Jianren Wang

Neus Vilà

Alain Walcarius

Redox-active vertically-aligned mesoporous silica thin films as transparent surfaces for energy storage applications

Keywords: Mesoporous silica film, transparent electrode, energy storage material, organic-inorganic hybrid, redox-active membrane, ferrocene, cobaltocenium, graphene oxide

Organic-inorganic hybrid membranes made of a high density of redox active moieties covalently bonded to the internal surfaces of vertically-aligned mesoporous silica thin films are relevant for applications in transparent energy storage devices. This is demonstrated here on the basis of functionalized transparent mesoporous silica thin films prepared on indium-tin oxide electrode from the combination of an electrochemically-induced self-assembly method (to generate azide functionalized silica) and a copper-catalyzed azide-alkyne click reaction (to derivatize the material with electroactive groups). The very small thickness (105 nm) and the uniformly distributed vertical mesochannels with ultranarrow diameter (2 nm) make the hybrid film a promising substrate that not only achieves transparency of 82%, but also provides large surface area to accommodate a high density of redox active species such as ferrocene. In such rigid and insulating porous membranes, the charge transfer reactions take place through a pure electron-hopping mechanism between adjacent redox sites, which are favored by the ordered and oriented mesostructure containing large amounts of uniformly distributed ferrocene functions in the mesochannels. Their performance results from both high charge transfer rates (electron hopping) and easy mass transport (fast diffusion of counter ions). The most effective system is the ferrocenefunctionalized silica film prepared from 40% organosilane, which is able to deliver a capacity of 105 C cm -3 (1.10 mC cm -2 ) at the current density of 0.4 A cm -3 . (with up to 48% capacity retention achieved at a charging time as short as 2.8 s). Such electrode can be associated to an electrodeposited graphene cathode in a solid-state battery-capacitor hybrid device which can deliver 0.74 mC cm -2 at a potential scan rate of 20 mV s -1 . The azide-functionalized mesoporous silica film is actually a versatile platform that can be functionalized with different redox molecules, as shown here for cobaltocenium moieties, which may broaden its application field.

INTRODUCTION

Transparent electronic systems have drawn considerable attention in recent years, representing the cutting-edge technology for next-generation electronic devices, such as touch screens, optical circuits, [START_REF] Wu | Transparent, Conductive Carbon Nanotube Films[END_REF] displays, [START_REF] Ju | Transparent Active Matrix Organic Light-Emitting Diode Displays Driven by Nanowire Transistor Circuitry[END_REF][START_REF] Park | Printed Assemblies of Inorganic Light-Emitting Diodes for Deformable and Semitransparent Displays[END_REF] and solar cells, [START_REF] Wang | TiO2 Nanotube Arrays Based Flexible Perovskite Solar Cells with Transparent Carbon Nanotube Electrode[END_REF][START_REF] Jeon | High-Performance Solution-Processed Double-Walled Carbon Nanotube Transparent Electrode for Perovskite Solar Cells[END_REF] etc. The in-situ integration of energy storage components acting as power source is a crucial step to developing such devices, which puts forward a transparency requirement for energy storage devices. [START_REF] Zhang | Graphene and MXene-Based Transparent Conductive Electrodes and Supercapacitors[END_REF][START_REF] Ma | A Silk Fabric Derived Carbon Fibre Net for Transparent Capacitive Touch Pads and All-Solid Supercapacitors[END_REF] Among various energy storage systems, supercapacitors featuring high power density, fast charge-discharge rates and long cycling life, are among the most prominent candidates for this emerging field. [START_REF] Zhang | Constructing Ultrahigh-Capacity Zinc-Nickel-Cobalt Oxide@Ni(OH)2 Core-Shell Nanowire Arrays for High-Performance Coaxial Fiber-Shaped Asymmetric Supercapacitors[END_REF] One of the most decisive factors to prepare the transparent energy storage electrode is to precisely control the thickness of active materials to keep high transparency. [START_REF] Yu | Improved Chemical Stability of ITO Transparent Anodes with a SnO2 Buffer Layer for Organic Solar Cells[END_REF] This could be basically always achievable by decreasing the thickness of the materials constituting the active layer, but meanwhile, the resulting energy storage performance would be also limited by too low amounts of active materials. Therefore, the main difficulty lies in how to improve the energy storage ability of materials to strike the right balance between the transparency and the energy/power density. Despite extensive efforts, the rational construction of electrode materials combining both excellent transparency and superior energy/power density remains a challenge.

According to the energy storage mechanisms, the "capacitive" materials can be classified into two major types: carbon-based materials for classical capacitors and faradic materials for so-called supercapacitors. As one representative of the former, graphene, a 2D carbon material (~0. 34 nm) with huge surface area (~2630 m 2 g -1 ) and outstanding conductivity (~2000 S m -1 ), is widely used to construct transparent energy storage devices based on ultra-thin coatings. [START_REF] Sawangphruk | Synthesis and Antifungal Activity of Reduced Graphene Oxide Nanosheets[END_REF] For example, Chou et al. prepared 4-layers graphene film with ultra-high transparency of ~90% by utilizing a chemical vapor deposition method. [START_REF] Xu | Laminated Ultrathin Chemical Vapor Deposition Graphene Films Based Stretchable and Transparent High-Rate Supercapacitor[END_REF] However, the capacity of this film was only 4.27 μF cm -2 at a scan rate of 20 mV s -1 , because charges/ions can only be stored on its surface via a pure physical electrical double-layer process. In contrast, faradic materials can always gain much higher energy density at similar thickness by involving additional chemical redox reactions. [START_REF] Patil | Flexible Asymmetric Solid-State Supercapacitors by Highly Efficient 3D Nanostructured α-MnO2 and h-CuS Electrodes[END_REF] Recently, the Yang's group reported a transparent Co3O4 nanoparticles electrode with the transparency of ~70% and a much higher energy density of 6.03 mF cm -2 at a scan rate of 1 mV s -1 . [START_REF] Liu | A Flexible, Transparent and Super-Long-Life Supercapacitor Based on Ultrafine Co3O4 Nanocrystal Electrodes[END_REF] Nevertheless, the rate performance of this electrode was severely restricted by its poor charge transfer kinetics and slow solid-diffusion process of counterions during the redox reaction. The capacity retention of this electrode was only 57% at a moderate scan rate of 20 mV s -1 , with unstable charges output at different rates which would make difficult its adaptation for practical applications. Therefore, seeking new redox systems likely to accelerate the electron transfer reactions is of great importance for the electrode material to gain high energy density while maintaining excellent rate performance.

On the basis of our understanding of these technical needs, a system based on an electron-hopping mechanism between redox adjacent sites inside an ordered, large surface area and transparent matrix may have the potential to improve the charge transfer processes. Different from the traditional electron transfer mechanism where the physical contact between the redox sites is required, the electrons can freely transfer between the close but isolated adjacent redox centers through an electron-hopping process (as that operating in redox polymers, [START_REF] Blauch | Effects of Long-Range Electron Transfer on Charge Transport in Static Assemblies of Redox Centers[END_REF] for instance). As investigated for self-assembled monolayers on electrodes, the kinetics of this electron hopping mechanism has been proven efficient enough as far as the active sites are close enough. [START_REF] Finklea | Electron-Transfer Kinetics in Organized Thiol Monolayers with Attached Pentaammine(Pyridine)Ruthenium Redox Centers[END_REF] However, the limited amount of active sites of monolayer-based systems restrict their energy density, and the implementation of redox molecular junctions in large-scale and three-dimensional assemblies remains challenging, especially in view of avoiding other possible charge transfer ways (e.g., translational motion of polymer chains in redox-active supramolecular systems [START_REF] Sato | Diffusion-Cooperative Model for Charge Transport by Redox-Active Nonconjugated Polymers[END_REF] ). Therefore, to best our knowledge, except for "between-particle" charge transport phenomena in metal oxidebased electrodes, [START_REF] Scibioh | Materials for Supercapacitor Applications[END_REF] there is no related work that systematically assesses the value of molecular electron-hopping processes in the energy storage field.

