Yanhui Wang

Cédric Leuvrey

Emilie Delahaye

Fabrice Leroux

Pierre Rabu

Christine Taviot-Gueho
email: christine.taviot-gueho@uca.fr

Guillaume Rogez
email: rogez@unistra.fr

Christine Taviot-Guého

Tuning the organization of the interlayer organic moiety in a hybrid layered perovskite

Keywords: Hybrid materials, Layered perovskites, Aurivillius phase, Microwave, Phenylbutylamine, 1D electron density profile, Interdigitation

Introduction

Besides graphene and transition metal dichalcogenides (TMDs), other 2D nanosheet-based materials have attracted a lot of attention in the last years. [START_REF] Ganter | Vapor-Phase Amine Intercalation for the Rational Design of Photonic Nanosheet Sensors[END_REF][START_REF] Ziegler | Artificial Solids by Design: Assembly and Electron Microscopy Study of Nanosheet-Derived Heterostructures[END_REF][START_REF] Wang | Titanium Oxide Nanosheets: Graphene Analogues with Versatile Functionalities[END_REF][START_REF] Coronado | Coexistence of superconductivity and magnetism by chemical design[END_REF][START_REF] Khan | Two-Dimensional (2D) Nanomaterials towards Electrochemical Nanoarchitectonics in Energy-Related Applications[END_REF][START_REF] Tan | Recent Advances in Ultrathin Two-Dimensional Nanomaterials[END_REF] Among them one may quote oxide and hydroxide nanosheets, with applications in the field of energy (fuel cells, artificial photosynthesis, photovoltaic b …) c c ( - dielectrics, f c c f c …). [START_REF] Wang | Titanium Oxide Nanosheets: Graphene Analogues with Versatile Functionalities[END_REF][START_REF] Li | Coexistence of Magnetic Order and Ferroelectricity at 2D Nanosheet Interfaces[END_REF][START_REF] Ma | Nanosheets of Oxides and Hydroxides: Ultimate 2D Charge-Bearing Functional Crystallites[END_REF][START_REF] Uppuluri | Soft chemistry of ion-exchangeable layered metal oxides[END_REF] One important characteristic of these oxide and hydroxide nanosheets is that they are obtained from bulk layered 2D materials which can be functionalized by various kind of reactions, insertion, insertion grafting or post-synthesis modification reactions. [START_REF]P by : A b f -State Reactions[END_REF][START_REF] Sanjaya Ranmohotti | Topochemical Manipulation of Perovskites: Low-Temperature Reaction Strategies for Directing Structure and Properties[END_REF][START_REF] Gopalakrishnan | f f[END_REF] This field, very recently reviewed, [START_REF] Uppuluri | Soft chemistry of ion-exchangeable layered metal oxides[END_REF] has experienced a renewed interest in the past few years, thanks to the development of microwave-assisted reactions. [START_REF] Wang | Efficient Microwave-Assisted Functionalization of the Aurivillius-Phase Bi 2 SrTa 2 O 9[END_REF][START_REF] Akbarian-Tefaghi | Rapid Topochemical Modification of Layered Perovskites via Microwave Reactions[END_REF][START_REF] Boykin | Rapid Microwave-Assisted Grafting of Layered Perovskites with n-Alcohols[END_REF][START_REF] Akbarian-Tefaghi | Microwave-assisted routes for rapid and efficient modification of layered perovskites[END_REF][START_REF] Wang | Post-Synthesis Modification of the Aurivillius Phase Bi 2 SrTa 2 O 9 via In Situ Microwave-A " c c[END_REF] Yet, despite the interest of this approach and notably the considerable gain in reaction time, it appears that the possible differences in the obtained products using microwave-assisted reactions or more classical ones have not been really explored. This could be of utmost importance since it has been shown for instance for the functionalization of layered double hydroxides that interesting and complex mechanisms could arise below apparently simple reaction processes. [START_REF] Hy | L Insight into the Structure of Layered Zinc Hydroxide Salts Intercalated with Dodecyl Sulfate Anions[END_REF][START_REF] O' | H Ob f c L y α-Cobalt Hydroxides: A Synthetic and Kinetic Study[END_REF][START_REF]O'H f c -Stage Intermediates in Anion-Exchange Intercalation Reactions of the Layered Double Hydroxide [LiAl 2 (OH) 6 ]Cl•H 2 O As Observed by Time-Resolved, in Situ X-ray Diffraction[END_REF] Based on our experience on the microwave-assisted functionalization and post-synthesis modification of an Aurivillius phase of formula Bi 2 SrTa 2 O 9 (BST), we describe in this article one example of the strong influence of the reaction conditions on the obtained product of the functionalization of HST with a particular amine, phenylbutylamine (PBA) (scheme 1).

Scheme 1. Phenylbutylamine (PBA).

