excess at Interfaces by Second Harmonic Generation[END_REF][START_REF] Castro | Energetics of Adsorption of Neutral and Charged Molecules at the Air/water Interface by Second Harmonic Generation: Hydrophobic and Solvation Effects[END_REF] Furthermore, the pKa of the carboxyphenyl groups may differ from their bulk values, observed previously for other acid/base equilibria at the water|air interface. [START_REF] Eisenthal | Liquid Interfaces Probed by Second-Harmonic and Sum-Frequency Spectroscopy[END_REF][START_REF] Tamburello-Luca | Optical Surface Second Harmonic Generation Study of the Two Acid/base Equilibria of Eosin B at the Air/water Interface[END_REF] MD simulations show that the hydrophilic, anionic [ZnTPPc] 4-species prefer to sit in water at a distance of ~3-5 Å above the interface due to their hydrophilic nature (Figure 1b), and suggest that initially, hydrophobic neutral H4[ZnTPPc] species accumulate at the interface driven by the hydrophobicity gradient at the immiscible liquid|liquid interface (Figure 1b and Figure S7). This interfacial layer acts as a template structure for the [ZnTPPc] 4-species to adsorb via carboxylic acid-carboxylate hydrogen-bonding and π-π interactions. In this manner, a crystalline film of ZnTPPc nanostructures builds up layer-by-layer at the interface. Akin to the clathrate crystals of ZnTPPc developed by Goldberg and coworkers, [START_REF] Diskin-Posner | From Porphyrin Sponges to Porphyrin Sieves: A Unique Crystalline Lattice of Aquazinc tetra(4-Carboxyphenyl)porphyrin with Nanosized Channels[END_REF][START_REF] Diskin-Posner | Crystal Engineering of 2-D and 3-D Multiporphyrin Architectures -The Versatile Topologies of Tetracarboxyphenylporphyrin-Based Materials[END_REF][START_REF] Diskin-Posner | New Effective Synthons for Supramolecular Self-Assembly of Meso-Carboxyphenylporphyrins[END_REF][START_REF] George | Self-Assembly of Supramolecular Porphyrin Arrays by Hydrogen Bonding: New Structures and Reflections[END_REF][START_REF] Goldberg | Crystal Engineering of Metalloporphyrin Molecular Sieves and Zeolite Analogues[END_REF] the strength of each individual hydrogen bond or π-π interaction may be insubstantial, but the cooperative effect allows the net enthalpies of these multivalent interactions to cumulatively rival the strength of a covalent bond and stabilise the Por-IN. Ex situ scanning electron microscopy (SEM) images reveal the thickness of the film of ZnPor-INs to be approximately 135 (± 5) nm (Figure 1c). Control experiments and MD simulations demonstrated that ZnTPPc and free-base H2TPPc molecules are kinetically stable in solution at pH 5.8, and do not undergo spontaneous bulk aggregation in the concentration range studied (Figure S8). This indicates that the Por-INs form only by self-assembly in situ at the water|TFT interface. Thus, to achieve selective Por-IN formation, the pH of the aqueous solution must be controlled across a narrow pH range between 5.1 and 6.0 (Figure 1d). More alkaline conditions inhibit formation of the Por-INs due to electrostatic repulsion between tetra-anionic porphyrins. More acidic conditions induce the spontaneous formation of aggregates in the bulk aqueous phase (Figure S9). This pH dependency of Por-IN formation indicates that cooperative H-bonding is key for self-assembly of the nanostructure. This finding is in line with a detailed examination of the photoelectrochemistry of ZnTPPc and decamethylferrocene at the water|1,2-dichloroethane (DCE) interface by Girault and co-workers. [START_REF] Jensen | Photoinduced Electron Transfer at Liquid|liquid Interfaces. Part IV. Orientation and Reactivity of Zinc tetra(4-Carboxyphenyl) Porphyrin Self-Assembled at the water|1,2-Dichloroethane Junction[END_REF] Based on the photocurrent dependence on the angle of polarisation under total internal reflection, they concluded that the spontaneous 2D coverage of ZnTPPc molecules at the interface is pH dependent, sharply decreasing above pH 6. While no interfacial nanostructures were observed by Girault and co-workers, [START_REF] Jensen | Photoinduced Electron Transfer at Liquid|liquid Interfaces. Part IV. Orientation and Reactivity of Zinc tetra(4-Carboxyphenyl) Porphyrin Self-Assembled at the water|1,2-Dichloroethane Junction[END_REF] their data strongly correlates with our observations that spontaneous 2D assembly is the initial step in the generation of 3D interfacial nanostructures.

Furthermore, nanostructure self-assembly is not restricted to a single immiscible biphasic system, with experiments showing that Por-INs also form selectively at the water|DCE interface (Figure S10).

The interfacial nanostructures represent a kinetically trapped metastable state rather than a thermodynamically stable state. The interfacial concentration of Por-INs was measured

as a function of the solution concentration of ZnTPPc and H2TPPc, respectively. Over the concentration range studied (0.5-100 µM), ZnTPPc adsorption followed a Brunauer-Emmet-Teller (BET) isotherm behaviour, whereas H2TPPc adsorption followed a linear isotherm behaviour (Figure 3). Using the BET model for liquid phase adsorption reported by Ebadi et al., [START_REF] Ebadi | What Is the Correct Form of BET Isotherm for Modeling Liquid Phase Adsorption?[END_REF] the isotherm obtained for ZnTPPc adsorption was fit to Eqn. 1:

Г = Г 𝑚 𝐾 1 𝐶 𝑒𝑞. (1-K 2 C eq. )(1-𝐾 2 𝐶 𝑒𝑞. +𝐾 1 𝐶 𝑒𝑞. ) (1)
in which 𝐾 1 and 𝐾 2 are the pseudo-equilibrium constants of adsorption to form the first and second layers of the ZnPor-INs, Г and Г 𝑚 are the equilibrium and monolayer porphyrin surface concentrations, respectively, and 𝐶 𝑒𝑞. is the equilibrium concentration of the porphyrin in solution.

Non-linear fitting determined Г 𝑚 as 13.08 (±0.94) nmolcm -2 , and 𝐾 1 and 𝐾 2 as 4.55 x 10 -2 (±0.25) and 8.79 x 10 -3 (±2.21 x 10 -4 ) Lµmol, respectively (95% confidence). Thus, the Gibbs free energy of adsorption (ΔG ads. ) of ZnTPPc molecules to form the first and second layers of the ZnPor-INs are 7.65 kJmol -1 and 11.72 kJmol -1 , respectively. These values suggest that the interfacial nanostructures represent a kinetically trapped metastable state rather than a thermodynamically stable state. In other words, the interfacial molecular self-assembly process is under kinetic control, and such out-of-equilibrium self-assembly systems are known to yield porphyrin nanostructures that are inaccessible through the spontaneous thermodynamic process. [START_REF] Fukui | Control over Differentiation of a Metastable Supramolecular Assembly in One and Two Dimensions[END_REF] The latter kinetic control has also been observed in supramolecular polymerization of porphyrins, [START_REF] Ogi | Living Supramolecular Polymerization Realized through a Biomimetic Approach[END_REF] and the self-assembly of polyelectrolytes, [START_REF] Wu | Non-Equilibrium Phenomena and Kinetic Pathways in Self-Assembled Polyelectrolyte Complexes[END_REF] perylenes, [START_REF] Tidhar | Pathway-Dependent Self-Assembly of Perylene Diimide/peptide Conjugates in Aqueous Medium[END_REF] and other types of nanostructures. 44 The difference in the adsorption isotherms for ZnTPPc and H2TPPc further emphasises the influence of the metal centre during porphyrin interfacial adsorption. Previous studies at an aqueous|dodecane interface demonstrated that the adsorption of an oil-soluble metalloporphyrin was highly dependent on the nature of the central metal ion due to the different water coordination geometries around different metals. [START_REF] Nagatani | Specific Adsorption of Metal Complexes of Tetraphenylporphyrin at Dodecane-Water Interface[END_REF] Also, the study by Girault and co-workers [START_REF] Jensen | Photoinduced Electron Transfer at Liquid|liquid Interfaces. Part IV. Orientation and Reactivity of Zinc tetra(4-Carboxyphenyl) Porphyrin Self-Assembled at the water|1,2-Dichloroethane Junction[END_REF] noted vide supra, concluded that there is a direct link between the surface coverage and orientation of ZnTPPc adsorbed at the interface. A dihedral angle between the porphyrin ring and the surface normal in the range of 60 to 75° was observed, depending on the bulk ZnTPPc concentration and the application of a Galvani potential difference at the liquid|liquid interface, and rationalized in terms of minimizing the contact of deprotonated carboxyl groups to the hydrophobic phase. [START_REF] Jensen | Photoinduced Electron Transfer at Liquid|liquid Interfaces. Part IV. Orientation and Reactivity of Zinc tetra(4-Carboxyphenyl) Porphyrin Self-Assembled at the water|1,2-Dichloroethane Junction[END_REF] We used MD simulations to quantify the influence of the presence or absence of Zn 2+ , and the protonation state, on the adsorption of ZnTPPc and H2TPPc at the water|TFT interface. For all model variants, there is a significant degree of orientational freedom (Figure S11), with all molecules adopting a range of orientations relative to the interface. This is reflected in the orientational angle probability distributions which have peaks at a low (<15°) angle but remain non-zero for all angles. (Figure S11). This suggests that the difference in the adsorption behaviour stems from different supramolecular packing modes within the Por-INs and not their interaction with the liquid|liquid interface.

Influence of nanostructure formation on the photophysical properties of the assembly.

In situ UV/vis absorption spectra of ZnTPPc in solution and ZnPor-INs are shown in Figure 4a and Additionally, in solution, ZnTPPc presented one main peak centred at 422 nm (Figure 4a and Table S1). This peak further indicates the monomeric nature of ZnTPPc under the experimental conditions used during this work. The latter is supported with the adherence of ZnTPPc in solution to the Beer-Lambert law across an extensive concentration range (Figure S12), and with previous studies. 46 S2 for peak assignments).

In situ steady-state fluorescence spectra was measured for an excitation wavelength (λexc.) of 413 nm (Figure 4b). Comparison of the emission profiles of ZnTPPc in solution and the ZnPor-INs shows that the Q(0,0) transient is slightly blue-shifted and Q(0,1) transition slightly red-shifted upon nanostructure formation. Following Kasha's exciton model, the ZnPor-INs can be classified as H-type structures (with a blue shifted max., see Figure 4a) where strong - overlap is expected.