Our group has established an Electro-Assisted Self-Assembly (EASA) method to generate thin layers of vertically-aligned mesoporous silica on various conductive substrates, [START_REF] Walcarius | Electrochemically Assisted Self-Assembly of Mesoporous Silica Thin Films[END_REF][START_REF] Goux | Oriented Mesoporous Silica Films Obtained by Electro-Assisted Self-Assembly (EASA)[END_REF] and further achieved their surface functionalization by click chemistry (following Copper-catalyzed Azide-Alkyne Cycloaddition, CuAAC)) to introduce large amounts of organic groups onto the internal silica walls. [START_REF] Vilà | Electrochemically Assisted Generation of Highly Ordered Azide-Functionalized Mesoporous Silica for Oriented Hybrid Films[END_REF][START_REF] Vilà | Clickable Bifunctional and Vertically Aligned Mesoporous Silica Films[END_REF] If introducing redox active centers in such system, the resulting rigid organicinorganic hybrid becomes electroactive and electron-hopping becomes the only possible way to conduct the electrons among the redox centers due to the isolating nature of the silica walls. [START_REF] Vilà | Electrochemical Response of Vertically-Aligned, Ferrocene-Functionalized Mesoporous Silica Films: Effect of the Supporting Electrolyte[END_REF] Furthermore, the density of the redox molecules in the film can be adjusted by varying the components of the precursor solution. [START_REF] Vilà | Electrochemically Assisted Generation of Highly Ordered Azide-Functionalized Mesoporous Silica for Oriented Hybrid Films[END_REF][START_REF] Vilà | Clickable Bifunctional and Vertically Aligned Mesoporous Silica Films[END_REF] Both features make such thin, ordered and oriented, transparent organic-inorganic hybrid film an ideal platform to evaluate the interest of the electronhopping process in offering possibilities to construct new types of energy storage materials. This is thus what we have investigated here on the basis of electrodes modified with mesoporous silica films generated by EASA and functionalized with metallocene redox centers (ferrocene, cobaltocenium), going up to their association to a graphene counter electrode to form a solid-state battery-capacitor hybrid device.

EXPERIMENTAL

Chemicals and reagents

Tetraethoxysilane (TEOS, 98%), 3-chloropropyltrimethoxysilane (Cl-PTES, 97%) and graphite were purchased from Alfa Aesar and cetyltrimethylammonium bromide (CTAB, 99%) from Acros. Acetonitrile (ACN, 99%), dimethylformamide (DMF, 99%) and cyclohexane (99%) were obtained from Merck. Ethynylferrocene (97%), lithium perchlorate (95%), lithium chloride (95%), copper sulfate (99%), Sodium diethyldithiocarbamate trihydrate, and HCl (37%) were purchased from Sigma-Aldrich. Sodium nitrate (99%), ascorbic acid (97%), tetrabutylammonium bromide (99%) and poly(vinyl alcohol) (PVA, MW = 72000 g/mol) originated from Fluka. All compounds were used directly without further purification.

Preparation of 3-azidopropyltrimethoxysilane (AzPTMS). The AzPTMS precursor was synthesized by adding 2 g of Cl-PTES, 1.08 g sodium azide and 1.29 g tetrabutylammonium bromide) in 100 mL ACN, which was stirred and refluxed at 90 o C for 24 h under nitrogen atmosphere. Then, the ACN solvent was removed by rotary evaporation at 80 o C under a vacuum of 0.2 bar. The residual mixture was suspended in cyclohexane and filtered with filter paper. The residual cyclohexane was further removed by rotary evaporation at 80 o C under pressure of 0.2 bar and the oily Az-PTMS can be finally obtained. Yield: ~1.3 g, 65 %. [START_REF] Wu | Transparent, Conductive Carbon Nanotube Films[END_REF] H NMR (400 MHz, CDCl3): δ (ppm) 3.82 (s, 9H), 3.65 (t, 2H, 8.25 Hz), 1.24 (m, 2H), 0.75 (t, 2H, 8.31 Hz).

Thin films synthesis, electrodes and devices fabrication

The indium-tin oxide (ITO) plates (surface resistivity of 8~12 Ω) was obtained from Delta Technologies.

Preparation of azide-functionalized mesoporous silica films (Az-MS). The azide-functionalized vertically-aligned mesoporous silica thin films were prepared through the EASA method. [START_REF] Vilà | Electrochemically Assisted Generation of Highly Ordered Azide-Functionalized Mesoporous Silica for Oriented Hybrid Films[END_REF] Firstly, the silane sol was prepared by adding 120 mM TEOS and Az-PTMS (at Az-PTMS/TEOS molar ratios equal to ranging 20%, 40% or 60%) in a solution containing 20 mL ethanoland 20 mL water with 0.1 M NaNO3. Then, 1.35 mmol CTAB was added under stirring and the pH was adjusted to 3 by using 0.1 M HCl aqueous solution. The resulting sol was stirred at room temperature for 2.5 h for ageing. The mesoporous functionalized silica films were grown on ITO electrode in the potentiostatic mode by applying a cathodic potential of -1.3 V vs. Ag wire pseudo-reference for 20 s. The electrode surface was immediately rinsed with water and dried overnight at 130°C. The CTAB surfactant was extracted from the film by dipping it into an ethanolic solution containing 0.1 M HCl with moderate magnetic stirring for 10 min and the obtained sample was labeled as Az-

MS.

Preparation of the ferrocene functionalized mesoporous silica (Fc-MS) thin films. The above Az-MS films were further derivatized with ferrocene molecules by covalent bonding through click chemistry. [START_REF] Vilà | Electrochemically Assisted Generation of Highly Ordered Azide-Functionalized Mesoporous Silica for Oriented Hybrid Films[END_REF] Az-MS was first dipped into a mixed DMF/H2O (12/8 mL) solvent containing ethynylferrocene (10.0 mg, 4.6×10 -2 mmol), copper sulfate (3.5 mg, 1.4×10 -2 mmol), and ascorbic acid (7.5 mg, 4.3×10 -2 mmol) and the resulting mixture was protected from light and allowed to react for 24 h in order to perform the click reaction between the ethynylferrocene and the azido groups on the silica surface. After reaction, the film was collected and soaked in 1 mM sodium diethyldithiocarbamate in ethanol solution to remove the remaining copper catalyst. Followed by rinsing with water and drying at room temperature, the ferrocene functionalized mesoporous silica film can be finally obtained. The samples derived from 20%, 40%, 60% Az-PTMS in precursor sol were labelled as Fc-MS-20, Fc-MS-40, Fc-MS-60, respectively. Fc-MS-40 was primarily exploited in this work, the other samples Fc-MS-20 and Fc-MS-60 being only used for comparison purposes.

Preparation of a cobaltocenium functionalized mesoporous silica (Co + -MS) thin film. A Co + -MS-40 film was prepared similarly as the Fc-MS-40 sample, but by replacing ethynylferrocene by the ethynylcobaltocenium molecule, [START_REF] Wildschek | Ethynylcobaltocenium Compounds as Precursors for Bridged, Heteronuclear Oligometallocenes. Preparation and Reactions of Ethynyl-, Trimethylsilylethynyl-and Ferrocenylethynylcobaltocenium Salts[END_REF] to get the surface of the silica channels covered with covalentlybonded cobaltocenium moieties.

Preparation of the graphene counter-electrode. The graphene oxide (GO) was prepared from graphite powder following the classical Hummers' method. [START_REF] Hummers | Preparation of Graphitic Oxide[END_REF] The graphene electrode was prepared by electrophoretic deposition of GO onto ITO, by applying -1.2V (vs. Ag/AgCl) to the ITO electrode for 30s in 30 mL solution containing 50 mg GO and 0.32 g LiClO4 as supporting electrolyte.

Preparation of the asymmetric solid-state device. A PVA/LiCl gel was prepared by dissolving 6.37 g LiCl and 3 g PVA in 37 mL water at 90 o C. [START_REF] Xu | Approaching the Lithium-Manganese Oxides' Energy Storage Limit with Li2MnO3 Nanorods for High-Performance Supercapacitor[END_REF] The device was assembled with a sandwich configuration by using the Fc-MS-40 (1 cm -2 ) as the cathode, the graphene electrode (1 cm -2 ) as the anode and a PET plate (thickness ~1 mm) with a hole in the center as separator. Several droplets of the PVA/LiCl gel were added between the two electrodes and several clamps were used to fix the device.

Apparatus

Electrochemical measurements. Cyclic voltammetry experiments and recording charge and discharge curves were performed using µAutolab potentiostat with a three-electrode system. The as-prepared redox active films, an Ag/AgCl (3.5 M KCl) electrode and a stainless steel, were used as the working electrode, reference and counter electrodes, respectively.

Other characterization techniques. Transmission electron microscopy (TEM) investigation was performed with a JEOL ARM 200F -Cold FEG TEM/STEM equipped with a GIF Quantum ER.

Fourier transform infrared spectroscopy (FTIR) measurement was carried out using a Nicolet 8700 apparatus equipped with a diffuse reflectance accessory (Smart Collector). Grazing-Incidence Small-Angle X-ray Scattering (GISAXS) pattern was obtained using the Nanoviewer from Rigaku (CuK α radiation). The angle of incidence of the X-ray beam with respect to the film surface was set at 0.22°. The profilometry measurements were performed using the Dektak XT apparatus.