This amine is characterized by a flexible alkyl chain bearing the amino group, and a rigid moiety at the other side of the molecule. We investigate here the reaction between this amine and a protonated Aurivillius phase of formula H 2 Bi 0.1 Sr 0.85 Ta 2 O 7 (HST) as a function of the synthesis conditions: using short reactions times by means of microwave assisted reactions and longer reaction times using classical solvothermal reaction. The compounds are characterized using ancillary techniques (XRD, IR, 13 C CP/MAS solid state NMR spectroscopy). 1D electron density profiles extracted from XRD patterns allow to precise the organization of the molecules within the interlayer space. Finally, using a slightly different starting compound (HST prefunctionalized by ethyl amine), a compound with another molecular organization could be isolated as a pure phase. The possibility to realize a mutual conversion between these phases by post-synthesis solvent treatment was finally explored.

Experimental Section Syntheses

Microwave syntheses were performed with a microwave synthesis reactor Monowave 300 (Anton Paar) (non-acidic conditions) and with a microwave synthesis reactor MultiwaveGO (Anton Paar) for the synthesis of HST (acidic conditions). 1.4H 2 O (PBA1-HST): 100 mg (0.17 mmol) of HST were dispersed in a solution of 20 mmol of PBA (3 mL) in a mixture of 5 mL of THF and 1 mL of water. The mixture was placed in a 30 mL vial and heated by microwave irradiation at 130°C during 1.5 h (maximum incident power: 70 W). In these conditions, the mean incident power was about 10 W, and the autogenous pressure was about 6 bars. The obtained white powder was collected after three centrifugations (14000 rpm (18400 g), 5 min each) (the supernatant was replaced after each centrifugation by distilled water (twice) and ethanol (last) in order to wash the product) and air-dried. O (PBA3-HST): 100 mg (0.17 mmol) of HST were dispersed in a solution of 20 mmol of PBA (3 mL) in a mixture of 5 mL of THF and 1 mL of water. The mixture was placed in a sealed Teflon-lined autoclave (volume 50 mL) and was heated in oven at 130°C during 18 h. The obtained white powder was collected after three centrifugations (14000 rpm (18400 g), 5 min each) (the supernatant was replaced after each centrifugation by distilled water (twice) and ethanol (last) in order to wash the product) and air-dried. The results of elemental analyses and TGA and the proposed formulae are given in Table 1.

Caution: even though no problem was encountered in the course of this research, special care must be taken while working with sealed glass containers. Containers must be carefully checked for any defect, and replaced if necessary to avoid possible explosion. Amines and concentrated hydrochloric acid are corrosive and irritating.

Characterization procedures

Elemental analyses for C, H and N were carried out at the Service Commun d'Analyses of the University of Strasbourg. The SEM images were obtained with a JEOL 6700F microscope equipped with a field emission gun, operating at 3 kV in the SEI mode. FT-IR spectra were collected in ATR mode on a SpectrumII spectrometer (Perkin-Elmer). TGA-TDA experiments were performed using a TA instrument SDT Q600 (heating rates of 5°C•min -1 under air stream, using alumina crucibles). NMR spectra in solution were recorded using a Bruker AVANCE 300 (300 MHz) spectrometer. Solid state NMR 13 C (I = 1/2) experiments were performed with a 300 Bruker spectrometer at 75.47 MHz, using magic angle spinning (MAS) condition at 10 kHz and a 4 mm diameter size zirconia rotor. 13 C spectra obtained by proton enhanced cross-polarization method (CP) were referenced to the carbonyl of the glycine calibrated at 176.03 ppm. Recycling and Hartman-Hahn contact times were 5 s and 1250 µs, respectively. Spinal 64 1 H phase-decoupling was applied during 13 C channel acquisition.