- interactions are reported to lower exciton diffusion and fluorescence rates. [START_REF] Huijser | Exciton Diffusion and Interfacial Charge Separation in Meso-tetraphenylporphyrin/TiO2 Bilayers: Effect of Ethyl Substituents[END_REF][START_REF] Verma | Exciton Energy and Charge Transfer in Porphyrin Aggregate/semiconductor (TiO2) Composites[END_REF] However, energy transfer within these types of molecular assemblies is also affected by other parameters such as intermolecular distance, molecular packing orientation or molecular crystallinity. [START_REF] Ya-Rui | Theoretical Study of the Charge Transport Mechanism in π-Stacked Systems of Organic Semiconductor Crystals[END_REF] The importance of the latter is highlighted in several works where high carrier mobility is observed in structures with strong - stacking interactions. [START_REF] Ya-Rui | Theoretical Study of the Charge Transport Mechanism in π-Stacked Systems of Organic Semiconductor Crystals[END_REF][START_REF] Vohra | Efficient Inverted Polymer Solar Cells Employing Favourable Molecular Orientation[END_REF] For example, Vohra et al. [START_REF] Vohra | Efficient Inverted Polymer Solar Cells Employing Favourable Molecular Orientation[END_REF] reported crystalline lamellar structures of thiophene polymers with short - stacking distance as highly efficient active layers in solar cells. Thus, the retention of fluorescence emission after nanostructure formation demonstrates how the long-range molecular order in the nanostructure leads to diminished concentration quenching, a key attribute when designing light-harvesting antennae in artificial photosynthetic technologies. Additional dependence of the emission properties of these nanostructures on the excitation wavelength is presented in Figures S13 andS14.

Since ZnTPPc self-assembles in mild pH conditions, expulsion of Zn 2+ was avoided, as confirmed by analysis of the effect of nanostructure formation on the vibrational modes of the ZnPor-and H2Por-INs by ex situ Raman spectroscopy (Figure 4c). Prominent differences between the two are entirely consistent with previous comparisons of metallo-and free-base 4carboxyphenyl-substituted porphyrin Raman spectra (Table S2). [START_REF] Vlčková | Surface-Enhanced Resonance Raman Spectra of Free Base 5,10,15,20-tetrakis(4-Carboxyphenyl)porphyrin and Its Silver Complex in Systems with Silver Colloid: Direct Adsorption in Comparison to Adsorption via Molecular Spacer[END_REF][START_REF] Cotton | Surface-Enhanced Resonance Raman Scattering from Water-Soluble Porphyrins Adsorbed on a Silver Electrode[END_REF] The retention of Zn 2+ is a key advantage of the interfacial self-assembly method described over common aggregation methods in bulk solution at acidic conditions. The presence of the metal increases the inter-system crossing (ISC) rate constant, kISC, due to the heavy atom effect, increasing the probability of the forbidden S1→ T1 transition. From the T1 state, relaxation may occur via phosphorescence or charge transfer.

The long-lived (up to millisecond) excited triplet state lifetimes provides sufficient time for the excited state to efficiently interact with ground state quencher molecules. [START_REF] Fermín | Photoinduced Electron Transfer at Liquid/liquid Interfaces Part II. A Study of the Electron Transfer and Recombination Dynamics by Intensity Modulated Photocurrent Spectroscopy (IMPS)[END_REF] As a proof-of-concept, the photoactivity of the ZnPor-INs was demonstrated by mediating interfacial photo-induced electron transfer (PET) between redox species chemically confined to different sides of a liquid|liquid interface using the methodology pioneered by Girault and coworkers (Figure 5a). [START_REF] Jensen | Photoinduced Electron Transfer at Liquid|liquid Interfaces. Part IV. Orientation and Reactivity of Zinc tetra(4-Carboxyphenyl) Porphyrin Self-Assembled at the water|1,2-Dichloroethane Junction[END_REF][START_REF] Fermín | Photoinduced Electron Transfer at Liquid/liquid Interfaces Part II. A Study of the Electron Transfer and Recombination Dynamics by Intensity Modulated Photocurrent Spectroscopy (IMPS)[END_REF][START_REF] Fermín | Photoinduced Electron Transfer at Liquid/liquid Interfaces. Part III. Photoelectrochemical Responses Involving Porphyrin Ion Pairs[END_REF][START_REF] Fermín | Photoinduced Electron Transfer at Liquid/Liquid Interfaces. 1. Photocurrent Measurements Associated with Heterogeneous Quenching of Zinc Porphyrins[END_REF] The multi-layers of porphyrin in the ZnPor-INs floating at the interface function as light harvesters. Upon illumination, the generated triplet excited state was reductively quenched by electron transfer from decamethylferrocene, and the ground state regenerated by hole transfer from O2. The charge separation was accompanied by an electrical photocurrent through an external circuit, and the latter increased on application of a more positive Galvani potential difference (Figure 5b-c).

Control cyclic voltammograms (CVs) were carried out, in the dark and with chopped LED illumination, in the presence of the ZnPor-INs at the interface and decamethylferrocene in the TFT phase (Figure S15). Charge transfer peaks due to the adsorbed ZnPor-INs precluded the study of applied Galvani potentials less than -0.1 V (Figure S15). Decamethylferrocene is a relatively strong electron donor, capable of reducing dissolved O2, and consequently leading to a dark current at positive Galvani potentials with an onset of ~+0.15 V. The mechanism underlying this dark current has been identified previously as the reduction of O2 by first proton transfer to the organic phase, followed by formation of a decamethylferrocene-hydride. The latter then reacts with dissolved O2 in the TFT to generate a peroxyl radical species, HO2 • . [START_REF] Stockmann | Mechanism of Oxygen Reduction by Metallocenes near Liquid|liquid Interfaces[END_REF] However, as seen in Figure S15, the photocurrents obtained are superimposed upon this dark current and can be resolved by background subtraction of the dark current (as is the case for the photocurrent transients shown in Figures 5b andc).

A major future perspective outside the scope of this article is an in-depth exploration of PET at electrified liquid|liquid interfaces using this photoactive film. In particular, a detailed analysis on the potential dependence of the magnitude and line-shape of the photocurrent transient measurements is required and will lead to an elegantly simple system to realise a new approach to artificial photosynthesis entirely based on a self-assembled system.

CONCLUSIONS

The defect-free nature of the water|organic interface provides an ideal platform to self-assemble interfacial nanostructures with unique structural arrangements. In this Article, we report the selfassembly of interfacial nanostructures of zinc(II) meso-tetrakis(4-carboxyphenyl)porphyrin. The nanostructures are stabilised by cooperative hydrogen bonding and, due to the templating interaction of the interface with adsorbed porphyrin molecules, possess a highly ordered structure.

This approach uniquely harnesses the difference in hydrophobicity between the neutral protonated and tetra-anionic non-protonated versions of the porphyrin at pKa conditions, combined with the introduction of a hydrophobicity gradient to trigger interfacial self-assembly. We open a new avenue to kinetically stable porphyrin nanostructure formation under mild experimental conditions without the need for acidic pH, designer amphiphilic porphyrin molecules, aggregation-inducing additives or external triggers. The feasibility of using such nanostructures for light collection and harvesting was demonstrated in situ by measuring photocurrents associated with interfacial photoinduced electron transfer across the water|TFT interface.

ASSOCIATED CONTENT Supporting Information

Supporting experimental methods (in situ microscopy, molecular dynamic simulations, photocurrent transient measurements), methodology of porphyrin interfacial nanostructure

2. 5 .

 5 Photocurrent measurements with a DC illumination were performed using the LED driver

Figure 1 .

 1 Figure 1. Formation of porphyrin interfacial nanostructures. a) Optical images of the formation of Por-INs at the interface between water and TFT. b) Computed potential of mean force or free energy profiles for translation of ZnTPPc molecules across the water|TFT interface, averaged over

3. 2 .

 2 Molecular structure of the Por-INs. Transmission electron microscopy (TEM) and X-ray diffraction (XRD) analysis confirm the crystalline and layered nature of the porphyrin interfacial nanostructures. Ex situ TEM images with corresponding selected area electron diffraction (SAED) analysis, and patterns from ZnPor-INs (Figure 2a-c) were acquired after immobilization of the film on an amorphous hydrophilized glass substrate. The diffraction pattern for ZnPor-INs bears astriking resemblance to NAFS (nanofilm on a solid surface) structural models for liquid phase interfacial synthesis of highly ordered molecular nanosheets (Figure2a).[START_REF] Motoyama | Highly Crystalline Nanofilm by Layering of Porphyrin Metal-Organic Framework Sheets[END_REF] SAED and XRD estimate a 0.55-0.57 nm interplanar spacing between {110} planes, with a ~1.1 nm periodicity measured from HRTEM lattice fringes. The intense 220 reflection is consistent with a lack of layer-on-layer stacking order (due to non-registered stacking), but a highly crystalline nanosheet material is formed. The (200) reflections indicate strong axial coordination that is a distinct feature of layering along the c-axis normal to the support. Clearly discernible (hk0) reflections are consistent with a tetragonal unit cell with preferred growth orientation along the plane of the liquid|liquid interface (Figure2b,c). Experiments with nanostructures of the free base H2TPPc (H2Por-INs) demonstrate the key role of the central metal ion in enhancing the crystallinity of the film of porphyrin interfacial nanostructures. H2Por-INs grown in the same way also form a crystalline 2D layered material, and show a similar XRD pattern but with a more pronounced amorphous background and suppressed reflection intensity compared to the ZnPor-INs (Figure 2d,e). A schematic representation of the crystalline and interdigitated layered clathrate-type structure of the ZnPor-INs is illustrated in Figure 2f. This structure assumes interdigitation to account for the reduced unit cell spacing compared to a NAFS-1 or NAFS-2 tetragonal metalorganic nanosheet crystalline structure.

Figure 2 .

 2 Figure 2. Characterisation of ZnPor-INs and H2Por-INs ex situ by transmission electron microscopy (TEM) and X-ray diffraction (XRD). a) TEM image of ZnPor-INs transferred from the water|TFT interface to a copper grid with a holey carbon substrate, taken at 185 k magnification. The profile of the lattice fringing shown on the right is taken from the area marked by the red box in the TEM image. b) TEM image of ZnPor-INs, taken at 185 k magnification at an accelerating voltage of 300 kV and c) the corresponding diffraction pattern with a 40 μm selected area aperture and camera length of 580 mm. d) XRD pattern of the ZnPor-INs transferred to a silicon substrate. Patterns were acquired in ω-2θ geometry and clearly discernible (hk0) reflections confirms a tetragonal unit cell and dominant c-axis layering. e) XRD of the H2Por-INs. f) Schematic representation of the crystalline and layered clathrate-type structure of the ZnPor-INs. Black balls represent carbon, pink is zinc, blue are nitrogen, and green are oxygen.

Figure 3

 3 Figure 3 Adsorption isotherms obtained at 20 °C of ZnTPPc and H2TPPc at the water|TFT interface. Inset: Adsorption isotherm data for ZnTPPc at the water|TFT interface, highlighting the Brunauer-Emmet-Teller (BET) behavior

  Figure S12. The λmax. value of the Soret band for the ZnPor-INs is blue-shifted (from 422 to 413 nm with respect to ZnTPPc in solution). Perturbation of the electronic absorption spectra in terms of shifts and broadening of the Soret band, indicate the presence of multiple structural domains within the ZnPor-INs. Analogous to ZnTPPc in solution, ZnPor-INs display two Q bands, indicating that the porphyrin ring remains metallated with Zn 2+ and thus retains the D4h symmetry.