RESULTS AND DISCUSSIONS

Main characteristics of the ferrocene-functionalized mesoporous silica films

The overall preparation of ferrocene-functionalized vertically-aligned mesoporous silica thin films is schematically illustrated in Figure 1, involving the EASA generation of an azide-functionalized silica membrane onto the ITO surface, which is further derivatized with ferrocene units by coppercatalyzed azide-alkyne Huisgen reaction. 2) indicate a highly porous film composed of fairly ordered hexagonal mesopores, as also identified by the hexagonal diffraction spots of the corresponding Fast Fourier Transformation (FFT) diffractogram. The cross-section view of the sample (Fig. 2c) further reveals the vertical channels run directly from the top to the bottom of the film, and the thickness of the film is ~105 nm, a value which was also confirmed by profilometry.

Grazing Incidence Small Angle X-ray Scattering (GISAXS) is further used to analyze the internal structure of the film in order to obtain the statistically averaged lattice information. Two sharp diffraction spots can be observed in the equatorial plane of the scattering in (Fig. 2e), indicating the long-range ordered perpendicular structure of the film, along with a very shallow ring in-plane arising from few silica beads with wormlike mesostructured on the film surface (as commonly observed for films prepared by EASA [START_REF] Gamero-Quijano | Vertically Aligned and Ordered One-Dimensional Mesoscale Polyaniline[END_REF][START_REF] Guillemin | Electrogeneration of Highly Methylated Mesoporous Silica Thin Films with Vertically-Aligned Mesochannels and Electrochemical Monitoring of Mass Transport Issues[END_REF] Moreover, the qy value of 1.81 nm -1 can be obtained by measuring the distance between the light source and the diffraction spots (Fig. 2f). This value can be used to calculate the lattice spacing, d, of the structure using the equation d = 2π/q, 28 giving rise to a d value equal to 3.47 nm, which is consistent with TEM results. Similar mesostructure characteristics can be observed for the less functionalized Fc-MS-20 film, in agreement with previous observations, [START_REF] Vilà | Electrochemically Assisted Generation of Highly Ordered Azide-Functionalized Mesoporous Silica for Oriented Hybrid Films[END_REF] whereas the film generated from a sol containing 60% of the organosilane AzPTMS (Fc-MS-60) was no more ordered (because of interference in the self-assembly of the mesophase structure [START_REF] Lim | Comparative Studies of Grafting and Direct Syntheses of Inorganic-Organic Hybrid Mesoporous Materials[END_REF] ), as illustrated in Supporting Information (Fig. S1). Besides, benefiting from the its small thickness and vertical channels, the film is highly transparent, and the UV-Vis spectrum demonstrates a transmittance of 82% that can be achieved at the wavenumber of 550 nm (Fig. 2d), which makes possible its use in the transparency-required devices. The incorporation of the organic groups and effectiveness of click coupling with ferrocene moieties can be checked by infrared spectroscopy (IR). Comparing the evolution of the azide signature (stretching asymmetric band located at 2095 cm -1 ) in the IR spectra for samples prepared from sols containing 20 to 60% AzPTMS (Fig. S2a) clearly indicates the presence of increasing amounts of azide groups. After the click reaction (Fig. S2b), the area of this absorption band decreased by 85% (for Fc-MS-20), by 79% (for Fc-MS-40) and by 75% (for Fc-MS-60), confirming that a large part of the azido groups took part in the click reaction (100% yield cannot be reached for steric hindrance reasons). The formation of triazole units incorporating ferrocene functions can be also noticed on the figure, via their characteristic band located at 1610 cm -1 , 30 yet located in a noisy region of the spectra.

Electrochemical characterization of the ferrocene-functionalized films

The energy storage performance of the as-prepared Fc-MS samples was first evaluated by cyclic voltammetry (CV) in 1 M LiCl aqueous solution (see parts a & b in Figure 3). Comparing the CV curves recorded at a scan rate of 5 mV s -1 for samples bearing various ferrocene contents (Fig. 3a) reveals the absence of any noticeable signal for the azide-functionalized film (i.e., without ferrocene grafted onto the silica walls) while well-defined redox peaks, yet significantly distinct in shape, can be observed in the potential window from 0.0 to +0.6 V vs. Ag/AgCl for the Fc-MS films. Clearly the best performance is achieved for Fc-MS-40 (i.e., prepared from 40% AzPTMS and 60 %TEOS as silane precursors), from both view points of smaller anodic-to-cathodic peaks separation and larger peak currents. This can be explained taking into accounts the electron transfer mechanism involving the electron hopping between adjacent ferrocene sites attached to the insulating silica film and associated counter-anions transport through the mesopore channels to maintain electroneutrality (Eq. 1): [START_REF] Vilà | Electrochemical Response of Vertically-Aligned, Ferrocene-Functionalized Mesoporous Silica Films: Effect of the Supporting Electrolyte[END_REF] Fc -1 e -+ X - (sol)  Fc + ,X -

(film) (1)
(with Fc = ferrocene; Fc + = ferricinium; X -= counter-anion; sol = solution)

In doing so, the Fc-MS-40 sample is characterized by the most attractive energy storage behavior, exhibiting an almost ideal surface-controlled redox reaction (with a pair of intense bell-shaped peaks located at +0.35 V vs. Ag/AgCl, with a peak-to-peak separation of 12 mV). This is due to the large density of redox-active sites in the material enabling efficient electron hopping between close together ferrocene molecules uniformly distributed on the mesopore walls and easy transport of charge-compensating ions thanks to the regular and oriented mesostructure. By contrast, the Fc-MS-20 sample containing a lower amount of ferrocene (nearly twice as less as Fc-MS-40) not only resulted in (expected) lower peak currents (by almost two times) but also resulted in less reversible diffusion-limited signals (peak-to-peak separation equal to 70 mV) arising from redox sites located at a longer distance between each other (slower pseudo-diffusion of the electrons in the hopping mechanism). [START_REF] Compton | Understanding Voltammetry[END_REF] On the other hand, the sample with more ferrocene loading amount (i.e., Fc-MS-60 prepared from 60% AzPTMS and 40 %TEOS as silane precursors) also exhibited a smaller current response and the redox peaks shifted to higher potential values (i.e., +0.38 V, with a peak-to-peak separation of 28 mV). This can be explained by the loss of mesostructural order (Fig. S1) and steric hindrance limiting the number of electrochemically accessible ferrocene molecules in the film and slowing down the charge compensation process (Eq. 1). Similarly, a nonordered film prepared by drop coating did not give any noticeable signal for ferrocene, confirming again the interest of EASA to provide highly electroactive mesoporous films. The Fc-MS-40 film was thus chosen as the most appropriate for energy storage application, and used exclusively afterwards. Note however that increasing the potential scan rate contributed to induce charge transfer limitations in the Fc-MS-40 film (Fig. 3b), the fast thin-layer behavior being maintained up to 20 mV s -1 and then becoming more and more rate-limited at faster scan rates (Fig. S3).

In order to evaluate the energy storage performance, the galvanostatic charge-discharge curves of Fc-MS-40 were recorded at various current densities from 0.4 to 20 A cm -3 (Fig. 3c). Different from the typical triangular curve of double-layer capacitance, the charge/discharge curves are distorted but with no clear platform (the sign of a battery behavior). This phenomenon can be explained by the fast surface redox reaction without limitation of the diffusion process. [START_REF] Jiang | Definitions of Pseudocapacitive Materials: A Brief Review[END_REF] Besides, the similar charge and discharge time also proves that there is no side reaction, and almost all stored charges can be released during the discharge process. The related coulombic efficiency of Fc-MS-40 is given in Supporting Information (Fig. S4). The capacities are calculated based on the galvanostatic charge-discharge curves and expressed as a function of current density (Fig. 3d). The Fc-MS-40 film can deliver a capacity of 105 C cm -3 (1.10 mC cm -2 ) at the current density of 0.4

A cm -3 and the capacity retention can achieve 48% (50.4 C cm -3 ) at the current density of 20 A cm -3 (2.8 s charging time). By contrast, the capacities of Fc-MS-20 and Fc-MS-60 were lower than that of Fc-MS-40 at the same current density (Fig. S5), consistent with the CV results and confirming the most attractive features of Fc-MS-40. The Fc-MS-40 film electrode also exhibits a robust cycling performance with 75% capacity retention after 1000 successive CV cycles at a scan rate of 200 mV/s (Fig. 3e) owing to the durability of covalently bonded ferrocene molecules.