X-ray diffraction analysis

The powder XRD patterns were collected in Bragg Brentano geometry with a Bruker D8 diffractometer (CuKα1 = 0.1540598 nm) equipped with a LynxEye detector discriminating in energy. For Le Bail refinement, z f 0.02° (2θ) c f 14 were used. The Le Bail method consisting in the full pattern decomposition of the XRD pattern of the powdered sample was used to determine the cell parameters assuming the I4/mmm space group, typical for BST and HST materials, [START_REF] Rae | Structure refinement of commensurately modulated bismuth strontium tantalate, Bi 2 SrTa 2 O 9[END_REF][START_REF] Tsunoda | Preparation and HREM Characterization of a Protonated Form of a Layered Perovskite Tantalate from an Aurivillius Phase Bi 2 SrTa 2 O 9 via Acid Treatment[END_REF] and to extract the intensities of diffraction peaks. The refinement was carried out using Fullprof suite program. [START_REF] Rodriguez-Carvajal | Recent developments of the program FULLPROF[END_REF] The pseudo-Voigt profile function of Thompson, Cox and Hastings was selected to analyze peak profiles. [START_REF] Thompson | Rietveld refinement of Debye-Scherrer synchrotron X-ray data from Al 2 O 3[END_REF] The instrumental contribution to peak broadening was estimated by measuring the standard corundum reference sample leading to the following Caglioti half-width parameters: U= 0.0111 V= -0.0041 and W = 0.0016. It should be noted that two phases of same composition but with different profile parameters had to be considered to reach a good fit and this is due to the presence of two different particle sizes as evidenced by SEM analysis (Fig. S3 shows a representative SEM image of the compounds obtained in this study). The one-dimensional electron density distributions along the c-stacking axis were calculated for PBA3-HST and PBA2-HST samples from the intensity of the 00l diffraction lines according to the following equation: where c is the unit cell parameter, z is the fractional coordinate along the c-stacking axis, and F 00l are the structures factors. [START_REF] Jacobson | Intercalation Chemistry[END_REF] Thirteen isolated 00l diffraction lines were used for calculating the 1D plots. The signs of the structure factors were obtained from the scattering contributions of the perovskite layers assuming a relatively small contribution of the intercalated molecules. For comparison, the 1D electron density profile along the z-direction for BST was simulated (Fig. S4); it was calculated from the crystal structure data taken from Crystallography Open Datatabase (entry 1521701). The difference Fourier map for PBA3-HST was calculated using GFourier program by employing the observed structure factors F obs extracted from the Le Bail whole powder diffraction pattern decomposition and plotted in the form of contour plots summed from 0 to 1 along the a-axis. The known part of the structure i.e. the perovskite layer was entered and the corresponding calculated structure factors were substracted from the F obs thus emphasizing the electron density in the interlayer space. A simple structural model was proposed for PBA3-HST obtained by treating the intercalated PBA anion as a quasi rigid body and comparing the resulting calculated Fourier map with the above experimental difference Fourier map. The molecular structure of phenylbutylamine was built using ChemBio 3D Utra 13.0 Suite program; energy minimization and geometric optimization were applied using the MM2 forced field implemented in the program. The nitrogen atom of the amine group was chosen to express the rigid body in orthonormal coordinates; it was positioned on x c = 0.0, y c = 0.0, z c = 0.10568 corresponding to 4e position in I4/mmm space group with a site occupancy of 1/4. This absolute position and the Euler angles theta = 65°, phi = 70°, chi = 175° describe the molecular body orientation. The profile parameters and unit cells used were those determined from Le Bail profile fitting.

Results and discussion

Synthesis and structural analysis

The syntheses of the three PBA-HST hybrids were performed using a large excess (ca. 100) of PBA with respect to HST or C 2 N-HST, in a THF/H 2 O mixture (5:1 v:v). The PBA1-HST and PBA2-HST phases were obtained by reaction between PBA and HST and C 2 N-HST respectively using microwave irradiation (130°C, 1.5 h) following the method established previously for aliphatic amines. [START_REF] Wang | Efficient Microwave-Assisted Functionalization of the Aurivillius-Phase Bi 2 SrTa 2 O 9[END_REF][START_REF] Akbarian-Tefaghi | Rapid Topochemical Modification of Layered Perovskites via Microwave Reactions[END_REF] The PBA3-HST phase was obtained using classical solvothermal heating in a Teflon-lined autoclave under autogenous pressure (130 °C, 18 h) (see Experimental).