Figure 4 .

 4 Figure 4. Spectroscopic characterisation of the ZnPor-INs by in situ UV/vis absorbance and steady-state fluorescence, and ex situ Raman spectroscopy. Deconvolution of the Soret absorbance bands for a) ZnTPPc in solution and ZnPor-INs. b) Comparison of the fluorescence emission spectra of ZnTPPc in solution and ZnPor-INs for an excitation wavelength (λexc.) of 418 nm. c) Comparison of the Raman spectra of films of ZnPor-and H2Por-INs transferred from the water|TFT interface to a silicon substrate (see TableS2for peak assignments).

Figure 5 .

 5 Figure 5. Photoconversion at the interface between two immiscible liquids. a) Schematic of "soft-

2.3. Adsorption isotherms. Vials containing biphasic systems of ZnTPPc or H2TPPc in lithium

  Prior to imaging, the samples were sequentially rinsed with water and TFT, and dried under a stream of nitrogen gas. TEM images were acquired on a Thermo Fisher double aberration

	2.4. Molecular dynamics simulations. Simulations of interfacial adsorption and assembly were
	performed using a pre-equilibrated water|TFT interface, consisting water and TFT regions with
	Aldrich Bis(triphenylphosphoranylidene)ammonium tetrakis(pentafluorophenyl)borate (BATB) was and Boulder Scientific Company, respectively. -2 geometry with a Phillips Xpert PW3719 diffractometer using Cu Kα radiation (λ = 0.15418 nm, operation voltage 40 kV, current 40 mA). Patterns were also acquired in -2 geometry using a PANalytical X'pert PRO XRD. UV/vis absorbance spectra were collected in a Thermo Scientific Evolution 60S UV/vis spectrophotometer with illumination provided by a Xenon light source (accuracy ±0.8 nm). Steady-state fluorescence experiments were performed in a LS 55 Perkin Elmer Fluorescence spectrometer. The experimental configuration implemented to obtain UV/vis and steady-state fluorescence spectra of the Por-INs is illustrated in Figure S1. Raman measurements were carried out using a Horiba Jobin Yvon T64000 spectrometer equipped with a nitrogen cooled CCD detector. The laser wavelength was 532 nm with a power of 13 mW. Raman spectra were obtained in 10 acquisitions of 30 seconds duration. citrate buffer (10 mM ionic strength, pH 5.8) at different initial bulk concentrations and TFT as the organic phase were prepared and left to stand for 24 hours. After this time, the Por-INs formed and all remaining unadsorbed porphyrin in the bulk aqueous phase was extracted by thoroughly rinsing with porphyrin-free buffer solution. The solutions containing unadsorbed porphyrin were collected and analysed by UV/vis absorbance spectroscopy to quantify the porphyrin concentration 4000 water molecules and 1226 TFT molecules, respectively. Full details are provided in the prepared by metathesis of BACl and Li(OEt2)]TB, as described previously. corrected Titan Themis, spot size 6, on a Gatan Oneview detector. SEM images were obtained on a FEI Quanta 650 FEG high resolution SEM. X-ray diffraction patterns (XRD) were acquired in Supporting Information.
	therein (final bulk concentration). By subtracting the final from the initial bulk concentrations, the
	surface concentration (number of moles adsorbed per geometric area of aqueous|organic interface)
	was determined. All quantifications were performed using a calibration curve.

The ionic strength of each lithium citrate buffer solution containing either porphyrin was maintained at 10 (±2) mM. For the photoelectrochemistry experiments, bis(triphenylphosphoranylidene) ammonium chloride (BACl, 97%) and lithium tetrakis(pentafluorophenyl)borate diethyletherate ([Li(OEt2)]TB) were obtained from Sigma-26

2.2. Characterisation methodology. The films of Por-INs were gently transferred to a silicon substrate for SEM, XRD and Raman characterisation, or an Agar Scientific TEM grid (Holey Carbon film 300 Mesh Cu) for TEM imaging, by bringing the solid supports into contact with the interface.

ACKNOWLEDGMENTS

This publication has emanated from research by M.D.S. and A.F.M.-O. supported by the European Research Council through a Starting Grant (agreement no. 716792) and in part by a research grant from Science Foundation Ireland (SFI) (grant number 13/SIRG/2137). M.D.S. and A.F.M.-O. acknowledge funding through Irish Research Council New Foundations Awards (2014 and 2015) to facilitate the research. A.M.O., M.D.S., G.H. and M.D. are grateful to the support of the Irish Research Council and Campus France for travel support between the French and Irish groups through their joint ULYSSES programme. G. H. is grateful to the French Programme Investissement d'Avenir (PIA) "Lorraine Université d'Excellence" (Reference No. ANR-15-IDEX-04-LUE) for the partial financial support of this work. C.O.D. acknowledges support from Science Foundation Ireland (SFI) under Grant Numbers 13/TIDA/E2761, 14/IA/2581 and 15/TIDA/2893. Computational facilities and support for the molecular dynamics simulations were provided by the Irish Centre for High-End Computing (ICHEC). Ivan Robayo-Molina (University of Limerick) is acknowledged for assistance in carrying out the potentiometric titrations.

formation, potentiometric titration data, molecular dynamic studies of porphyrins in the bulk (dimerisation free energy calculations) or at the liquid|liquid interface (mechanism of layer-bylayer formation of the porphyrin interfacial nanostructures, determining probability distributions of the angle of orientation of adsorbed porphyrins), spectroscopic studies of the porphyrins in bulk aqueous solutions (influence concentration and pH on dimerisation or aggregation, respectively), in situ spectroscopic studies of porphyrin interfacial nanostructures (UV/vis, fluorescence and Raman).

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

TOC graphic

Zn porphyrins self-assemble at the interface between two immiscible liquids driven by a hydrophobicity gradient and hydrogen bond interactions.


Alonso Gamero-Quijano

Grégoire Herzog
email: gregoire.herzog@univ-lorraine.fr

Micheál D Scanlon
email: micheal.scanlon@ul.ie

Bioelectrochemistry of Cytochrome c in a Closed Bipolar Electrochemical Cell

Keywords: Bipolar electrochemistry, four electrode configuration, bipolar electrode, Cytochrome c, bioelectrochemistry

The reversible oxidation and reduction of Cytochrome c (Cyt c) is demonstrated with a closed bipolar electrochemical cell (CBPEC). Herein, a 4-electrode configuration was studied with the opposite poles of the bipolar electrode resting in separate aqueous and organic electrolyte solutions, respectively. Using biocompatible indium tin oxide (ITO) slides as the bipolar electrode poles, we investigated the influence of the redox potential of the reductant (decamethyferrocene or dimethylferrocene) in an trifluorotoluene organic phase on the observed voltammetry. Reversible electron transfer was only observed between Cyt c and decamethylferrocene. Use of the weaker dimethylferrocene as the reductant required a larger external bias of the driving electrodes to initiate the electron transfer reaction between the two poles of the bipolar electrode. Consequently, the surface of the ITO slide at the aqueous pole experienced a significant negative cathodic potential and underwent irreversible reduction. The biphasic setup using the 4-electrode CBPEC provides insights into electron transfer processes at an interface between two immiscible electrolyte solutions (ITIES), highlighting the strong probability of observing interfacial electron transfer between decamethylferrocene (but not dimethylferrocene) and Cyt c within the short ~ 1 V polarisable potential window available at an ITIES.

Introduction

Direct electron transfer (DET) to redox proteins from conducting materials is of fundamental interest in mechanistic studies in biochemistry [START_REF] Léger | Direct Electrochemistry of Redox Enzymes as a Tool for Mechanistic Studies[END_REF][START_REF] Armstrong | Direct electrochemistry of redox proteins[END_REF]. Optimisation of such charge transfer processes is central to the development of nanobiotechnological devices, such as biosensors for health monitoring or enzymatic fuel cells [START_REF] Bostick | Protein bioelectronics: a review of what we do and do not know[END_REF][START_REF] Walcarius | Nanomaterials for bio-functionalized electrodes: Recent trends[END_REF][START_REF] Leech | Enzymatic fuel cells: Recent progress[END_REF]. Cytochrome c (Cyt c) is a particularly well studied, relatively small (104 amino acid residues), redox protein due to its essential role in both mitochondrial electron transport and intrinsic type II apoptosis [START_REF] Hüttemann | The multiple functions of cytochrome c and their regulation in life and death decisions of the mammalian cell: from respiration to apoptosis[END_REF]. Cyt c is capable of DET between its heme co-factor and suitably modified electron conducting materials [START_REF] Seyedi | Theory and Electrochemistry of Cytochrome c[END_REF]. Bipolar electrochemistry involves the generation of simultaneous asymmetric electrochemical reactions at the opposite ends of isolated conductive objects, known as bipolar electrodes (BPEs), immersed in electrolyte solutions in the presence of an applied electric field [START_REF] Bradley | Creating electrical contacts between metal particles using directed electrochemical growth[END_REF][START_REF] Fosdick | Bipolar electrochemistry[END_REF][START_REF] Loget | Bipolar electrochemistry: from materials science to motion and beyond[END_REF][START_REF] Sequeira | Bipolar Electrochemistry, a Focal Point of Future Research[END_REF][START_REF] Koefoed | Bipolar electrochemistry-A wireless approach for electrode reactions[END_REF]. In this study, the bioelectrochemistry of Cyt c was investigated using immiscible aqueous-organic electrolyte solutions in a 4-electrode closed bipolar electrochemical cell (CBPEC) configuration [START_REF] Plana | The voltammetric response of bipolar cells: Reversible electron transfer[END_REF][START_REF] Hotta | Electron-conductor separating oil-water (ECSOW) system: A new strategy for characterizing electron-transfer processes at the oil/water interface[END_REF][START_REF] Hotta | Clarification of the Mechanism of Interfacial Electron-Transfer Reaction between Ferrocene and Hexacyanoferrate(III) by Digital Simulation of Cyclic Voltammograms[END_REF][START_REF] Gründer | Solids at the liquid-liquid interface: Electrocatalysis with pre-formed nanoparticles[END_REF][START_REF] Wu | Integrating bipolar electrochemistry and electrochemiluminescence imaging with microdroplets for chemical analysis[END_REF][START_REF] Uehara | Au Electrodeposition at the Liquid-Liquid Interface: mechanistic aspects[END_REF][START_REF] Nishi | Dendritic nanofibers of gold formed by the electron transfer at the interface between water and a highly hydrophobic ionic liquid[END_REF][START_REF] Smirnov | Gold Nanofilm Redox Catalysis for Oxygen Reduction at Soft Interfaces[END_REF][START_REF] Zhang | High-efficient preparation and screening of electrocatalysts using a closed bipolar electrode array system[END_REF][START_REF] Gamero-Quijano | Closed bipolar electrochemistry in a four-electrode configuration[END_REF].