Moreover, the comprehensive plots between areal capacity vs. transmittance at 550 nm for Fc-MS-40 and materials reported in recent years (Fig. 3f) reveal that Fc-MS-40 can achieve a good balance in terms of high capacitance and excellent transparency. This material also exhibits a good volumetric energy density, when compared with similar thin-film systems reported in literatures (Fig. S6), [START_REF] Niu | A Repeated Halving Approach to Fabricate Ultrathin Single-Walled Carbon Nanotube Films for Transparent Supercapacitors[END_REF][START_REF] Byon | Thin Films of Carbon Nanotubes and Chemically Reduced Graphenes for Electrochemical Micro-Capacitors[END_REF][START_REF] Chen | Transparent and Stretchable High-Performance Supercapacitors Based on Wrinkled Graphene Electrodes[END_REF][START_REF] Song | General Method for Large-Area Films of Carbon Nanomaterials and Application of a Self-Assembled Carbon Nanotube Film as a High-Performance Electrode Material for an All-Solid-State Supercapacitor[END_REF][START_REF] Higgins | Avoiding Resistance Limitations in High-Performance Transparent Supercapacitor Electrodes Based on Large-Area, High-Conductivity PEDOT:PSS Films[END_REF][START_REF] Zhang | Highly Flexible and Transparent Solid-State Supercapacitors Based on RuO2/PEDOT:PSS Conductive Ultrathin Films[END_REF] due to the high density of redox molecules in Fc-MS-40 (1 mmol cm -3 ). RuO2/PEDOT, [START_REF] Zhang | Highly Flexible and Transparent Solid-State Supercapacitors Based on RuO2/PEDOT:PSS Conductive Ultrathin Films[END_REF] SWCNT, [START_REF] King | Percolation Effects in Supercapacitors with Thin, Transparent Carbon Nanotube Electrodes[END_REF] carbon nanocup [START_REF] Jung | Flexible Supercapacitors from Nano-Engineered Carbon Films[END_REF] and Ti3C2Tx. [START_REF] Zhang | Transparent, Flexible, and Conductive 2D Titanium Carbide (MXene) Films with High Volumetric Capacitance[END_REF] Electron-hopping is indeed the only way to transfer electrons in our system because of the insulating character of the silica walls (the resistivity of silica is very huge, ranging from 10 12 ~10 14 /cm 42 ). This is also confirmed by a control experiment showing the absence of signal for redox species in solution when the electrode surface is covered with a surfactant-templated mesoporous silica film (Fig. S7). In the goal to characterize the electron-hopping processes in a more quantitative way, one can go deeper in the analysis of the CV curves of ferrocene-functionalized silica films recorded at different scan rates (Fig. 3b, Fig. S8). Illustrative results are shown on (0.90) over a wide sweep rate range varying from 5 mV s -1 to 50 mV s -1 and then tended to decrease (Fig. 4a). This suggests that the energy storage of Fc-MS-40 is an almost exclusive surfacecontrolled process and that diffusion starts to become a bottleneck only at larger scan rates. By contrast, b values calculated for Fc-MS-20 (Fig. S8a&b) are only around 0.4 (probably due to slow electron diffusion in the hopping process between more distant ferrocene moieties), whereas the ferrocene-functionalized film with higher density of redox centers but lacking mesostructural order (i.e., Fc-MS-60), gave rise to b values around 0.8 (Fig. S8c&d) indicating an overall charge transfer mechanism mainly governed by surface-controlled redox processes but also limited by the slow diffusion of counter ions. As the result, Fc-MS-40 shows the best rate performance among the three Fc-MS samples even if suffering from some diffusional limitations at higher potential scan rates but much less than Fc-MS-20 and Fc-MS-60 samples (Fig. S9). Besides, to quantitatively evaluate the contribution of surface-controlled current at a fixed scan rate, the electrochemical data extracted from CV measurements for Fc-MS-40 can be further fitted with the following equation:

𝑖(𝑉) = 𝑘 𝑣 + 𝑘 𝑣 .
; where i(V) is the current at a given potential and k1v and k2v 0.5 represent the surface-controlled current and diffusion-controlled current, respectively. [START_REF] Tian | Metal-Organic Frameworks Mediated Synthesis of One-Dimensional Molybdenum-Based/Carbon Composites for Enhanced Lithium Storage[END_REF] The results (Fig. 4b) demonstrate that the greatest part of the total faradic current comes from a surface-controlled contribution (91.2% at the scan rate of 20 mV s -1 ), pointing out further that the existence of a high density of the redox species contributes to facilitate the electron hopping process (as reported for other densely packed redox-active systems [START_REF] Blauch | Effects of Long-Range Electron Transfer on Charge Transport in Static Assemblies of Redox Centers[END_REF][START_REF] Finklea | Electron-Transfer Kinetics in Organized Thiol Monolayers with Attached Pentaammine(Pyridine)Ruthenium Redox Centers[END_REF] ) and the suitable pore structure to reduce the ions diffusion resistance (Fig. 4c). This scheme also illustrates that no electron transfer can occur through the isolating silica walls, but only via electron hopping between adjacent ferrocene moieties (thanks to the flexibility of the alkyl arm linking them to the silica) and concomitant charge neutralization by the electrolyte ions, of course after a first electron transfer between the electrode surface and the ferrocene group located close to this surface. Finally, one can estimate the charge transfer resistance for the electron-hopping process by impedance spectroscopy (see the impedance spectrum of the Fc-MS-40 electrode in Fig. S10), from which a value of ~ 60 Ω can be observed, confirming the possible long-range charge transport in a film, however, made of an insulating material. [START_REF] Park | Printed Assemblies of Inorganic Light-Emitting Diodes for Deformable and Semitransparent Displays[END_REF] . The reason could be the existence of trace amount of oxygen in solution (even after bubbling with nitrogen) which would chemically oxidize back the cobaltocene (generated during the electrochemical reduction process) into cobaltocenium. Based on the discharge curves, the capacity of Co + -MS-40 can reach 105 C cm -3 at the current density of 4 A cm -3 and still be able to retain 68% at the current density of 40 A cm -3 (Fig. 5c). The solid-state hybrid-device is further assembled in a two-electrode set-up, by using the Fc-MS-40 as the anode, polyvinyl alcohol/lithium chloride gel as separator electrolyte, and electrodeposited graphene as the cathode material. The capacity of the electro-deposited graphene electrode is adjusted by controlling the deposition time in order to get a value equal to the charge storage capacity of the Fc-MS-40 electrode (see the corresponding CV curves in different potential window with 1 M LiCl electrolyte in Supporting Information, Fig. S11). The energy storage characteristics of the as-prepared Fc-MS-40//Graphene hybrid-capacitor device are illustrated in Figure 6. As shown, it is able to work over the potential window from 0V to 1.2V at various scan rates ranging from 20 mV/s to 1000 mV/s (Fig. 6a). Despite the hybrid nature of the asymmetrical device, a pair of broad voltammetric signals corresponding to the redox processes related to ferrocene can still be observed in this potential window. The CV curves are well defined and symmetrical even at the scan rate of 1000 mV s -1 , indicating the good rate performance. According to the literature, it is better to use the unit of battery (C) rather than that of capacitor (F), especially because the present system is actually a battery-capacitor hybrid device. [START_REF] Zuo | Battery-Supercapacitor Hybrid Devices: Recent Progress and Future Prospects[END_REF] By integrating these CV curves, the specific capacities are calculated for all scan rates investigated here, based on the total areas of the two electrodes (Fig. 6b). This device can deliver 0.74 mC cm -2 at the scan rate of 20 mV s -1 , and it can still maintain 57% at a scan rate of 500 mV/s (2.4 s for the charging/discharging time) and even 45% at a scan rate of 1000 mV s -1 (only 1.2 s for the charging/discharging time). This outstanding performance demonstrates that the redox reaction rate of the assembled redox molecules system through the electron-hopping process is fast and comparable to its double-layer type graphene counter electrode. The charge-discharge curves (Fig. 6c) exhibit, in contrast to battery, a distorted triangle charge-discharge shape without obvious plateau, also revealing the pseudo-supercapacitor nature of the Fc-MS-40//Graphene device, while the symmetrical curves indicate a good coulombic efficiency. The cycling stability of the device, as also determined by the CV method (see the corresponding results in Fig. S12) is quite robust as 74% capacity retention is maintained after 500 successive cycles. Ragone plots (Fig. 6d) further reveal that a high energy density of 0.13 μWh cm -2 can be achieved at a power density of 12.8 μW cm -2 , a value still remaining as high as 0.07 µWh cm -2 at higher power density (180 μW cm -2 ).

Figure 1 .

 1 Figure 1. Schematic illustration of the preparation process of the ferrocene-functionalized

Figure 2 .

 2 Figure 2. Structural characterization of the Fc-MS-40 film. (a-c) TEM micrographs: top views (a-

Figure 3 .

 3 Figure 3. Energy storage performance of the ferrocene-functionalized film electrodes. (a)

Figure 4 .