Fig. 1 shows the powder XRD patterns (raw data) of HST, C 2 N-HST and the three reaction products. The layered structure is evidenced by the series of intense (00l) reflections at low angles. After reactions the interlayer distances increase from that of HST (0.98 nm) to 2.20 nm for PBA1-HST and to 3.00 nm for PBA3-HST. For PBA2-HST, the interlayer distance increases from that of C 2 N-HST (1.57 nm) to 2.67 nm. The (00l) reflection lines of the starting products are not visible in the diagrams of the reaction products. In contrast, the characteristic out-of-plane reflections assigned to (100) and (110) in the XRD pattern of HST, are observed at the same positions ( 22.68° (0.392 nm) and 32.59° (0.274 nm) respectively) for the three phases. Therefore, after the intercalation reactions, the structures of the inorganic perovskite-like slabs are retained. In Fig. 2 are reported the Le Bail whole-powderdiffraction-pattern profile fittings for PBA2-HST and PBA3-HST samples. As can be seen, satisfactory refinements were obtained by considering a tetragonal cell with the space group I4/mmm. On the other hand, in the case of PBA1-HST sample, we were not able to do similar refinement due to an anisotropic broadening of X-ray diffraction peaks, thus the interlayer distance was estimated from the position of the 00l diffraction lines. After reactions, the interlayer distances increase from that of HST (0.98 nm) to c.a. 2.20 nm for PBA1-HST and to 2.98 nm for PBA3-HST. For PBA2-HST, the interlayer distance increases from that of C 2 N-HST (1.57 nm) to 2.67 nm. By subtracting the perovskite layer thickness i.e. 0.765 nm from these interlayer distances, one can estimate the distance available for PBA molecule in the interlayer space: 2.215 nm for PBA3-HST, 1.905 nm for PBA2-HST and 1.420 nm for PBA1-HST. Comparing these values to the estimated length of the guest species c.a. 0.85 nm suggests a bilayer arrangement of PBA, probably interdigitated in the case of PBA1-HST, with the amino groups nearby the perovskite layers. The observed interlayer distances are in total agreement with a bilayer insertion of the phenylbutylamine, with different interdigitation rates and/or different angles between the molecule and the inorganic layer. The elemental analyses and thermogravimetric analyses show that there is no obvious difference in the insertion rates and in the water content between the three phases (Tab. 1 and Fig. S1). The three different phases present identical thermal behaviours (Fig. S1). The first mass losses (4%, 4% and 3% for PBA1-HST, PBA2-HST and PBA3-HST, respectively) occur before 125°C and are ascribed to the removal of water. The TGA curves show very obvious mass losses (11.1%, 12.6% and 10.3% for PBA1-HST, PBA2-HST and PBA3-HST, respectively) between 125 °C and 500 °C, associated to exothermic events. This second mass loss is thus attributed to the removal of PBA. The mass losses (ca. 3%) between 600°C and 700°C are ascribed to the loss of protons in the form of water for the final phase are presented in In order to precise the arrangements of the PBA molecules which lead to different basal spacings, one-dimensional (1D) electron density profiles along the c-staking direction were determined for PBA2-HST and PBA3-HST. Indeed, the large number of 00l diffraction lines, up to thirteen visible, allowed us to probe the structure of the interlayer space by the calculation of a one-dimensional electron density maps from the analysis of integrated intensities extracted from the above Le Bail profile decompositions and Fourier transformation. This approach has been applied successfully to the structural characterization of many intercalative nanohybrids. [START_REF] Choy | Intracrystalline Structure of Molecular Mercury Halide Intercalated in High-Tc Superconducting Lattice of Bi 2 Sr 2 CaCu 2 O[END_REF][START_REF] Choy | HRTEM and Micro-c c -c c Nanohybrid[END_REF][START_REF] Marangoni | Organic inorganic dye filler for polymer: Blue-coloured layered double hydroxides into polystyrene[END_REF][START_REF] Fujita | Intercalation characteristics of rhodamine 6G in fluor-taeniolite; orientation in the gallery[END_REF][START_REF] Itoh | Reversible Color Changes in Lamella Hybrids of Poly(diacetylenecarboxylates) Incorporated in Layered Double Hydroxide Nanosheets[END_REF][START_REF] Park | X-ray diffraction and X-ray absorption spectroscopic analyses for intercalative nanohybrids with low crystallinity[END_REF][START_REF] Liu | Synthesis, Structure, and Reactions of Poly(ethylene oxide)/V 2 O 5 Intercalative Nanocomposites[END_REF] As shown in Fig. 3a, peaks on the 1D plot for PBA3-HST match perfectly with a bilayer arrangement of PBA molecule, in particular the minimum in the center. The most intense peaks are due to the perovskite layers containing Sr, Ta and Bi elements (Fig. S4). The amino groups together with water molecules cause small maxima at the outer parts of the interlayer space at a distance of ~ 3.0 Å from the inorganic layers which is consistent with hydrogen bond interactions. The central minimum framed by two maxima supports the bilayer arrangement with the two phenyl rings positioned on either side of the center. For PBA2-HST (Fig. 3b), peaks of higher electron densities are observed in the interlayer space indicating more compactly arranged PBA molecules. The two maxima are nearer of the center which suggests closely spaced phenyl rings. Considering all the integrated intensities extracted from Le Bail fitting for PBA3-HST, we attempted to calculate the difference Fourier map after entering the known part of the structure i.e. the perovskite layer and assuming that the phases are controlled by the heavy atoms present in these layers. Such a difference map may help to highlight regions of low electron density associated with light atoms such as interlayer species. [START_REF] Monteiro | Investigation of Layered Double Hydroxides Intercalated by Oxomolybdenum Catecholate Complexes[END_REF]36] As can be seen in Figure 4b, the electron density in the interlayer space of PBA3-HST sample is quite low but nevertheless shows a distribution in different planes perpendicular to the c stacking direction and the z coordinates are consistent with the positions of the maxima noted on the 1D plot. A more or less important spreading of the density is observed for each of these planes indicating an orientation disorder of PBA molecule. This result is then compared to a calculated Fourier map (Fig 4d) considering a structural model based on PBA molecules arranged head to tail with the butylamine chain inclined of ~30° with respect to the normal of the perovskite layers, which is typical of the angle observed in the case of aliphatic amines. [START_REF] Wang | Efficient Microwave-Assisted Functionalization of the Aurivillius-Phase Bi 2 SrTa 2 O 9[END_REF]The nitrogen atom of the amine function chosen to express the rigid body was placed on the 4-fold axis (4mm) in vertical alignment with the position of Sr atoms with a site occupancy of 1/4 and at a distance of ~3.7 Å from the HST layer, consistently with the formation of moderate hydrogen bonds. Phenyl rings of adjacent molecules are separated by ~ 3.5 Å, in agreement with the observed interchain distance in the case of purely bilayer arrangement of aliphatic chains. [START_REF] Rösner | Interlayer reactions of the silver molybdate Ag 6 Mo 10 O 33[END_REF] The comparison between this calculated Fourier map derived from this structural model and the difference Fourier map discussed above derived from F obs shows strong similarities which somehow confirms the structural model proposed. Of course, water molecules must be added and are most certainly located near the HST layer forming hydrogen bonding therewith and with the surrounding amino groups.