The BPE studied consisted of two indium tin oxide (ITO) slides, known to be electrochemically biocompatible with Cyt c [START_REF] Kasmi | Adsorptive immobilization of cytochrome c on indium / tin oxide ( ITO ): electrochemical evidence for electron transfer-induced conformational changes[END_REF][START_REF] Matsuda | Spectroelectrochemical studies on surface immobilized cytochrome c on ITO electrode by slab optical waveguide spectroscopy[END_REF], connected by an electric wire. The lipophilic species were dimethylferrocene (DiMFc), a weak reductant, and decamethyferrocene (DcMFc), a strong reductant. To successfully achieve bioelectrochemistry in a 4-electrode CBPEC configuration, we demonstrate that considerations such as the stability of the BPE across the polarisable potential window must be taken into account. We show that when the weaker DiMFc reductant is used, the large external biasing of the driving electrodes, i.e., changing the applied Galvani potential difference ( o w ), required to initiate the electron transfer reaction establishes an equilibrium Galvani potential difference ( o w  eq. ) of sufficient magnitude to reduce the ITO surface at the aqueous pole.

The interaction of Cyt c with lipophilic redox species is interesting as Cyt c undergoes redox chemistry with species present in a lipophilic membrane environment in the electron transport chain of mitochrondria. The latter involves Cyt c shuttling electrons between Complexes III (Coenzyme Q -Cyt c reductase) and IV (Cyt c oxidase). Thus, in line with previous uses of this technique [START_REF] Plana | The voltammetric response of bipolar cells: Reversible electron transfer[END_REF][START_REF] Hotta | Electron-conductor separating oil-water (ECSOW) system: A new strategy for characterizing electron-transfer processes at the oil/water interface[END_REF][START_REF] Hotta | Clarification of the Mechanism of Interfacial Electron-Transfer Reaction between Ferrocene and Hexacyanoferrate(III) by Digital Simulation of Cyclic Voltammograms[END_REF][START_REF] Gründer | Solids at the liquid-liquid interface: Electrocatalysis with pre-formed nanoparticles[END_REF][START_REF] Wu | Integrating bipolar electrochemistry and electrochemiluminescence imaging with microdroplets for chemical analysis[END_REF][START_REF] Uehara | Au Electrodeposition at the Liquid-Liquid Interface: mechanistic aspects[END_REF][START_REF] Nishi | Dendritic nanofibers of gold formed by the electron transfer at the interface between water and a highly hydrophobic ionic liquid[END_REF][START_REF] Smirnov | Gold Nanofilm Redox Catalysis for Oxygen Reduction at Soft Interfaces[END_REF][START_REF] Zhang | High-efficient preparation and screening of electrocatalysts using a closed bipolar electrode array system[END_REF][START_REF] Gamero-Quijano | Closed bipolar electrochemistry in a four-electrode configuration[END_REF], our primary motivation is to acquire mechanistic insights for future studies of electron transfer events involving Cyt c at the interface between two immiscible electrolyte solutions (ITIES), considered a simple model system that mimics biological interfacial processes. Thus, the work herein highlights the under-utilised technique of 4-electrode closed bipolar electrochemistry with immiscible aqueous-organic solutions to study redox reactions between lipophilic redox species and aqueous redox active proteins. The latter reactions are inaccessible using typical 3-electrode bioelectrochemical experiments carried out in homogeneous aqueous conditions.

Experimental methods

3-electrode electrochemical cell experiments

The materials used in this work are detailed in section S1.1, Supplementary Information (SI). All electrochemical measurements were obtained using an Autolab PGSTAT204 potentiostat from Metrohm, Switzerland. All 3-electrode electrochemistry experiments were performed in a home-made electrochemical cell designed for low volumes (< 5 mL). The working electrode was an ITO slide (area of 1 cm 2 and resistivity of 120 ); the electron conductor used to create the BPE. The ITO slides were rinsed and sonicated in acetone prior to electrochemical measurements. The counter electrode was a Pt wire and the reference electrode was a silver/silver chloride (Ag/AgCl) wire. Cyclic voltammograms (CVs) obtained in aqueous phosphate buffer solutions with a 3-electrode cell using Ag/AgCl reference electrodes were calibrated to the SHE scale as described in Fig. S1, section S1.2, SI.

Electrochemistry in the organic solvent -trifluorotoluene (TFT) required a slight modification of the electrochemical cell, with the Ag/AgCl electrode immersed in an "organic reference solution" that established a liquid junction with the organic phase. The organic reference solution was an aqueous solution of 1 mM LiCl and 10 mM BACl. CVs obtained in TFT solutions with a 3-electrode cell using Ag/AgCl reference electrodes were also calibrated to the SHE scale using the ferrocene (Fc + /Fc) redox couple in TFT. The redox potential of the latter in TFT versus SHE has been determined previously as 0.720 V [START_REF] Smirnov | Interfacial Redox Catalysis on Gold Nanofilms at Soft Interfaces[END_REF]. All experiments were carried out under aerobic conditions.

Results and Discussion

Voltammetry of Cyt c in a 4-electrode closed bipolar electrochemical cell

The configuration of the 4-electrode CBPEC used for the study of Cyt c bioelectrochemistry with aqueous-organic electrolyte solutions is illustrated in Scheme 1. Overall, the heterogeneous redox reaction is:

DcMFc o 0 + Cyt 𝑐-Fe w (III) ⇌ DcMFc o + + Cyt 𝑐-Fe w (II) (1)
In the organic compartment, DcMFc o 0 will be oxidised at Po, see Equation ( 2), with the concurrent reduction of molecule X (e.g., a solvent molecule) at the surface of the organic driving electrode maintaining electroneutrality. Simultaneously, in the aqueous compartment, Cyt 𝑐-Fe w (III) will be reduced at Pw, see Equation ( 3), with oxidation of molecule M at the surface of the aqueous driving electrode (see Scheme 2).

DcMFc o 0 + P o 0 ⇌ DcMFc o + + P o - (2)
Cyt 𝑐-Fe w (III) + P w -⇌ Cyt 𝑐-Fe w (II) + P w 0

The current convention for electrochemical experiments with a 4-electrode CBPEC is explained in section 1.4, SI. Furthermore, the theoretically expected separation between the redox signals of +0.477 V was not observed, with a smaller separation of 0.432 V measured (Fig. 1(A), solid red line).

Comparing the voltammetry obtained using a conventional 3-electrode cell to that obtained with a 4-electrode closed bipolar electrochemical cell

By comparison with the redox response of Cyt c at an ITO working electrode using a conventional

Conclusions

Herein, direct electron transfer between Cyt c and the aqueous pole of a bipolar electrode in a 4electrode closed bipolar electrochemical cell (CBPEC) with aqueous-organic electrolyte solutions is demonstrated. The stability of the aqueous ITO pole was linked to the redox potential of the organic electron donor species (either decamethylferrocene or dimethylferrocene). The larger the difference between the redox potentials of the ferrocene-derivative and Cyt c, the greater the required external bias of the driving electrodes. Thus, the large external bias required when using the weaker reductant, dimethylferrocene, led to irreversible reduction of the ITO at the aqueous pole during the 4-electrode CBPEC experiment. The latter is demonstrated as a viable alternative experimental approach to the conventional 3-electrode cell to explore the bioelectrochemistry of Cyt c, especially using lipophilic reductants or oxidants. In this sense, ample opportunities exist to test the biocompatibility of surface modifications to the aqueous pole of the bipolar electrode, such as through the use of self-assembled monolayers on gold, lipid membranes, advanced carbon nanomaterials, or conductive polymers, to prevent protein denaturation on contact with the electrode surface. A major perspective is to confirm the predicted observation of interfacial electron transfer at the ITIES between decamethylferrocene and Cyt c within the available potential window. The use of the soft interface at an ITIES, mirroring soft biomembranes, to monitor bioelectrochemical reactions with redox active proteins would be of major interest.

Scheme 1 .

 1 Scheme 1. Schematic of the 4-electrode CBPEC with aqueous-organic electrolyte solutions. The BPE consisted of two ITO slides connected by an electric wire, one in each compartment acting as the aqueous pole (Pw) or organic pole (Po), respectively. The ferrocene derivatives studied in the TFT organic phase were DiMFc and DcMFc.

Scheme 2 . 7 versus)

 27 Scheme 2. Schematic of the 4-electrode CBPEC configuration with aqueous-organic electrolyte solutions used for the study of Cyt c bioelectrochemistry as  o w  is biased sufficiently positively by the potentiostat to facilitate electron transfer along the BPE from the organic electron donor DcMFc at Po to the aqueous Cyt c molecule at Pw.

Fig. 1 .

 1 Fig. 1. (A) CVs obtained using the 4-electrode CBPEC configuration with aqueous-organic electrolyte solutions outlined in Scheme 1, in the absence of Cyt c and both ferrocene derivatives (solid black line), in the presence of 14 M DcMFc only (dashed black line), in the presence of 14 M DiMFc only (dashed red line), and in the presence of 8 M Cyt c, 14 M DiMFc and 14 M DcMFc (solid red line). The scan rate used was 20 mVs -1 . (B) CVs obtained using the 4-electrode CBPEC configuration outlined in Scheme 1, as described in (A), using a smaller polarisable potential window to clearly observe the redox response at  o w  1/2 = -0.161 V in the presence of 8 M Cyt c and 14 M DcMFc (solid red line). The scan rate used was 20 mVs -1 .

3 -

 3 Fig.2(A), the aqueous Ag/AgCl reference electrode potential scale was linked to the DcMFc potential scale in the TFT organic electrolyte, against which all reduction processes are occurring in the 4-electrode

Fig. 2 .

 2 Fig. 2. Comparison of CVs recorded using the 4-electrode CBPEC with aqueous-organic electrolyte solutions outlined in Scheme 1 in the presence of (A) 8 M Cyt c and 14 M DcMFc (solid black line) or (B) 8 M Cyt c, 14 M DiMFc and 14 M DcMFc (solid black line) and a CV recorded using a 3-electrode cell with 8 M Cyt c in phosphate buffer solution at pH 7 (solid red line). The aqueous Ag/AgCl reference electrode potential scale was linked to the DcMFc potential scale in the TFT organic electrolyte, as

Acknowledgements

This publication has emanated from research by M. D. S. supported by the European Research Council through a Starting Grant (agreement no. 716792) and in part by a research grant from Science Foundation Ireland (SFI) (grant number 13/SIRG/2137). A. G.-Q. acknowledges funding received from an Irish Research Council Government of Ireland Postdoctoral Fellowship Award (grant number GOIPD/2018/252). G. H. is grateful to the French Programme Investissement d'Avenir (PIA) "Lorraine Université d'Excellence" (Reference No. ANR-15-IDEX-04-LUE). for the partial financial support of this work. The authors are grateful to the support of the Irish Research Council and Campus France for travel support between the French and Irish groups through their joint ULYSSES programme.