 4 Figure 4. First of all, the redox peak currents (𝑖) vs. scan rates (𝑣) are fitted with the equation of

Figure 4 .

 4 Figure 4. Analysis of the redox process of Fc-MS-40. (a) Variation of the anodic and cathodic

3. 3 .

 3 Extension to cobaltocenium-functionalized mesoporous silica thin films Not only limited to ferrocene, further derivatization of the azide-functionalized mesoporous silica films can be extended to other species with the only requirement to contain an alkyne function in their structure. In this sense, a cobaltocenium-functionalized vertically-aligned silica thin film (Co + -MS-40) has been prepared by following an analogous procedure to the one previously described in the case of the Fc-MS films. As shown in Figure5(part a), the CV curves of the Co + -MS-40 exhibits a pair of well-defined redox peaks at potentials around -1 V, proving that redox reactions of the immobilized cobaltocenium/cobaltocene system can also occur through the electron-hopping process in the mesoporous film. Similar to those of Fc-MS-40, the redox curves are symmetrical even at the scan rate of 500 mV s -1 , illustrating the fast charge transfer and associated ion transport processes. The galvanostatic charge-discharge curves of Co + -MS-40 were also recorded at various current densities from 4 to 20 A cm -3 and the obtained results (Fig.5b) are indicative of fast surface-controlled redox reactions as no obvious platform can be observed during the charge-discharge processes. However, compared with that of the Fc-MS-40 sample, the coulombic efficiency of Co + -MS-40 is relatively low, only ~36% at the current density of 4 A cm -

Figure 5 .

 5 Figure 5. Energy storage performance of the cobaltocenium-functionalized film electrodes. (a)

Figure 6 .

 6 Figure 6. Energy storage performance of the asymmetrical solid-state device (Fc-MS-40 as the

) at a current density of 0.4 A cm-3 , and retain 48% of its capacity at a short charging time of 2.8

ACKNOWLEDGMENT

JW acknowledges a financial support from the China Scholarship Council for his doctoral studies.

s. A solid-state battery-capacitor hybrid device was also prepared by using graphene as counterelectrode, and it can deliver an energy density of 0.07 µWh cm -2 at the high power density of 180 μW cm -2 . Moreover, such transparent nanoporous silica membranes can be manufactured with other ethynyl-containing redox molecules (e.g., ethynylcobaltocenium), which broadens their possible application scenarios in the field of electrochemical energy storage.

ASSOCIATED CONTENT Supporting Information.

The following file as available free of charge.

Additional experimental data (characterization and electrochemical measurements), especially for

Fc-MS-20 and Fc-MS-60 samples, as well as the asymmetric solid-state device (PDF)

AUTHOR INFORMATION

Corresponding Author * E-mail: alain.walcarius@univ-lorraine.fr (A.W.) ORCID Alain Walcarius: 0000-0003-3633-200X

Notes

The authors declare no competing financial interest.


Hossein Derakhshankhah

Atiyeh Hosseini

Fereshteh Taghavi

Samira Jafari

Alireza Lotfabadi

Mohammad Reza Ejtehadi

Sahba Shahbazi

Ali Fattahi

Atiyeh Ghasemi

Ebrahim Barzegari

Mohammad Javad Hajipour

Mina Evini

Ali Akbar Saboury

Eng-Poh Ng

Seyed Mehdi

Kamali Shahri

Hussein Awala

Svetlana Mintova
email: mintova@ensicaen.fr

Rassoul Dinarvand
email: dinarvand@tums.ac.ir

Morteza Mahmoudi
email: mmahmoudi@bwh.harvard.edu

Molecular interaction of fibrinogen with zeolite nanoparticles

Keywords: Zeolite nanoparticles, Fibrinogen, Conformational change, Protein-nanoparticles interactions

Introduction

Nanoparticles (NPs) surfaces are instantaneously covered by a layer of biomolecules (so-called protein corona), when they come into contact with physiological fluids such as blood plasma [1][2][3][4] .

The competitive adsorption of proteins on the NP surface is dependent on the physicochemical properties of NPs, incubation condition (time and temperature), and plasma protein alterations (protein concentration/structure) mediated by diseases [4][5][6][7][8][9][10] . In fact, the formation of protein corona changes the surface properties of NPs and it provides a biological mask, which is being "seen" by the biological systems such as cells 2,[11][12][13][14][15] . The type, concentration and configuration of the participated proteins in the corona layer can give useful information for predicting the biological fate of NPs including their pharmacokinetics and biodistribution 5,9,[16][17][18][19] .

During last few years, many studies were devoted on achieving in-depth information on the structural integrity of proteins after participating in the corona structure; As expected, the preliminary results revealed that the degree of structural rearrangement in protein is dependent on the protein type and physicochemical properties of NPs. 9,20,21 . As an example of the role of protein type, fibrinogen demonstrated higher structural changes compared to albumin when interacting with graphene oxide 9 and silica NPs 21 . Regarding the NPs' properties, hydrophobic surfaces induce more structural changes to both albumin and fibrinogen compared to hydrophilic surfaces 21 .

Moreover, the structural variations of proteins can change their physiological functions. For example, we demonstrated that transferrin experiences irreversible structural changes and loose the main functionality (i.e., transport of iron among cells) after adsorption to the surface of iron oxide NPs 22 . Along with the functional changes, the structural changes in some proteins may activate inflammatory responses. For instance, structural changes in fibrinogen (i.e., exposure of its C-terminous of γ chain (γ [377][378][379][380][381][382][383][384][385][386][387][388][389][390][391][392][393][394][395] )), after interaction with poly(acrylic acid)-coated gold NPs, can provoke the inflammation response and downstream unwanted cascade pathways 18 .

The structural changes in proteins, after participating in the corona layer, can affect the cellular uptake mechanism of nanoparticles. For example, bovine serum albumin (BSA) proteins adsorbed on the surface of cationic polystyrene NPs experienced structural changes, while the bounds to anionic polystyrene remained intact 23 . These variations in the protein structure at the corona layer led to different interactions of the NPs with the cell receptors, i.e. the BSA-coated cationic and anionic NPs bounded to scavenger and native albumin receptors, respectively. In a similar study, Minchin group 24 demonstrated that the silica NPs have capability to change the albumin's structure, leading to exposure of a typical hidden epitope, which is exclusively recognized by macrophages expressing class A receptor. Prapainop et al. 25 demonstrated that apolipoprotein conformational was changed after binding to quantum dots and it substantially increased their uptakes by macrophages. Thus the challenges associated with the structural integrity of corona proteins raised serious concerns about the in vivo fate/behavior of NPs.

Zeolite NPs have promising potential medical applications including drug delivery, imaging, and microbial infection and neurodegenerative diseases therapy [26][27][28][29] . Probing the structure of adsorbed proteins on the surface of zeolite NPs is of great interest as it helps scientists in the field to maximize the therapeutic efficacy of these particles while maintaining the toxic effects at minimal level. Recently, we demonstrated that fibrinogen has the most contribution, among other plasma proteins, in the corona structure of the zeolite NPs 30,31 . However, the molecular interactions between zeolite NPs and fibrinogen are inadequately understood. The aim of this work is to understand the nature of interaction of zeolite nanoparticles with fibrinogen through probing the binding kinetics, thermodynamic parameters and structural changes using a wide range of experimental approaches including fluorescence and UV-visible spectroscopy, and circular dichroism (CD) spectroscopy.

Experimental Section

Synthesis and characterization of EMT zeolite NPs

The EMT NPs were synthesized as follows: 32 Aluminate solution was prepared by mixing 9.07 g of sodium aluminate (Strem Chemicals), 1.61 g of sodium hydroxide (Prolabo, 97%) and 100.00 g of double distilled water. The mixture was stirred for 10 min before another 44.00 g of sodium hydroxide was added to the solution. The solution was continuously stirred until a resulting clear aluminate suspension was obtained. The silicate solution was prepared by dissolving 57.69 g of sodium silicate (Prolabo, 27% SiO2, 8% Na2O) and 20.00 g of sodium hydroxide in 80.00 g of double distilled water. The preparation of both solutions involved exothermic reaction and hence both solutions should be cooled down in an ice bath (4 C). The aluminate solution was then slowly poured into the silicate solution under magnetic stirring (800 rpm); a white colloidal suspension with a molar composition of 1Al2O3:5.15SiO2:18.45Na2O:240H2O was obtained. The resulting suspension was stirred for additional 5 min before it was crystallized at 30 C for 36 h under static condition. The resulting EMT zeolite nanocrystals were then centrifuged (20000 rpm for 1 h) and purified with distilled water until the pH of the suspension reached 7.