Spectroscopic investigation

IR spectra are very similar for the three phases (Fig. 5). The sharp N-H stretching bands around 3300 cm -1 for the free ligand are greatly reduced in the hybrid compounds and a new N-H stretching band appears at 2520 cm -1 , which indicates the protonation of the amine group. The signals of the aromatic group at 3062 and 3026 cm -1 and the signals of C-C bond and C-N bond between 1500 cm -1 and 1700 cm -1 are clearly observed. The antisymmetric and symmetric stretching vibration bands of CH 2 groups can also clearly be observed. The values of ν s (CH 2 ), ν as (CH 2 ) and δ(CH 2 ) for the three compounds are collected in Table 2. The antisymmetric stretching bond ν as (CH 2 ) decreases from 2929.6 cm -1 to 2925.5 cm -1 and further to 2920.5 cm -1 for PBA1-HST, PBA2-HST and PBA3-HST respectively. In the present case, as seen above, there is no clear difference in the packing densities of the methylene chains. Therefore, the only explanation for a variation in the position of ν as (CH 2 ) is the gauche/trans conformation ratio. [START_REF] Vaia | Interlayer Structure and Molecular Environment of Alkylammonium Layered Silicates[END_REF] It has indeed been shown that the lower ν as (CH 2 ) frequencies correspond to highly ordered all-trans conformations whereas higher frequencies indicate a larger amount of gauche conformations along the hydrocarbon chain, and an increase of chain disorder. [START_REF] Vaia | Interlayer Structure and Molecular Environment of Alkylammonium Layered Silicates[END_REF] Therefore, the above results indicate that the number of all-trans conformations increases and the number of gauche conformations (disorder) decreases along with the increase of the interlayer distance of PBA-intercalated products (i.e. in the order PBA1-HST, PBA2-HST, PBA3-HST), in line with the XRD study. The bending vibration δ(CH 2 ) hardly changes for PBA1-HST, PBA2-HST and PBA3-HST, neither in position nor in intensity. The high frequency of this band (ca. 1472 cm -1 ) indicates that PBA molecules in the three phases have at least partially ordered methylene chains. [START_REF] Vaia | Interlayer Structure and Molecular Environment of Alkylammonium Layered Silicates[END_REF] 13 C CP/MAS solid state NMR spectroscopy was also performed. The spectra display a high degree of similarity (Fig. 6). Signals attributed to carbon A and carbon B appear at 40 ppm and 29 ppm respectively for the three compounds whereas they appear at 42. ' G G' b c se of the fast averaging of the chemical shifts due to electron delocalization). This may come from slightly different arrangements of the molecules in the interlayer spacing. Despite the partial interdigitation evidenced for PBA3-HST by 1D electron density map, no effect of this interdigitation (likely due to π-stacking of the aromatic rings) could be observed on the 13 C CP/MAS solid state NMR spectra. It thus appears that the arrangement of the PBA molecules is strongly dependent of the synthesis conditions. Partial interdigitation of the phenylbutylamine is favoured for short reaction times, whereas when a longer reaction time (and longer heating time, the two parameters being difficult to decorrelate) is used, the obtained phase is better crystallized, with a complete bi-layer arrangement of the interlayer molecules, without any interdigitation. The choice of another precursor, C 2 N-HST instead of HST, and short reaction times leads to a somehow intermediate phase.