Supplementary Information

Supplementary Information can be found online at ???????????????


Ming Xue

Qianrong Fang
email: qrfang@jlu.edu.cn

Yushan Yan

Valentin Valtchev
email: valentin.valtchev@ensicaen.fr

Shilun Qiu

Xinyu Guan

Hui Li

Yunchao Ma

Chemically stable polyarylether covalent organic frameworks

Chemically stable polyarylether-based covalent organic frameworks

Introduction

Covalent organic frameworks (COFs) are an emerging class of crystalline porous materials (CPMs), which are composed of organic building blocks linked by strong covalent bonds. [START_REF] Côté | Porous, crystalline, covalent organic frameworks[END_REF][START_REF] Colson | Rationally synthesized two-dimensional polymers[END_REF][START_REF] Huang | Covalent organic frameworks: a materials platform for structural and functional designs[END_REF][START_REF] Diercks | The atom, the molecule, and the covalent organic framework[END_REF][START_REF] Ding | Covalent organic frameworks (COFs): from design to applications[END_REF][START_REF] Jin | Tessellated multiporous two-dimensional covalent organic frameworks[END_REF] Over the past decade, COFs have attracted wide attention in various application fields including gas adsorption and separation, [START_REF] Kuhn | Porous, covalent triazine-based frameworks prepared by ionothermal synthesis[END_REF][START_REF] Furukawa | Storage of hydrogen, methane, and carbon dioxide in highly porous covalent organic frameworks for clean energy applications[END_REF][START_REF] Guan | Fast, ambient temperature and pressure ionothermal synthesis of three-dimensional covalent organic frameworks[END_REF] catalysis, [START_REF] Ding | Construction of covalent organic framework for catalysis: Pd/COF-LZU1 in suzuki miyaura coupling reaction[END_REF][START_REF] Fang | 3D microporous base-functionalized covalent organic frameworks for size-selective catalysis[END_REF][START_REF] Li | 3D covalent organic frameworks with dual linkages for bifunctional cascade catalysis[END_REF][START_REF] Han | Chiral covalent organic frameworks with high chemical stability for heterogeneous asymmetric catalysis[END_REF] electrochemistry, [START_REF] Wan | A belt-shaped, blue luminescent, and semiconducting covalent organic framework[END_REF][START_REF] Bertrand | Thiophene-based covalent organic frameworks[END_REF][START_REF] Calik | Extraction of photogenerated electrons and holes from a covalent organic framework integrated heterojunction[END_REF] and a number of others. [START_REF] Fang | 3D porous crystalline polyimide covalent organic frameworks for drug delivery[END_REF][START_REF] Du | Ionic covalent organic frameworks with spiroborate linkage[END_REF][START_REF] Wang | Exfoliation of covalent organic frameworks into few-layer redox-active nanosheets as cathode materials for lithium-ion batteries[END_REF][START_REF] Sun | Postsynthetically modified covalent organic frameworks for efficient and effective mercury removal[END_REF][START_REF] Li | Three-dimensional ionic covalent organic frameworks for rapid, reversible and selective ion exchange[END_REF][START_REF] Lu | Postsynthetic functionalization of three-dimensional covalent organic framework for selective extraction of lanthanide ions[END_REF] Nonetheless, the linkages of available COFs are limited to B-O, C=N, C-N, C=C and B=N bonds. Furthermore, low chemical stabilities, especially in acid and base, present a considerable hindrance to the further development and application of COFs. Although structural stabilities of some COFs can be improved by hydrogen bonds, [START_REF] Kandambeth | Self-templated chemically stable hollow spherical covalent organic framework[END_REF] keto-enol tautomerism, [START_REF] Mallick | Construction of crystalline 2D covalent organic frameworks with remarkable chemical (acid/base) stability via a combined reversible and irreversible route[END_REF] enhanced interlayer interactions [START_REF] Xu | crystalline, porous, covalent organic frameworks as a platform for chiral organocatalysts[END_REF] or conversion of the imine linkages, [START_REF] Wei | Benzoxazole-linked ultrastable covalent organic frameworks for photocatalysis[END_REF] these materials do not survive harsh chemical environments, e.g., enhanced acid/base concentration. Design and preparation of a new class of robust COFs that are stable under extreme conditions thus remains a major challenge for bringing these exceptional materials to practical applications.

Polyarylether (PAE) is a highly stable engineered plastic with high heat resistance, excellent chemical stability, good flame retardant properties, and high mechanical robustness. [START_REF] Hergenrother | The use, design, synthesis, and properties of high performance/high temperature polymers: an overview[END_REF] PAEs can be easily modified with various functional groups [START_REF] Gao | Comparison of PEM properties of copoly(aryl ether ether nitrile)s containing sulfonic acid bonded to naphthalene in structurally different ways[END_REF] including cyano, carbonyl, carboxyl, and sulfo groups, and thus have broad application prospects in the aerospace, rail transportation, automobile manufacturing, machinery industry and many other technological fields. [START_REF] Gotham | Poly(ether sulphone) as an engineering material[END_REF] Polymers of intrinsic microporosity (PIMs), a new kind of porous material connected by ether bonds, have recently been reported. 30 However, PIMs are amorphous phases without uniform and ordered pores, which limits their potential applications. The synthesis of crystalline porous PAEs will grant this material with all the advantages of crystalline porous solids, such as a defined channel system that is able to perform recognition and separation of close in size molecules, and an extended micro-and/or mesoporous volume and functional surface with crystallographically defined positions.

Herein, we address the above issues by synthesizing a new kind of polyarylether-based covalent organic frameworks (PAE-COFs) that combine high crystallinity, porosity, and outstanding stability.

Two novel PAE-COFs with micropores of 16.8 Å and mesopores of 28.4 Å, denoted as JUC-505 and JUC-506 (JUC = Jilin University China), were successfully synthesized. JUC-505 features excellent carbon dioxide (CO 2 ) adsorption (231.3 mg g -1 at 273 K and 189.0 mg g -1 at 298 K) and separation (48.9 for CO 2 /CH 4 and 97.2 for CO 2 /N 2 ), which are among the highest reported for COF and PIM materials. Remarkably, PAE-COFs maintain their crystallinity after the treatment under harsh chemical environments, including boiling water, strong acids (HCl, H 2 SO 4 and HF) and bases (NaOH and MeONa), oxidation (chromic acid lotion) and reduction (LiAlH 4 ) medium, which outstrips all known CPMs including the aluminosilicate zeolites, metal-organic frameworks (MOFs) and COFs. These COFs with high stability, crystallinity and porosity provide a material platform for structural design and functional development. PAE-COFs with carboxyl or amino functional groups, denoted JUC-505-COOH and JUC-505-NH 2 , were subsequently obtained by the post-synthetic functionalization, and showed a remarkable performance in the removal of antibiotics from water over a wide pH range (pH = 1-13).

Results and discussion

Design of PAE-COFs. Our strategy for preparing PAE-COFs involves aromatic nucleophilic substitution between the extended o-difluoro benzene and catechol building units catalyzed by anhydrous potassium carbonate. In the model condensation reaction of tetrafluoroterephthalonitrile (TFTPN) with 1,2-dihydroxybenzene (DHB), fluoride ion and proton are eliminated to yield a dioxin product, 3,13-dicyanobenzo-1,2,4',5'-bis(1,4-benzodioxane) (DCBD, Fig. 1a). On the basis of this reaction, DHB is extended to a triangular building unit, 2,3,6,7,10,11-hexahydroxytriphenylene hydrate (HHTP), whereas TFTPN can be employed directly or replaced by other linear building blocks, such as 2,3,6,7-tetrafluoroanthraquinone (TFAQ). As shown in Fig. 1b, the condensation reaction of HHTP and TFTPN produces a two-dimensional (2D) microporous crystalline JUC-505 with hexagonal pores of 16.8 Å. On the other hand, replacing TFTPN with a longer linker, TFAQ, gives 2D mesoporous JUC-506 with larger hexagonal pores of 28.4 Å (Fig. 1c). By linking the linear and triangular building units through the aromatic nucleophilic substitution reaction, the products form 2D structures based on the boron nitride net (bnn, Fig. 1d).

Synthesis and characterization.

Typically, PAE-COFs were synthesized by suspending anhydrous potassium carbonate, HHTP with TFTPN or TFAQ in a mixed solution of N-methyl-2-pyrrolidone (NMP) and mesitylene followed by heating at 120 °C and 160 °C for 3 days, giving crystalline solids at yields of 84% for JUC-505 and 78% for JUC-506, respectively. Scanning electron microscopy (SEM) inspection revealed that both materials exhibit fiber-like particles with a cross section diameter of about 100 nm. The length of fiber-like particles is several hundred nanometers in the case of JUC-505, while up to 10 micron fibers can be seen in the case of JUC-506 (Figs. S1 andS2). Fourier transform infrared (FTIR) spectra displayed adsorption peaks at 1264 cm -1 and 1021 cm -1 for JUC-505 and 1270 cm -1 and 1031 cm -1 for JUC-506 corresponding to asymmetric and symmetric vibration modes of the ether bonds, respectively, which is consistent with those from the model compound DCBD (1274 cm -1 and 1030 cm -1 , Figs. S5-7). Noteworthy, the significant decrease at 3432 cm -1 (referring to OH stretching of the starting material HHTP) was observed, indicating a high-conversion polymerization. Solid-state [START_REF] Han | Chiral covalent organic frameworks with high chemical stability for heterogeneous asymmetric catalysis[END_REF] C cross polarization magic-angle-spinning (CP/MAS) nuclear magnetic resonance (NMR) analysis resulted in the exact attribution for each peak and further confirmed the formation of PAE-COFs (Figs. S9 andS10). Elemental analysis of PAE-COFs was in good agreement with the expected formulas of C 10 H 2 O 2 N for JUC-505 and C 13 H 4 O 3 for JUC-506, respectively. The content of residue fluorine was further determined by energy dispersive spectrometer (EDS) as 0.94% for JUC-505 and 0.49% for JUC-506 (Figs. S13 andS15). Furthermore, the reaction conversions were calculated from residue fluorine content as 95.3% for PAE-505 and 96.9% for PAE-506. S1 andS3). 31 After a geometrical energy minimization using the universal force field, the unit cell parameters were obtained (a = b = 24.6865 Å, c = 3.5002 Å, α = β = 90° and γ =120° for JUC-505; a = b = 33.7458 Å, c = 3.4702 Å, α = β = 90° and γ =120° for JUC-506). The simulated PXRD patterns were in good agreement with the experimental ones (Fig. 2, a andb). Furthermore, full profile pattern matching (Pawley) refinements were carried out on the experimental PXRD patterns. Peaks at 2 = 4.12, 7.13, 8.24, 10.93 and 14.88° for JUC-505 and 3.02, 5.23, 6.04, 8.00 and 10.91° for JUC-506 correspond to the (100), ( 110 S2 andS4). The simulated PXRD pattern for this arrangement does not match the experimental data (Figs. S19 andS22). On the basis of these results, JUC-505 and JUC-506 were proposed to have the eclipsed stacking architectures with hexagonal micropores of about 16.8 Å and hexagonal mesopores of 28.4 Å, respectively (Fig. 2, c andd). The formation of the eclipsed structure can be attributed to the strong tendency for aromatic units to form cofacial aggregates between adjacent layers.