The XRD patterns of zeolite NPs were obtained using a PANalytical X'Pert PRO XRD diffractometer (step size 0.01, 1.5 seconds per step, Cu-Kα radiation). The morphology and crystallite size of zeolite samples were inspected by a transmission electron microscope (TEM) (JEOL Model 2010 FEG system, 200 kV). The average size of zeolite NPs was determined by randomly counting 50 particles through TEM observations obtained in different regions. The hydrodynamic size and zeta potential, ξ, of colloidal solution of zeolite NPs (1 wt%, pH 7.5, 25 C) were measured by a Malvern Zetasizer Nano Series equipment. The surface charge density, σ, was calculated using the Grahame equation (Eq. 1):

           =  T k 2 e sinh T N k c 8 B 0 A B 0 0 (Eq. 1)
where c0 is the concentration of zeolite (0.1%) in suspension in unit m 3 , εε0 is the dielectric permittivity of EMT zeolite (1.3547 × 10 -11 AsV -1 m -1 ), kB is the Boltzmann constant (1.381 × 10 -23 J K -1 ), 0 is the surface potential or zeta potential of the zeolite suspension (-45.7 mV), e is the electronic charge (1.602 × 10 -19 C), and T is the absolute temperature (298 K).

The surface charge of EMT NPs, Q, was computed by using Eq. 2:

ratio Al / Si S Q BET   = (Eq. 2)
where SBET is the specific surface area (m 2 g -1 ) and Si/Al ratio is the silicon to aluminum ratio of the zeolite NPs.

The elemental analysis was characterized by using a Phillips X'Unique X-ray Fluorescence (XRF) spectrometer. The porosity of zeolite NPs was analyzed by a Micromeritics ASAP 2010 nitrogen adsorption analyzer. Prior to analysis, the powder was dehydrated at 250 °C under vacuum overnight. The specific surface area was calculated using the BET equation while the external surface area and micropore volume were computed using the t-plot technique. The pore sizes were calculated using the Density Functional Theory (DFT) model provided by Micromeritics software.

Fluorescence spectroscopy

Fluorescence quenching of fibrinogen was measured in the presence of increasing concentrations of zeolite NPs using spectrofluorometer (Hitachi MPF-4, equipped with a thermostatically controlled cuvette compartment). The fluorescent property of fibrinogen is related to its aromatic amino acids such as tryptophan (Excitation: 280 nm; Emission: 360 nm). This analysis was performed at different temperatures (25, 37, 40 and 42 °C) to determine the effect of temperature on quenching process. The protein and NPs concentrations were 0.588 µM and 17-102 µM, respectively.

The fluorescence quenching can be quantified by the following Eq. 3:

Q = (F0 -F) / F0 (Eq. 3)
where F0 and F are fluorescence intensities in the absence and presence of NPs, respectively 33 .

By assuming that the NPs-protein binding occurs at the equilibrium condition, the quenching data was fitted for Q to determine an association constant (Kα) to describe the NP-protein interaction. The association constant K is the reciprocal of the "dissociation constant", kD. The Stern-Volmer equation (SI: Eq. S1) is used to assess the efficiency and mechanism of fluorescence quenching. As fibrinogen potentially binds to NP surface with different domains/binding sites, exhibition of cooperativity in the binding equilibrium is expected. The binding kinetics and cooperativity in fibrinogen-zeolite interaction were determined using Hill equation (SI, Eq. S3).

Circular dichroism (CD) spectroscopy

Fibrinogen (1 mg mL -1 ) was incubated with different zeolite concentrations (concentration range from 4.25 to 119 µM) for 1 h at 37 °C. The CD spectra of resulting solutions were recorded at the wavelengths between 190 and 260 nm with an average of 20 scans using Aviv model 215 spectropolarimeter (Lakewood, NJ, USA). All CD measurements were performed at room temperature (25 °C), in a 1 mm path cuvette. The ellipticity was represented in millidegrees.

Results and discussion

Characterization of EMT zeolite nanoparticles

Highly crystalline hexagonal EMT type zeolite NPs was synthesized and stabilized in water suspensions. 32,34 The particle size and the morphology of NPs were determined by dynamic light scattering (DLS) and transmission electron microscopy (TEM) techniques. The EMT nanocrystals indicated unimodal particle size distribution (8-20 nm) and hexagonal morphology (SI, Figure S1a,b). The porosity of the EMT zeolite was also measured (SI, Figure S2). The EMT nanocrystals showed a combination of Types I and IV adsorption isotherm curves, indicating the presence of both micro-and textural meso-porosities (SI, Figure S2). 35 The size of the micropores determined using the DFT model was 0.73 nm, which is in agreement with the size of the hypocage (0.75 nm × 0.65 nm in diameter) and hypercage (0.73 nm × 0.73 nm in diameter) of the EMT-type zeolite. In addition, the size of the mesopores was 3.12 nm, which is due to the close packing of the nanoparticles resulting in textural (inter-particles) porosity. The EMT zeolite nanoparticles have a surface area of 720 m 2 g -1 , external surface area of 260 m 2 g -1 , and total pore volume of 1.32 cm 3 g -1 . The chemical composition of the EMT zeolite was analyzed using XRF spectroscopy. The unit cell composition was Na88(AlO2)88(SiO2)104, with the Si/Al ratio equal to 1.17, suggesting that the zeolite surface was highly negatively charged.

Hence, the high Na content was detected to counter balance the negative charge originated from the (Al-O-Si) -groups. The surface charge and the charge density of EMT NPs were also calculated (see Methods section). The result showed that the EMT zeolite NPs contained a surface charge of -4356 mC g -1 due to the high surface area and high alumina content in the framework. As a result, the high surface charge density (-6.05 mC m -2 ) was calculated (SI, Table S1). The high surface charge and high charge density of EMT NPs are thus in line with their hydrophilic and polar nature.

Interaction of fibrinogen with EMT zeolite nanoparticles

Fluorescence spectroscopy is widely used to study the interaction of NPs and proteins 36 . It is known that the intrinsic tryptophan (TRP) fluorescence have significant changes through protein unfolding, leading to the exposure of internal TRP that is typically hidden in the folded state [START_REF] Lakowicz | Principles of fluorescence spectroscopy[END_REF] .

We probed fibrinogen-folding variations during the interaction with zeolite NPs using the leverage of TRP via monitoring the protein folding changes. The fluorescence spectra of fibrinogen were measured in the presence of various concentrations of EMT NPs at different temperatures (25, 37, 40 and 42 °C) (Figure 1). The maximum fluorescence peak (λmax) of fibrinogens incubated with EMT NPs was observed at 342 nm, and the fluorescence intensity of fibrinogen gradually decreased as the zeolite NPs concentration increased. These results indicate that the EMT NPs, which act as a quencher, have strong interactions with the fibrinogen. In this case, one may speculate that zeolite-bounded fibrinogen may experience structural rearrangement leading to the change in microenvironment of internal TRP. Molecular interaction between protein and zeolite NPs assists to perceive the origins in addition to have safe zeolites with more predictable biological efficacy. Therefore, several involved parameters in the fibrinogen-zeolite interaction are probed as described in the following sections.

Quenching behavior of EMT zeolite nanoparticles

It is well-recognized that NPs quench the proteins in a dynamic or static manner [START_REF] Lakowicz | Principles of fluorescence spectroscopy[END_REF] . Depending on the incubation temperature, the fluorophore-quencher complex is formed before (static) and/or after (dynamic) fluorophore excitation [START_REF] Zhang | [END_REF] . The quenching behavior of NPs is strongly dependent on the temperature at which proteins incubate with NPs. The molecular mechanism of quenching is proposed using the Stern-Volmer equation (SI, Eq. S1). The Stern-Volmer plots and the constants of fibrinogen quenching induced by different concentrations of EMT NPs at various temperatures are shown in Figure 2 and Table 1, respectively. As can be seen, a decrease in both the Stern-Volmer quenching constants and the slope of the Stern-Volmer plot is observed at higher temperatures. This result indicates that fibrinogen is mainly quenched in a static manner.

Previously, we showed that slight change in incubation temperature affects the protein decoration on the surface of NPs and consequent biological responses 40 . Depending on the incubation temperature, different types and quantities of plasma proteins were adsorbed on the NPs 6 . This means that plasma proteins interact with NPs to different extent. Change in protein corona decoration is partly related to the protein conformational changes occurred at high temperature 7 . Thus, it can be suggested that newly exposed epitopes/sequences, as a result of protein unfolding, determine how proteins interact with the EMT zeolite NPs.