E H G G' F F' A D B C E G G' F F' H A D B C E G G' F F' H A D B C *

Influence of reaction parameters: temperature, reaction time, and quantity of PBA used

To study the influence of the reaction temperature, a series of reactions were performed using microwave assisted reactions, using the same experimental procedure as for PBA1-HST, at 80°C, 130°C (PBA1-HST) or 160°C. Fig. 7 shows the XRD patterns of HST and the obtained reaction products. The XRD pattern of the compound obtained at 80°C is essentially identical to the one of HST (with a slightly larger interlayer distance (1.00 nm) and a decreased crystallinity), indicating that the intercalation reaction did not take place. At higher temperatures, the interlayer distance increases from that of HST (0.98 nm) to 2.20 nm (130°C, PBA1-HST) and up to 2.65 nm for the compound obtained at 160°C. Yet for this last compound, diffraction lines corresponding to the ones of HST (indicated by an asterisk) indicate the presence of the starting material in the final compound, likely due to a partial degradation of the obtained compound, as marked also by the decreased intensity of the 00l diffraction lines with respect to the characteristic reflections (100) at 22.68°, (110) at 32.59°, and (200) at 46.32° (Cu Kα1)). Despite this degradation, it appears that higher reaction temperatures favour intercalation and an increase of the interlayer distance. Yet even at 160°C, the interlayer distance of the obtained product does not reach the one of PBA3-HST (3.0 nm), underlining the crucial role played by the reaction time. Figure [START_REF] Li | Coexistence of Magnetic Order and Ferroelectricity at 2D Nanosheet Interfaces[END_REF]. XRD patterns of HST and its reaction products with PBA at 80°C, 130°C and 160°C. Indexation of the peaks of HST comes from reference [START_REF] Tsunoda | Preparation and HREM Characterization of a Protonated Form of a Layered Perovskite Tantalate from an Aurivillius Phase Bi 2 SrTa 2 O 9 via Acid Treatment[END_REF]. For the indexation of the peaks of the other compounds, see Figure 2.

The same reaction was then performed, at 130°C, but using different reaction times (0.5 h, 1.5 h (PBA1-HST), 3 h and 4.5 h). Fig. 8 shows the XRD patterns of HST and the obtained reaction products. The XRD pattern of the compound obtained after 0.5 h shows an increase of the interlayer distance (2.20 nm, identical to that of PBA1-HST) but indicates also the presence of another compound with an interlayer distance of 1.00 nm, likely corresponding to hydrated HST. For longer reaction times, the interlayer distances of the reaction products increase from 2.20 nm (PBA1-HST) to 2.72 nm. Yet, for reaction times longer than 1.5 h, the crystallinity decreases and/or multiphasic compounds are obtained. Longer reaction times thus favour a larger interlayer distances, revealing that PBA1-HST (d = 2.20 nm) is in fact a kinetic product. Yet the reaction mechanism from PBA1-HST to the thermodynamic compound PBA3-HST (d = 3.00 nm) is not straightforward. One hypothesis is that interstratification (second staging) phenomenon is at stake during the reaction process, [START_REF] Hy | L Insight into the Structure of Layered Zinc Hydroxide Salts Intercalated with Dodecyl Sulfate Anions[END_REF][START_REF] O' | H Ob f c L y α-Cobalt Hydroxides: A Synthetic and Kinetic Study[END_REF][START_REF]O'H f c -Stage Intermediates in Anion-Exchange Intercalation Reactions of the Layered Double Hydroxide [LiAl 2 (OH) 6 ]Cl•H 2 O As Observed by Time-Resolved, in Situ X-ray Diffraction[END_REF] yet such a phenomenon could not be evidenced clearly in the present work. Finally, it is worth mentioning that PBA1-HST is stable for months in the solid state, without any reduction of the crystallinity nor any change of the interlayer distance. Finally, the influence of the molar ratio between HST and PBA and (HST:PBA) has been investigated (1, 2, 5, 10, 50, 100, and 200). The microwave assisted (130 °C, 1.5 h) reactions were always performed in a THF/H 2 O mixture (5:1 v:v) as for the synthesis of PBA1-HST. XRD patterns show that the interlayer distances of reaction products are always around 2.2 nm except for very low quantities of PBA (HST:PBA = 1:1 and 1:2) for which new different phases were obtained (at 1.25 nm for the ratio 1:1, and at around 3 nm for the ratio 1:2, most probably corresponding to swelling) (Fig. 9). In conclusion, there is not an obvious effect of the molar ration between PBA and HST on the interlayer distance. We retrieve here the result described for microwave-assisted insertion of aliphatic amines which requires a large excess of amine (about 100). [START_REF] Wang | Efficient Microwave-Assisted Functionalization of the Aurivillius-Phase Bi 2 SrTa 2 O 9[END_REF][START_REF] Akbarian-Tefaghi | Rapid Topochemical Modification of Layered Perovskites via Microwave Reactions[END_REF] Our experience shows that the crystallinity of HST influence the crystallinity of the reaction products, but not the reactivity of HST nor the nature of the obtained products. [START_REF] Hyb F Y | f y de Strasbourg[END_REF])