Gas adsorption and separation. Nitrogen (N 2 ) adsorption-desorption analysis of JUC-505 measured at 77 K showed a type I isotherm with a sharp uptake at a low pressure of P/P 0 < 0.05, which is characteristic of microporous material (Fig. 3a). Meanwhile, N 2 adsorption-desorption isotherm of JUC-506 showed a rapid uptake at a low pressure of P/P 0 < 0.1, followed by a sharp step between P/P 0 = 0.15 and 0.25 (Fig. 3b). This sorption profile is best described as a type IV isotherm, which is characteristic of mesoporous materials. The total pore volumes were evaluated at P/P 0 = 0.95 to be Vp = 0.51 cm 3 g -1 for JUC-505 and 0.71 cm 3 g -1 for JUC-506. Pore size distributions of PAE-COFs calculated on the basis of nonlocal density functional theory (NLDFT) showed pore with sizes of 16.2 Å for JUC-505 and 30.1 Å for JUC-506, respectively (Fig. 3 a and b, inset), which is in good agreement with that of the proposed model (16.8 Å for JUC-505 and 28.4 Å for JUC-506). The Brunauer-Emmett-Teller (BET) equation was applied over the 0.02 < P/P 0 < 0.20 range of the isotherms, revealing BET surface areas of 1584 m 2 g -1 for JUC-505 and 1655 m 2 g -1 for JUC-506 (Figs.

S28 and S29).

Considering the high crystallinity, excellent porosity and affinity to CO 2 of PIMs, 30 we studied the potential application of PAE-COFs for CO 2 adsorption and separation. The adsorption isotherms of CO 2 , N 2, and CH 4 were measured at 273 K and 298 K, respectively. As shown in Figs. 3, S32 and S33, the sorption amounts of CO 2 are as high as 231.3 mg g -1 for JUC-505 and 203.0 mg g -1 for JUC-506 at 273 K, and the CO 2 sorption analysis at 298 K shows the capacity of 189.0 mg g -1 for JUC-505 and 159.2 mg g -1 for JUC-506. Thus, JUC-505 exhibits the highest CO 2 uptake among COFs and PIMs reported to date, such as PyTTA-BFBIm-iCOF (177 mg/g at 273 K) [START_REF] Huang | Ionic covalent organic frameworks: design of a charged interface aligned on 1D channel walls and its unusual electrostatic functions[END_REF] and TZPIMs (132 mg/g at 273 K). [START_REF] Du | Polymer nanosieve membranes for CO 2 -capture applications[END_REF] Meanwhile, the CO 2 uptake is much higher with respect to other gases such as CH 4 (19.2 mg g -1 for JUC-505 and 18.3 mg g -1 for JUC-506 at 273 K; 15.2 mg g -1 for JUC-505 and 13.1 mg g -1 for JUC-506 at 298 K) and N 2 (10.4 mg g -1 for JUC-505 and 8.8 mg g -1 for JUC-506 at 273 K; 12.5 mg g -1 for JUC-505 and 8.9 mg g -1 for JUC-506 at 298 K). The ideal adsorption selectivity of PAE-COFs was calculated from the ratio of the initial slopes in the Henry region of the isotherms (Figs. S34-S37), [START_REF] Henry | Experiments on the quantity of gases absorbed by water, at different temperatures, and under different pressures[END_REF] and high selectivity of CO 2 /N 2 and CO 2 /CH 4 was observed (Table 1). For example, the ideal adsorption selectivity of JUC-505 is as high as 48.9 for CO 2 /CH 4 and 97.2 for CO 2 /N 2 at 273 K, respectively. Furthermore, preliminary breakthrough experiments for JUC-505 were carried out by exposing to streams containing binary mixtures of CO 2 /N 2 or CO 2 /CH 4 (50:50 v/v) at room temperature. The results clearly showed that in both cases only CO 2 was retained in the pores of JUC-505 while N 2 and CH 4 passed through without impediment (Fig. 3 e andf). Moreover, N 2 adsorption isotherms at 77 K after the treatment are very close to those of as-synthesized materials, which further confirmed the preserved porosity (Figs. S46 andS47). For the sake of comparison, we tested the chemical stability of the typically stable COFs reported to date, including DhaTab-COF, [START_REF] Kandambeth | Self-templated chemically stable hollow spherical covalent organic framework[END_REF] TpPa-1, [START_REF] Mallick | Construction of crystalline 2D covalent organic frameworks with remarkable chemical (acid/base) stability via a combined reversible and irreversible route[END_REF] TPB-DMTP-COF [START_REF] Xu | crystalline, porous, covalent organic frameworks as a platform for chiral organocatalysts[END_REF] , LZU-190 [START_REF] Wei | Benzoxazole-linked ultrastable covalent organic frameworks for photocatalysis[END_REF] , PI-COF-2 [START_REF] Fang | Designed synthesis of large-pore crystalline polyimide covalent organic frameworks[END_REF] , CS-COF [START_REF] Guo | Conjugated organic framework with threedimensionally ordered stable structure and delocalized p clouds[END_REF] and TpOMe-Pa1 [START_REF] Halder | Ultrastable Imine-based Covalent Organic Frameworks for Sulfuric Acid Recovery: An Effect of Interlayer Hydrogen Bonding[END_REF] (Figs. S48-S54). On the basis of the PXRD analysis, TpPa-1, PI-COF-2, LZU-190, CS-COF, TpOMe-Pa1 and DhaTab-COF are not stable in extreme alkaline medium whereas TPB-DMTP-COF, PI-COF-2 and DhaTab-COF cannot withstand strong acid. Noteworthy, none of these most stable COFs retains crystallinity after the treatment with concentrate H 2 SO 4 or chromic acid lotion. Furthermore, some typical MOFs (e.g., UiO-66 [START_REF] Katz | A facile synthesis of UiO-66, UiO-67 and their derivatives[END_REF] , ZIF-8 [START_REF] Pan | Rapid synthesis of zeolitic imidazolate framework-8 (ZIF-8) nanocrystalsin an aqueous system[END_REF] , HKUST-1 [START_REF] Stephen | A chemically functionalizable nanoporous material [Cu 3 (TMA) 2 (H 2 O) 3 ] n[END_REF] and MOF-5 [START_REF] Eddaoudi | Systematic design of pore size and functionality in isoreticular MOFs and their application in methane storage[END_REF] ) and zeolites (commercial Silicalite-1, X, Y and 4A) were also selected for the stability test, and none of these matters retained entire crystallinity after the treatment as can be seen in Table 2 and S5 andS6). Carboxyl or amino functionalized PAE-COFs, JUC-505-COOH and JUC-505-NH 2 , were obtained by refluxing JUC-505 in NaOH (20% in ethanol/water = 1/1) [START_REF] Du | High-performance carboxylated polymers of intrinsic microporosity (PIMs) with tunable gas transport properties[END_REF] and LiAlH 4 (2.4 M in THF), [START_REF] Peng | Diverse macroscopic helical motions of microribbons driven by electrons[END_REF] respectively. SEM images of functionalized JUC-505 derivatives exhibited similar morphology to the parent material (Figs. S3 andS4). Adsorption peaks of IR spectra at 1268 cm -1 and 1021 cm -1 for JUC-505-COOH as well as 1264 cm -1 and 1022 cm -1 for JUC-505-NH 2 , corresponding to asymmetric and symmetric vibration modes of the ether bonds, were consistent with those of the pristine structure (1264 cm -1 and 1021 cm -1 for JUC-505, Fig. S8). Peaks at 1716 cm -1 for JUC-505-COOH and 3293 cm -1 for JUC-505-NH 2 indicated the grafting of carboxyl and amino group on the respective COFs. A significant decrease of the peak at 2239 cm -1 demonstrated the high transformation ratios of cyano groups for both post-modifications. Furthermore, the peaks at 163 ppm for JUC-505-COOH and 25 ppm for JUC-505-NH 2 from the solid-state [START_REF] Han | Chiral covalent organic frameworks with high chemical stability for heterogeneous asymmetric catalysis[END_REF] C CP/MAS NMR analysis confirmed the presence of carboxyl and aminomethyl groups, respectively (Figs. S11 andS12). The PXRD patterns of JUC-505-COOH and JUC-505-NH 2 were identical to JUC-505 (Fig. S23), which showed that the framework was not affected by the grafting. Conversation rates were further determined by CHN elemental analysis as 98.6% for JUC-505-COOH and 94.6% for JUC-505-NH 2 . The porosities of JUC-505-COOH and JUC-505-NH 2 were evaluated by N 2 -sorption isotherm measurements. Both matters showed typical type I isotherms (Figs. S26 andS27), and the BET surface areas were reduced slightly with respect to the parent material (1392 m 2 g -1 for JUC-505-COOH and 1244 m 2 g -1 for JUC-505-NH 2 , Figs. S30 andS31).

Stability of PAE-

Adsorptive removal of antibiotics. Antibiotics, as one of the major classes of pharmaceuticals and personal care products, are widely used in hospitals, households, and veterinary applications, and their production and usage increase every year. [START_REF] Sholl | Seven chemical separations to change the world[END_REF] However, when released into the environment, they can promote the development of resistance in microbial populations and induce toxic effects on aquatic organisms. [START_REF] Ali | New generation adsorbents for water treatment[END_REF] Tetracycline antibiotics (TCA) are among the most widely used ones. As an experimental proof-of-concept, three common species of TCA including tetracycline (TC), oxytetracycline (OTC) and chlortetracycline hydrochloride (CTC) were chosen for the adsorption test. The parent JUC-505 and functionalized PAE-COFs (JUC-505-COOH and JUC-505-NH 2 ) were activated to remove guest molecules and then immersed in antibiotic solutions at pH = 1-13 for 3 days. The residual concentrations and the medical uptake were determined by UV-Vis spectroscopy and calculated according to the standard curve.