Binding sites of protein on EMT zeolite nanoparticles

Fibrinogen showed different orientations on the NPs surface, which is depending on the physicochemical properties of NPs (surface chemistry and size/curvature), the type of presorted proteins and the degree of NP surface coverage 21,41,42 . For example, fibrinogen tends to be adsorbed on small-sized gold (5.6-14 nm) and silica (15-60 nm) NPs via side-on configuration 21,42 . However, fibrinogen preferably attached to NPs surface through end-on configuration as the size of NPs increased 42 . The number of binding sites per protein was calculated using double-logarithm equation (Figure 3 and SI: Eq. S2) 43 . The proteins that adopt a side-on configuration wrap the NP surface and therefore, engage more binding sites compared to the end-on state. As expected, fibrinogen, which is a hydrophilic protein, has more than one, binding sites on EMT zeolite NPs (Table 2). It is suggested that fibrinogen is mainly adsorbed on EMT NPs through side-on configuration. The influence of incubation temperature on fibrinogen-NPs interactions is presented in Figure 3. The number of binding sites per protein decreased as the incubation temperature increased from 25 to 42 C. Thus, the temperature-induced conformational changes affect the orientation of proteins on the NPs. The proteins adopted in the side-on/end-on configuration may reorient to end-on/side-on state after structural rearrangement.

Depending on the protein unfolding extent and protein orientation model, the protein-NPs interaction may form different binding strengths. Therefore, various binding energies of the interaction at different temperatures can be explained by temperature-induced conformational changes.

Cooperativity assay of fibrinogen and EMT zeolite nanoparticles

Binding kinetics and cooperativity in fibrinogen-zeolite NPs interaction were assessed using Hill equation (SI: Eq. S3). Hill coefficient determines whether different binding sites involved through protein-NP interaction are self-governed or cooperative. The calculated Hill coefficient in physiological like condition is near to 1 implying that single or multiple self-governing binding sites mediate the fibrinogen-zeolite interaction (Figure 4) (SI: Figures S3-S5) 44 . For noncooperative protein binding, where Hill coefficient is near to 1, the affinity of proteins to NP surface is independent of the presorbed proteins. Fibrinogen has multiple domains that potentially interact with different surfaces. For example, domains D and E illustrated different affinities to the same surface 45 . They also attached to zeolite NPs independently. It can be suggested that the adsorption and subsequent unfolding of each domain do not affect neighboring domain adsorption/structure.

The particle size plays crucial role in determining the cooperativity in NPs-protein interaction 42 .

Considerable contradictories exist in the literatures regarding the effect of NPs size on the cooperativity in NPs-fibrinogen interaction. For example, Deng et al. 41 showed positive cooperativity occurred when fibrinogen interacted with the gold NPs larger than 7 nm. In contrast, Lacerda et al. 33 demonstrated that fibrinogen preferably bound to gold NPs larger than 5 nm in a negative cooperative manner.

Analysis of thermodynamic parameters involved in the EMT zeolite nanoparticles and fibrinogen interactions

The thermodynamic parameters involved in fibrinogen-EMT NPs interaction were measured using fluorescent spectroscopy. Thermodynamic parameters such as enthalpy (ΔH), Gibbs free energy (ΔG), and entropy changes (ΔS) were indirectly calculated using the Eqs. S4 and S5 (SI)

to determine the governing forces on the fibrinogen-EMT NPs interaction. The negative values

for ΔG indicate that the interaction occurs spontaneously. The negative values of ΔH (-2699.05 kJ mol -1 ) and ΔS (-787.16 J mol -1 K -1 ) imply that van der Waals forces and hydrogen bonds mainly drive the EMT-fibrinogen interaction 46 . Based on the exothermic reaction and the thermodynamic parameters obtained from van't Hoff equation and corresponded plots of fibrinogen-EMT interactions (Figure 5), it can be suggested that fibrinogen has high affinity towards EMT NPs

Secondary structure of bound fibrinogen on EMT zeolite nanoparticles

The secondary structure of fibrinogen was studied in the presence of various concentrations of EMT NPs using far-ultraviolet circular dichroism (CD) analytical approach. The CD spectra were recorded in the wavelength range 190-260 nm (SI: Figures S6-S9 and Tables S2-S11).

Two negative peaks appeared at 208 nm and 222 nm are known as characteristics of typical fibrinogen structure having α-helix (Figure 6a). The reduced ellipticity at 208 nm and 222 nm indicates that the α-helix content of fibrinogen treated with NPs decreased significantly. This means that the secondary structure of fibrinogen significantly changes in the presence of EMT zeolite NPs (Figure 6b). In addition, the degree of protein denaturation gradually increased with increasing the NP concentration.

Conclusion

The structural integrity of NPs bound proteins determines the in vivo fate of NP and biological responses. Understanding the NPs-protein interaction is crucial for predicting the therapeutic and/or toxic impacts of NPs in vivo. This study revealed that the interaction between zeolite NPs and fibrinogen was strong, thermodynamically favorable and occurred in a non-cooperative manner. The EMT zeolite NPs considerably changed the secondary structural of fibrinogen. It can be concluded that the zeolite-bound denatured fibrinogen can potentially trigger undesired biological responses in vivo. Therefore, the possible biological consequences resulted from zeolite-induced unfolding of proteins should be considered in future nanotoxicology studies. Star represents significant change compared to control (Fibrinogen alone) at p < 0.05. Alterations of different secondary structure types by increasing the EMT concentrations (μg mL -

Figure captions Figure 1 .

 captions1 Figure captions

Figure 2 .

 2 Figure 2. The Stern-Volmer plots of fibrinogen quenching caused by different concentrations of EMT zeolite NPs at different temperatures.

Figure 3 .

 3 Figure 3. The double-log plots Log ((F0 -F)/F) vs. Log EMT for fibrinogen-EMT zeolite NPs interactions at different temperatures.

Figure 4 .

 4 Figure 4. The Hill plots Ln (F0 -F)/F)) vs. Ln EMT for fibrinogen-EMT zeolite NPs interactions at physiological temperature (37 °C).

Figure 5 .

 5 Figure 5. The van't Hoff plots of fibrinogen-EMT zeolite NPs interactions.

Figure 6 .

 6 Figure 6. (a) CD spectra of fibrinogen molecules alone and in the presence of different concentrations of EMT NPs. (b) Secondary structural changes of fibrinogen in the presence of increasing concentrations of the EMT NPs. Results are mean ± standard error of mean (n = 20).

Figure 1 .

 1 Figure 1.

Figure 2 .

 2 Figure 2.

Figure 3 .

 3 Figure 3.

Figure 4 .

 4 Figure 4.

Figure 5 .

 5 Figure 5.

Figure 6 .

 6 Figure 6.

1 )

 1 , studied at various wavelength ranges, have been depicted in Figures S6-S9. Results are mean ± standard error of mean (n = 20). Asterisk means significant change compared to control (fibrinogen alone) at p < 0.05.

Fig. S6

 S6 Fig. S6 Fibrinogen secondary structure content changes upon binding EMT studied at 195-260 nm.

Fig. S7

 S7 Fig. S7 Fibrinogen secondary structure content changes upon binding EMT studied at 200-260 nm.

Fig. S8

 S8 Fig. S8 Fibrinogen secondary structure content changes upon binding EMT studied at 205-260 nm.

Fig. S9

 S9 Fig. S9 Fibrinogen secondary structure content changes upon binding EMT studied at 210-260 nm.

Table captions Table 1 .

 captions1 The Stern-Volmer constants calculated for fibrinogen-EMT zeolite NPs interactions.

Table 2 .

 2 Binding parameters of fibrinogen-EMT zeolite NPs interactions at different temperatures.

Table 3 .

 3 Thermodynamic parameter involved in fibrinogen-EMT zeolite NPs interactions.

Table 1 .

 1

	Zeolite	T (K)	Ksv (M -1 )	Kq (M -1 S -1 )	R 2
	EMT NPs	298.15	6280.4	6280.4×10 8	0.97
		310.15	4532.3	4532.3×10 8	0.99
		313.15	3980.3	3980.3×10 8	0.99
		315.15	3611.9	3611.9×10 8	0.98

Table 2 .

 2

	Zeolite	T (K)	Kα (M -1 )	n	R 2
	EMT NPs	298.15	1216746.20	1.56	0.98
		310.15	15595.52	1.13	0.99
		313.15	7184.55	1.06	0.99
		315.15	3322.76	0.99	0.98

Table 3 .

 3

	Zeolite	T (K)	ΔG (kJ mol -1 )
	EMT NPs	298.15	-34.72
		310.15	-24.88
		313.15	-23.11
		315.15	-21.22

Table S2 .

 S2 Percentage of different secondary structures in fibrinogen (0.25 mg mL -1 ).

		nm	nm	nm	nm
	Helix	48.00%	56.30%	55.40%	56.30%
	Antiparallel	3.50%	2.10%	2.20%	2.30%
	Parallel	6.80%	6.20%	5.80%	5.80%
	Beta-Turn	13.00%	11.80%	11.80%	12.20%
	Random Coil	29.50%	26.10%	26.80%	27.60%
	Total Sum	100.70%	102.40%	102.00%	104.10%
		195-260	200-260	205-260	210-260

Table S3 .