The possibility of mutual conversion between the three obtained products (PBA1-HST, PBA2-HST and PBA3-HST) has been investigated by using solvent treatment. When PBA2-HST (50 mg) is stirred in acetone (15 mL) at room temperature overnight, the interlayer distance increases from 2.67 nm to 2.95 nm (compound a), very close to the one of PBA3-HST (Fig. 10). The reverse conversion is achieved by stirring compound a in water (15 mL) at room temperature overnight. The obtained product, compound b, presents an interlayer distance of 2.62 nm, very close to the one of PBA2-HST. Yet this reverse process is accompanied by a partial hydrolysis of the compound. It thus appears that the mutual conversion between PBA2-HST and PBA3-HST can be realized by simple solvent treatments. Such solvent treatment-induced modification of the interlayer spacing has already been reported in a layered Zn hydroxide functionalized by dodecylsulfate. [START_REF] Hy | L Insight into the Structure of Layered Zinc Hydroxide Salts Intercalated with Dodecyl Sulfate Anions[END_REF] In this case, methanol induces a decrease of the number of intercalated molecules, which is accompanied by a modification of the structure of the inorganic layers. In the present case, the solvent treatment acts directly and only on the interactions between interleaved molecules, as reported by Fujita and Awaga in the case of a layered Cu hydroxide functionalized by 8-((p-(phentlazo)phenyl)oxy)octanoate. [START_REF] Fujita | Reversible Structural Transformation and Drastic Magnetic Change in a Copper Hydroxides Intercalation Compound[END_REF] It is worth noticing that in this latter case just like in the present work, the molecules at stake are made of a flexible aliphatic chain and terminal aromatic group. This scheme might indeed favour a controllable interdigitation phenomenon.

Unfortunately, up to now we have not found the proper conditions to realize the mutual conversion between PBA1-HST and PBA2-HST or between PBA1-HST and PBA3-HST.

Conclusion

We have described the synthesis of a layered Ta oxide perovskite functionalized by phenylbutylamine molecules. Depending on the reaction conditions, compounds with different interlayer spacings are obtained. Short reaction times, obtained using microwaveactivated reactions lead to compounds with a small interlayer spacing, whereas long reaction times, using classical solvothermal reactions provides compounds with a much larger interlayer spacing. Whereas no change in composition (organic loading) could be evidenced, careful analysis of the XRD patterns and calculation of the 1D electron density profiles allows to precise the degree of interdigitation of the molecules in the interlayer space, revealing an overlap of the aromatic cycles for short interlayer distances. Moreover, XRD analysis and IR spectroscopy show the higher disorder of the organic molecules in the interdigitated configuration. Using a different precursor, a phase possessing an intermediate interlayer distance could be isolated. Yet the reaction mechanism from the short interlayer distance phase (kinetic product) to the large interlayer distance phase (thermodynamic product) remains unknown. This work adds a new perspective to the ever-growing use of microwave irradiation for the synthesis of organic-inorganic layered hybrids. Indeed, microwave irradiation not only enables to synthesize much faster compounds that can be obtained classically, but also enables to obtain new pure kinetic phases, inaccessible by other means. Such an approach, reinforced by the careful design of the inserted molecules, may allow to tailor at will new hybrids layered compounds with precise applications.

Acknowledgements

de Strasbourg, the Université de Clermont-Ferrand and the Agence Nationale de la Recherche (ANR contract ANR-14-CE07-0004-01) for

Supplementary information

TGA and TDA curves, representative SEM image and calculated electron density profile for BST.

1

 1 Sr 0.85 Ta 2 O 7 1.6H 2 O (PBA2-HST): the same procedure as above was used, using 100 mg (0.15 mmol) of C 2 N-HST instead of HST. (C 6 H 5 (CH 2 ) 4 NH 3 ) 0.5 H 1.5 Bi 0.1 Sr 0.85 Ta 2 O 7 1.0H

Figure 1 .

 1 Figure 1. PXRD patterns of HST (black), C 2 N-HST (red), PBA1-HST (green), PBA2-HST (blue) and PBA3-HST(purple).

Figure 2 .

 2 Figure 2. Profile analysis of the XRD pattern of (a) PBA3-HST and (b) PBA2-HST: experimental X-ray diffraction (circles), calculated (line), Bragg reflections (ticks), and difference profiles. Refinement in a tetragonal cell with I4/mmm space group. Extended region 10-40/50° (2θ) showing all 00l diffraction lines. Reliability factors Rp/wp.

Figure 3 .

 3 Figure 3. One-dimensional electron density projected along the c-stacking axis for(a) PBA2-HST and (b)PBA3-HST

Figure 4 .