As shown in Fig. 4 a-c, carboxyl-decorated JUC-505-COOH reveals a larger uptake capacity in acidic solutions (e.g., 245.8 mg/g for TC, 213.8 mg/g for OTC and 238.9 mg/g for CTC at pH = 1) while amino-functionalized JUC-505-NH 2 shows better performance under strongly alkaline conditions (e.g., 247.3 mg/g for TC, 237.0 mg/g for OTC and 274.6 mg/g for CTC at pH = 13), which can be attributed to the hydrogen bond and electrostatic interactions at different pHs. These values prove high adsorbing capacities under extreme pH values, which is superior to the best performance materials reported for antibiotic assimilation, such as resins, clays, carbon nanotubes, zeolites, and MOFs. [START_REF] Wang | Adsorptive removal of antibiotics from water using magnetic ion exchange resin[END_REF][START_REF] Wang | Adsorption behavior and mechanisms of norfloxacin onto porous resins and carbon nanotube[END_REF][START_REF] Ali | Adsorption behavior of doxycycline antibiotic on NaY zeolite from wheat (Triticum aestivum) straws ash[END_REF][START_REF] Singh | Nanocuboidal-shaped zirconium based metal organic framework for the enhanced adsorptive removal of nonsteroidal anti-inflammatory drug, ketorolac tromethamine, from aqueous phase[END_REF] We also tested the reusability of PAE-COFs after antibiotics removal. After being immersed in concentrated HCl for 1 h, filtered and washed with water, PAE-COFs were reused for the antibiotics removal. The sorption and release process was repeated for five continuous cycles, and the procedure was reversible with almost no loss of capacity (Fig. 4 d-i). These results suggest that the extremely stable PAE-COFs with high adsorption capacities are excellent materials for antibiotics removal that can be used under extreme conditions, regardless of the pH value.

Conclusion

In summary, we have developed a novel series of crystalline, porous and highly stable COF materials, PAE-COFs, by regulating the crystallization of ultra-highly stable polyarylether. PAE-COFs are thermally stable up to 400 °C and extremely resistant to various chemical environments, including boiling water, strong acids and bases, oxidation and reduction atmosphere, which surpasses all known crystalline porous materials including zeolites, MOFs and COFs. PAE-COFs showed impressive CO 2 adsorption capacity and CO 2 /CH 4 and CO 2 /N 2 selectivities. A member of the family (JUC-505) recorded the highest CO 2 uptake (231.3 mg g -1 at 273 K) overpassing all COFs and PIMs reported to date. Carboxyl or amino functionalized PAE-COFs showed exceptional abilities for antibiotics removal from water covering the pH range of 1-13. These stable novel COFs are a perfect platform for the preparation of functional materials that can be used under extreme chemical environments. Finally, this study shows that the most significantdisadvantage of COF materials, their limited chemical stability, can be overcome by proper design of their skeletons based on new stable bonds.

Methods

Synthesis of TFAQ. [START_REF] Hayashi | Semiconducting self-assembled nanofibers prepared from photostable octafluorinated bisanthene derivatives[END_REF] A mixture of 4,5-difluorophthalic anhydride (5.0 g, 27.2 mmol) and 1,2-difluorobenzene (16.0 mL, 165.0 mmol) was heated to 60 ºC and stirred for 20 mins. After cooling to room temperature (RT), AlCl 3 (7.9 g, 54.0 mmol) was carefully added to the mixture in small portions. The resultant dark brown solution was stirred at RT for 4 hrs, and then the reaction mixture was cooled to 0 ºC and 1 M HCl was slowly added. The white precipitate obtained from the reaction was dissolved in Et 2 O, and the organic phase was washed with 1 M HCl and water and dried over

Synthesis of the model DCBD compound.

The model compound, 3,13-dicyanobenzo-1,2,4',5'-bis(1, 4-benzodioxane) (DCBD), was prepared as described previously in the thioamide PIM-1 paper. [START_REF] Mason | Polymer of intrinsic microporosity incorporating thioamide functionality: preparation and gas transport properties[END_REF] Anhydrous DMF (60.0 mL) was added to 1,2-dihydroxybenzene (2.2 g, 20.4 mmol), tetrafluoroterephthalonitrile (1.9 g, 9.3 mmol), and potassium carbonate (8.0 g, 57.8 mmol) under an inert atmosphere. The reaction was heated to 65 °C and kept at this temperature for 24 hrs. The mixture was cooled to RT, and the solid was collected via vacuum filtration. The crude product was then stirred in water (150.0 mL) for 1 hr and acetone (150.0 mL) for 1 hr before being dried in an oven at 110 °C overnight. DCBD was obtained as a yellow solid (2.9 g, 90% yield). [START_REF] Côté | Porous, crystalline, covalent organic frameworks[END_REF] H NMR (400 MHz, DMSO-d6) δ: 7.25-7.00 ppm (m, 8H). IR (KBr): 2240, 1607, 1498, 1446, 1099, 1029 cm -1 .

Synthesis of JUC-505.

A Pyrex tube measuring o.d. × i.d. = 10 × 8 mm 2 was charged with anhydrous K 2 CO 3 (138.2 mg, 1.0 mmol), HHTP (32.4 mg, 0.1 mmol) and TFTPN (30.0 mg, 0.15 mmol) in a solution of 0.30 mL mesitylene/0.60 mL NMP. The tube was flash frozen at 77 K (LN 2 bath), evacuated to an internal pressure of 0.15 mmHg and flame sealed. Upon sealing the length of the tube was reduced to ca. 13.0 cm. The reaction mixture was heated at 120 °C for 3 days and the obtained brown precipitate was isolated by filtration over a medium glass frit and washed with DMF (20.0 mL × 3), H 2 O (20.0 mL × 3) and acetone (20.0 mL × 3). The solvent was then exchanged with anhydrous acetone and removed under vacuum at 80 °C to afford JUC-505 as a brown powder (42.1 mg, 84%).

The degree of crystallinity was calculated from PXRD pattern by Jade 52 as 91.9%. Solid-state [START_REF] Han | Chiral covalent organic frameworks with high chemical stability for heterogeneous asymmetric catalysis[END_REF]

Synthesis of JUC-506.

In a manner similar to the preparation of JUC-505, treatment of K 2 CO 3 (138.2 mg, 1.0 mmol), HHTP (32.4 mg, 0.1 mmol) and TFAQ (42.0 mg, 0.15 mmol) in a solution of 0.30 mL mesitylene/0.60 mL NMP at 160 °C for 3 days yielded a brown precipitate (48.7 mg, 78%), which was subjected to purification by the above described procedure. The degree of crystallinity was calculated from PXRD pattern by Jade as 85.1%. Solid-state [START_REF] Han | Chiral covalent organic frameworks with high chemical stability for heterogeneous asymmetric catalysis[END_REF] C NMR (500 MHz) d: 177.4, 144.9, 138.9, 129. Synthesis of JUC-505-COOH. JUC-505 powder (200.0 mg) and 20% NaOH solution (H 2 O/ethanol = 1/1, 50.0 mL) were added to a one neck round-bottom flask equipped with a condenser. The mixture was heated to 120 °C and left to stir at this temperature under reflux for three days. Samples were filtrated and refluxed in water and 1 M HCl for 2 hrs, respectively. The solid was collected by vacuum filtration, then washed with water and THF. The product was then subjected to Soxhlet extraction with THF as the solvent for one day to remove the trapped guest molecules. The solvent was then exchanged with anhydrous acetone and removed under vacuum at 80 °C giving JUC-505-COOH as a brown powder (211.2 mg, 95%). The degree of crystallinity was calculated from PXRD pattern by Jade as 90.3%. Solid-state [START_REF] Han | Chiral covalent organic frameworks with high chemical stability for heterogeneous asymmetric catalysis[END_REF] (2.4 M in THF, 30.0 mL) were added to a one neck round-bottom flask equipped with a condenser. The mixture was heated to 90 °C and left to stir at this temperature under reflux for one day. After cooling down to room temperature, 1.0 mL 10% NaOH was added dropwise to quench the reaction. 10.0 mL HCl (HCl/H 2 O = 2/1) was then added to the solution and stirred for 1 hr. The solid was collected by vacuum filtration, then washed with water. The residue powder was immersed in 20.0 mL 10% NaOH to remove the extra acid and then exchanged with water until the solution became neutral. The solid was collected by vacuum filtration and washed with water and THF. The product was subjected to Soxhlet extraction with THF as the solvent for one day to remove the trapped guest molecules. The solvent was then exchanged with anhydrous acetone and removed under vacuum at 80 °C giving JUC-505-NH 2 as a brown powder (188.7 mg, 92%). The degree of crystallinity was calculated from PXRD pattern by Jade as 88.2%. Solid-state [START_REF] Han | Chiral covalent organic frameworks with high chemical stability for heterogeneous asymmetric catalysis[END_REF] Characterization. [START_REF] Côté | Porous, crystalline, covalent organic frameworks[END_REF] H NMR spectra were recorded on an AV400 NMR spectrometer. [START_REF] Han | Chiral covalent organic frameworks with high chemical stability for heterogeneous asymmetric catalysis[END_REF] C CP/MAS NMR spectra were recorded on an AVIII 500 MHz solid-state NMR spectrometer. The FTIR spectra (KBr) were obtained using a SHIMADZU IRAffinity-1 Fourier transform infrared spectrophotometer.

A SHIMADZU UV-2450 spectrophotometer was used for all absorbance measurements. TGA was carried out under nitrogen on a SHIMADZU DTG-60 thermal analyzer at a heating rate of 10 °C min -1 to 900 °C with N 2 flow rate of 30 mL min -1 . Element analysis was carried out on a Germany Elementar large sample volume element analyzer, vario MACRO cube CHNS. PXRD data were collected on a PANalytical B.V. Empyrean powder diffractometer using a Cu Kα source (λ = 1.5418 Å) over the range of 2θ = 2.0-40.0° with a step size of 0.02° and 2 s per step. The sorption isotherm for N 2 , CH 4 and CO 2 was measured by using a Quantachrome Autosorb-IQ analyzer with ultra-high-purity gas (99.999% purity). To estimate pore size distributions for PAE-COFs, nonlocal density functional theory (NLDFT) was applied to analyze the N 2 isotherm on the basis of the model of N 2 @77 K on carbon with slit pores and the method of non-negative regularization. The SEM images were obtained on JEOL JSM6700 scanning electron microscope. The TEM images and EDS spectra were obtained on JEM-2100 transmission electron microscopy. The gas-separation property was tested by breakthrough experiments using a CO 2 /CH 4 /He or CO 2 /N 2 /He (25:25:50 v/v/v) gas mixture at a flow rate of 2.0 mL min -1 under room temperature. The relative amounts of the gases passing through the column were monitored on a Thermo Star gas analysis system detecting ion peaks at m/z+ 44 (CO 2 ), 16 (CH 4 ), 28 (N 2 ), 4(He). [a] Largest pore diameter (LPD) obtained theoretically; [b] Calculated using the initial slope in the Henry region of isotherms at 273 K and 298 K based on Henry's law.

        MOF-5 41         Zeolites Silicalite-1 [b]         X [b]         Y [b]         4A [b]        
[a] The stabilities of the samples were determined by PXRD patterns. Green with the tick represents stable, and red with the cross represents unstable.

[b] The chemicals were obtained from commercial sources.