 S3 Percentage of different secondary structures in fibrinogen treated with EMT (25 μg mL -1 ).

		195-260	200-260	205-260	210-260
		nm	nm	nm	nm
	Helix	42.80%	45.50%	45.00%	45.30%
	Antiparallel	5.60%	4.40%	4.50%	4.60%
	Parallel	6.90%	6.40%	6.00%	5.90%
	Beta-Turn	14.10%	13.30%	13.30%	13.90%
	Random Coil 30.40%	31.20%	31.30%	31.50%
	Total Sum	99.90%	100.80% 100.10% 101.20%

Table S4 .

 S4 Percentage of different secondary structures in fibrinogen treated with EMT (50 μg mL -1 ).

		195-260	200-260	205-260	210-260
		nm	nm	nm	nm
	Helix	37.80%	43.70%	43.40%	44.00%
	Antiparallel	7.90%	4.80%	4.90%	5.00%
	Parallel	6.60%	6.30%	5.90%	5.80%
	Beta-Turn	15.10%	13.60%	13.60%	14.10%
	Random Coil 29.70%	31.30%	31.10%	31.40%
	Total Sum	97.10%	99.70%	98.90%	100.30%

Table S5 .

 S5 Percentage of different secondary structures in fibrinogen treated with EMT (100 μg mL -1 ).

		nm	nm	nm	nm
	Helix	41.50%	43.80%	42.40%	42.50%
	Antiparallel	7.00%	5.30%	5.60%	5.80%
	Parallel	7.20%	6.60%	6.10%	6.00%
	Beta-Turn	14.40%	13.60%	13.70%	14.50%
	Random Coil	31.30%	33.10%	34.10%	33.90%
	Total Sum	101.40% 102.40% 101.80% 102.70%
		195-260	200-260	205-260 210-260

Table S6 .

 S6 Percentage of different secondary structures in fibrinogen treated with EMT (200 μg mL -1 ).

		195-260	200-260	205-260	210-260
		nm	nm	nm	nm
	Helix	41.80%	43.40%	42.10%	42.10%
	Antiparallel	7.50%	5.50%	5.70%	6.00%
	Parallel	7.10%	6.70%	6.10%	6.00%
	Beta-Turn	14.70%	13.60%	13.70%	14.60%
	Random Coil	29.00%	33.80%	34.60%	34.30%
	Total Sum	100.10% 103.00% 102.20% 103.00%

Table S7 .

 S7 Percentage of different secondary structures in fibrinogen treated with EMT (300 μg mL -1 ).

		195-260	200-260	205-260	210-260
		nm	nm	nm	nm
	Helix	44.50%	43.30%	41.60%	41.40%
	Antiparallel	6.70%	5.60%	6.00%	6.40%
	Parallel	7.20%	6.80%	6.20%	6.00%
	Beta-Turn	14.50%	13.60%	13.80%	14.70%
	Random Coil 28.10%	34.20%	35.30%	35.00%
	Total Sum	100.80% 103.60% 102.80% 103.50%

Table S8 .

 S8 Percentage of different secondary structures in fibrinogen treated with EMT (400 μg mL -1 ).

		nm	nm	nm	nm
	Helix	42.50%	42.20%	40.20%	39.60%
	Antiparallel	8.00%	6.30%	6.70%	7.20%
	Parallel	7.30%	6.90%	6.20%	6.00%
	Beta-Turn	14.80%	13.80%	14.00%	15.10%
	Random Coil	29.10%	35.50%	36.80%	36.10%
	Total Sum	101.70%	104.70%	103.90%	104.00%
		195-260	200-260	205-260	210-260

Table S9 .

 S9 Percentage of different secondary structures in fibrinogen treated with EMT (500 μg mL -1 ).

		195-260	200-260	205-260	210-260
		nm	nm	nm	nm
	Helix	39.10%	41.50%	39.50%	38.50%
	Antiparallel	10.60%	6.90%	7.20%	8.00%
	Parallel	7.40%	7.10%	6.40%	6.10%
	Beta-Turn	15.40%	13.90%	14.10%	15.30%
	Random Coil 29.90%	37.10%	38.60%	37.40%
	Total Sum	102.50% 106.50% 105.80% 105.30%

Table S10 .

 S10 Percentage of different secondary structures in fibrinogen treated with EMT (600 μg mL -1 ).

		195-260	200-260	205-260	210-260
		nm	nm	nm	nm
	Helix	45.60%	41.40%	39.50%	38.10%
	Antiparallel	7.20%	7.10%	7.40%	8.30%
	Parallel	7.70%	7.20%	6.40%	6.10%
	Beta-Turn	14.30%	13.90%	14.00%	15.40%
	Random Coil 30.20%	38.20%	39.70%	38.10%
	Total Sum	105.00% 107.90% 107.00% 106.10%

Table S11 .

 S11 Percentage of different secondary structures in fibrinogen treated with EMT (700 μg mL -1 ).

		nm	nm	nm	nm
	Helix	45.60%	41.40%	39.50%	38.10%
	Antiparallel	7.20%	7.10%	7.40%	8.30%
	Parallel	7.70%	7.20%	6.40%	6.10%
	Beta-Turn	14.30%	13.90%	14.00%	15.40%
	Random Coil	30.20%	38.20%	39.70%	38.10%
	Total Sum	105.00% 107.90% 107.00% 106.10%
		195-260	200-260	205-260	210-260

Supplementary Information

Detailed results are presented in Supplementary Information.

Supplementray Information for

Molecular interaction of fibrinogen with zeolite nanoparticles

Hossein Derakhshankhah †, ‡, §, ι , Mohammad Javad Hajipour ∥ , ⊥, § , Fereshteh Taghavi # , Alireza Lotfabadi †, § , ‡ , Atiyeh Ghasemi # , Ebrahim Barzegari # , Mina Evini # , Ali Akbar Saboury # , Eng-Poh

S2. Quenching effects of EMT NPs at different temperatures

Si/Al ratio 1.17 Unit cell composition Na88(AlO2)88(SiO2)104 SBET (m 2 g -1 ) a 720 SExt (m 2 g -1 ) a 260 Vmicro (cm 3 g -1 ) a 0.24 Vmeso (cm 3 g -1 ) a 1.08 VTotal (cm 3 g -1 ) a 1.32 dmicro (nm) a 0.73 dmeso (nm) a 3.12 Mean particle size (nm) b 14.0 Charge density (mC m -2 ) c -6.05 Surface charge (mC g -1 ) d -4356 a SBET: BET specific surface area; Sext: external surface area; Vmicro: micropore volume; Vmeso: mesopore volume;

Vtotal: total pore volume; dmicro: micropore diameter; dmeso: mesopore diameter. In order to determine the molecular quenching mechanism, the fluorescence quenching results were analyzed by Stern-Volmer equation (Eq. S1). 1

(Eq. S1)

F0 and F reveal the fluorescence intensities at the steady-state of fibrinogen in the absence and presence of quencher (EMT-type zeolite NPs), respectively. KSV is the Stern-Volmer quenching constant and it is gained from the linear regression of Stern-Volmer equation; Kq is the quenching rate constant of protein whose maximum value is known to be 2.0 × 10 10 L mol -1 s -1 . 2

[Q] is the concentration of quencher (EMT-type zeolite NPs). τ is the average lifetime of the fluorophore/biomacromolecule in the absence of the quencher.

The following double-logarithm equation (Eq. S2) provides more information about binding equilibrium. 3 Log ((F0-F)/F) = Log K + nLog [Q] (Eq. S2)

In this equation, n is the number of binding sites per protein and K is the association constant (Kα).

S3. Cooperativity studies

The Hill coefficient was measured at different temperature using Hill equation (Eq. S3) through:

S4. Thermodynamic analysis

Free energy changes of the interaction between fibrinogen and NPs were calculated using following formula: 4 ΔG = -RTLn(Kα) (Eq. S4)

In this equation, Kα is the association/binding constants for EMT NPs. T is the temperature of experiments and R is the gas constant (8.314 J mol -1 K -1 ). With assuming that ΔH and ΔS values do not vary significantly over the temperature, the ΔG was calculated from Gibbs equation (Eq. S4) and other parameters can be obtained by plotting the binding constant according to van't Hoff equation (Eq. S5). 5 Ln (Kα) = -(ΔH/RT) + (ΔS/R) (Eq. S5)

S5. Circular Dichroism data

Tables S2-S11 and Figures S6-S9 give the detailed information obtained from the CD spectroscopy on the intereaction between fibrinogen and EMT zeolite NPs.