  Crystallinity and structural resolution. The crystallinity and unit cell parameters of PAE-COFs were determined by powder X-ray diffraction (PXRD) analysis. Data obtained with the Materials Studio software package based on of the eclipsed 2D bnn net show that both PAE-COFs crystallize in P6/mmm (No. 197) space group (Figs. S17, S20, Tables

  ), (200), (210) and (310) planes, respectively. The refinement results yielded unit cell parameters nearly equivalent to the predictions with good agreement factors (a = b = 24.7714 Å, c = 3.4624 Å, α = β = 90°, γ =120°, ωRp = 2.02% and Rp = 1.60% for JUC-505; a = b = 33.9363 Å, c = 3.5704 Å, α = β = 90°, γ =120°, ωRp = 5.71% and Rp = 3.07% for JUC-506). We also considered an alternative staggered 2D arrangement (AB stacking) in which the units of adjacent sheets are horizontally offset by a distance of a/2 and b/2 (Figs. S18, S21, Tables

  COFs. Similar to amorphous PAEs, crystalline PAE-COFs displays outstanding thermal and chemical stability. According to the thermogravimetric analysis (TGA), PAE-COFs are thermally stable up to 400 °C under nitrogen (Figs. S38 and S39). To investigate the chemical stability of PAE-COFs, we exposed JUC-505 and JUC-506 samples to different chemical environments for one week, including various organic solvents (e.g., NMP, N,N-dimethylformamide (DMF), tetrahydrofuran (THF), dichloromethane (DCM), ethanol (EtOH), acetone, n-hexane and m-cresol), boiling water, concentrated HCl (12 M), concentrated H 2 SO 4 (18 M), HF (40%), concentrated NaOH (14 M), MeONa (5 M in MeOH), chromic acid lotion (0.1 M K 2 Cr 2 O 7 in concentrate H 2 SO 4 ) and LiAlH 4 (2.4 M in THF). JUC-505 and JUC-506 retain their original skeleton and crystalline structure after the treatment under the above conditions as demonstrated by unaltered position and strength of the peaks in their PXRD patterns (Figs. 2e and S42) and the retained ether bond in the IR spectra (Figs. 2f and S45).

  Figs. S55-S62. These results unambiguously prove the outstanding chemical stability of PAE-COFs which surpasses all known crystalline porous materials. Functionalization of PAE-COFs. In order to extend the possible applications, JUC-505 was transformed into an open framework with functional sites by the chemical modification of cyano groups (Figs. 1b, S20, S21, Tables

Na 2 SO 4 ,

 24 and then concentrated under vacuum. Recrystallized from CH 2 Cl 2 /hexane gave pure TFAQ as a pale-brown solid (7.4 g, 91%) (Rf = 0.35 with CHCl 3 /MeOH 10:1); m.p. 123 °C; 1 H NMR (600 MHz, CDCl 3 ): d = 7.96 (t, J = 9.0 Hz, 1H), 7.63 (t, J = 8.4 Hz, 1H), 7.41 (br s, 1H), 7.23-7.19 ppm (m, 2H); IR (KBr): 3072-2581, 1685, 1611, 1590, 1519, 1435, 1321, 1285, 1180, 904 cm -1 .

Stability tests .

 tests The as-synthesized samples (10.0 mg) were kept for 7 days in 20.0 mL of NMP, DMF, THF, DCM, EtOH, acetone, n-hexane, m-cresol, boiling water, HCl(12 M), H 2 SO 4 (18 M), HF (40%), NaOH (14 M), MeONa (5 M in MeOH), chromic acid lotion (0.1 M K 2 Cr 2 O 7 in concentrate H 2 SO 4 ) and LiAlH 4 (2.4 M in THF). The samples were washed with HCl (HCl/H 2 O = 2/1, for samples treated in LiAlH 4 ), water (for samples treated in aqueous solutions), MeOH (for samples treated in MeONa) and THF (for samples treated in organic solvents) respectively, followed by solvent exchanged by acetone and dried under vacuum at 85 °C for 12 hrs and subjected to PXRD, IR and N 2 adsorption analyses. Sample activation. Before gas-/liquid-phase adsorption experiments, as-synthesized PAE-COFs (50.0 mg) were subjected to Soxhlet extraction with H 2 O and acetone as the solvents for one day to exchange the trapped guest molecules. Subsequently, it was collected by filtration and activated by drying under vacuum at 100 °C for 24 hrs. Antibiotic uptake measurement. At room temperature, activated PAE-COFs (10.0 mg) was transferred to aqueous solutions (20.0 mL) containing given concentrations of TC, OTC or CTC (200.0 mg/L) at each pH value between 1 to 13. After 3 days of soaking (for equilibrium adsorption), the solutions were filtrated and diluted to suitable concentration (among the linearity range of standard curves), and UV-Vis spectrophotometry of the solutions was performed to characterize the adsorption performances of PAE-COFs under specific conditions (360 nm for TC; 268 nm for OTC; 267 nm for CTC). The adsorbed amount Qe for antibiotics was calculated by using the mass balance Equation: Qe = (C 0 -Ce)V/M, where C 0 and Ce are the initial and equilibrium concentrations of solutions of antibiotics [mg L -1 ], V is the volume of solution [L], and M the mass of adsorbent [g].

Figure 1 |

 1 Figure 1 | Strategy for preparing stable porous crystalline PAE-COFs. a, The model reaction between TFTPN and DHB yields a dioxin product, DCBD. b, Condensation of TFTPN and HHTP gives a 2D microporous crystalline PAE-COF with pore size of 16.8 Å, denoted JUC-505. The functionalized PAE-COFs, JUC-505-COOH and JUC-505-NH 2 , can be obtained after the post-modification of JUC-505. c, TFAQ and HHTP forms a 2D mesoporous crystalline PAE-COF with pore size of 28.4 Å, denoted JUC-506. d, Condensation of linear and triangular building units constructs a 2D porous structure based on the boron nitride net (bnn).

Figure 2 |

 2 Figure 2 | Crystallinity and stability. a and b, PXRD profiles of JUC-505 and JUC-506, respectively. Experimentally observed (black), Pawley refined (red), their difference (blue) and calculated using the AA stacking mode (magenta). c and d, Structure representation of the AA stacking mode of JUC-505 and JUC-506, respectively (O, red; N, blue; C, grey; H, white). e and f, PXRD patterns and IR spectra of JUC-505 after treatments under different chemical environments for one week.

Figure 3 |

 3 Figure 3 | Gas adsorption and separation. a and b, N 2 adsorption-desorption isotherms at 77 K for JUC-505 and JUC-506, respectively. Inset: pore size distributions from fitting the NLDFT model to the adsorption data. c and d, CO 2 separation properties over CH 4 and N 2 at 273 K for JUC-505 and JUC-506, respectively. e and f, Breakthrough curves for JUC-505 using the CO 2 /CH 4 and CO 2 /N 2 gas mixture.

Figure 4 |

 4 Figure 4 | Study of antibiotic uptakes. a-c, TCN, OTC and CTC uptakes at different pH (initial concentration = 200 mg/L, contact time = 72 h, sorbent dosage = 0.5 g/L, temperature = 298 K). Inset: molecular structure of TCN, OTC and CTC. O, red; N, blue; C, grey; H, white; Cl, cyan. d-f, Recycling test of TCN, OTC and CTC uptakes of JUC-505-COOH at pH = 1. g-i, Recycling test of TCN, OTC and CTC uptakes of JUC-505-NH 2 at pH = 13.

  C NMR (500 MHz) d: 145.5, 137.8, 125.5, 109.7, 93.4. IR (KBr): 3077, 3028, 2239, 1681, 1624, 1506, 1460, 1264, 1185, 1021, 982, 877 cm -1 . Anal. Calcd: C, 71.44; H, 1.20; N, 8.33. Found: C, 70.76; H, 1.17; N, 8.14; F, 0.95.

  1, 125.0, 113.8, 109.2. IR (KBr): 3071, 1676, 1578, 1519, 1493, 1270, 1179, 1041, 982, 864 cm -1 . Anal.

	Calcd: C, 75.01; H, 1.94. Found: C, 74.64; H, 1.92; F, 0.49.

Synthesis of JUC-505-NH 2 . JUC

  -505 powder (200.0 mg) and LiAlH 4

	C NMR (500 MHz) d: 163.2, 143.8, 137.8, 124.7, 108.6. IR (KBr): 3178, 3063,
	2987, 2237, 1717, 1682, 1596, 1517, 1682, 1596, 1518, 1428, 1268,1190, 1021, 978, 921, 866 cm -1 .
	Anal. Calcd: C, 64.18; H, 1.62. Found: C, 63.70; H, 1.59.

  C NMR (500 MHz) d: 145.5, 138.7, 125.0, 109.1, 25.0. IR (KBr): 3300, 3078, 2941, 2237, 1634, 1587, 1515, 1264, 1186, 1144, 1022, 977, 867 cm -1 . Anal. Calcd: C, 69.77; H, 3.51; N, 8.14.

	Found: C, 69.33; H, 3.38; N, 7.94.

Table 1 | Experimental and modelling results of gas adsorption on PAE-COFs.

 1

	PAE -COFs	BET	Pore volume	LPD [a] Temperature		Uptake mg g -1		Ideal adsorption selectivity [b]
		m 2 g -1	cm 3 g -1	Å	K	CO 2	CH 4	N 2	CO 2 /CH 4 CO 2 /N 2
					273	231.3 19.2 10.4	48.9	97.2
	JUC-505 1584	0.51	16.8	298	189.0 15.2	8. 8	33.8	65.6
					273	203.0 18.3 12. 5	33.5	69.0
	JUC-506 1655	0.71	28.4	298	159.2 13.1	8. 9	24.2	44.1

Table 2 | Stability after the treatment under various chemical environments for one week. [a]

 2

		CPMs	water	Boiling	HCl	H 2 SO 4	HF	NaOH	MeONa	LiAlH 4	acid	Chromic
		JUC-505									
		JUC-506									
		DhaTab-COF 23									
		TpPa-1 24									
	COFs	TPB-DMTP-COF 25									
		LZU-190 26									
		PI-COF-2 35									
		CS-COF 36									
		TpOMe-Pa1 37									
		UiO-66 38									
	MOFs	ZIF-8 39 HKUST-1 40									

Acknowledgements

Q.F., V.V., S.Q. and M.X. acknowledge the support of National Natural Science Foundation of China (21571079, 21621001, 21390394, 21571076 and 21571078), 111 project (B07016 and B17020), Guangdong and Zhuhai Science and Technology Department Project (2012D0501990028), and the program for JLU Science and Technology Innovative Research Team. Q.F and V.V. acknowledge the support from Thousand Talents program (China). V.V., Q.F., and S.Q. acknowledge the collaboration in the framework of China-French joint laboratory "Zeolites".

Data availability. The authors declare that the data supporting the findings of this study are available within the Article and its Supplementary Information files, or from the corresponding author on reasonable request.

Author contributions

Q.F., V.V., Y.Y. and S.Q. were responsible for the overall design, direction and supervision of the project. X.G. performed the experimental work. H.L. and Y.M. took SEM images and helped with the

Additional information

Supplementary information is available in the online version of the paper. Reprints and permissions information is available online at www.nature.com/reprints. Correspondence and requests for materials should be addressed to Q.F., V.V., and Y.Y.

Competing financial interests

The authors declare no competing financial interests.