 4 Figure 4. Structural model for PBA3-HST deduced from: (a) one-dimensional electron density projected along the c-stacking axis and (b) Difference Fourier (contoured) map summed from 0 to 1 along the a-axis; the electron density scale(e-/Å) is given on the right. Both were calculated from the profile refinement of the XRD data; the phases and structure factors were computed from the known structure of the perovskite layers. (d) Theoretical Fourier map calculated from the structural model proposed(c)

Figure 5 .

 5 Figure 5. Zoom of the infrared spectra of PBA1-HST (green), PBA2-HST (blue) and PBA3-HST (purple) in the 4000-800 cm -1 region.

1 ppm and 33 . 4

 334 ppm respectively products for PBA in solution in D 2 O. This up-field shifts of A and B carbon signals confirm the conversion of -NH 2 groups to -NH 3 + groups.[START_REF] Tahara | Intercalation of a,w-Diaminoalkanes in the Interlamellar Space of the Protonated Form of the Layered Perovskite H 1.8 Bi 0.2 Sr 0.8 Ta 2 O 7[END_REF][START_REF]f . z b w Z. zw w A. z y-ł c The 15 N and 13 y f c y b c ff' bases[END_REF][START_REF] Komori | Intercalation of alkylamines and water into kaolinite with methanol kaolinite as an intermediate[END_REF][START_REF] Wallau | Room-temperature synthesis of crystalline solids in the system ZnO-P 2 O 5 -R-H 2 O, with R being an alkylamine or an alkylammonium ion[END_REF] On the contrary, the position of the signals of the aliphatic carbons C and D and of the aromatic carbons E, it is worth noticing that all signals are slightly split (except the aromatic F,

Figure 6 .

 6 Figure 6. Solid-state 13 C CP/MAS NMR spectra of PBA1-HST (green, bottom), PBA2-HST (blue, middle) and PBA3-HST (purple, top) (asterisk indicate the presence of a small residual amount of ethanol).

1 Figure 9 .

 19 Figure 9. XRD pattern of HST and its reaction products at 130 °C with various HST:PBA ratios. (The relatively low crystallinity of the compounds obtained here is due to a low crystallinity of the HST batch used for this set of experiments.Our experience shows that the crystallinity of HST influence the crystallinity of the reaction products, but not the reactivity of HST nor the nature of the obtained products.[START_REF] Hyb F Y | f y de Strasbourg[END_REF])

Figure 10 .

 10 Figure 10. XRD patterns illustrating the mutual conversions between PBA2-HST and PBA3-HST. Product (a): PBA2-HST was stirred in acetone at room temperature overnight. Product (b): Product (a) was stirred in water at room temperature overnight.

  . grant of Y.W. The authors thank Dr. M. Lenertz for fruitful discussions and D. Burger and C. Kiefer for technical assistance.

Bi 2 SrTa 2 O 9

(BST)

, H

2 Bi 0.1 Sr 0.85 Ta 2 O 7 (HST) and (CH 3 CH 2 NH 3 ) 0.8 H 1.2 Bi 0.1 Sr 0.85 Ta 2 O 7 3 H 2 O

	(C 2 N-
	HST) were synthesized according to published procedures.[13]
	(C 6

H 5 (CH 2 ) 4 NH 3 ) 0.5 H 1.5 Bi 0.1 Sr 0.85 Ta 2 O 7

  [START_REF] Ziegler | Artificial Solids by Design: Assembly and Electron Microscopy Study of Nanosheet-Derived Heterostructures[END_REF]

Table 1 .

 1 Mass

losses ascribed to the removal of water and of organic phase, elemental micro-analyses (C, H, N) and consistent proposed formulae.

  . By analogy with what has been described on H 2 SrTa 2 O 7 ,[26,27] the obtained phase is a three-dimensional perovskite of formula Bi 0.1 Sr 0.85 Ta 2 O

	Compound	Formula	Mass losses	Mass losses	Mass losses	C content	H content	N content
			(water+organic	(organic)	(water)	Found	Found	Found
			)	found	Found	(calcd)	(calcd)	(calcd)
			found	(calcd)	(calcd)	(%)	(%)	(%)
			(calcd)	(%)	(%)
			(%)
	PBA1-HST	(C10NH16)0.5H1.5Bi0.1Sr0.85Ta2O71.4H2O	15.1 (14.9)	11.1 (11.1)	4.0 (3.8)	11.06 (9.0)	1.97 (1.8)	1.28 (1.0)
	PBA2-HST	(C10NH16)0.6H1.4Bi0.1Sr0.85Ta2O71.6H2O	16.6 (17.2)	12.6 (13.0)	4.0 (4.2)	13.55 (10.4)	2.24 (2.4)	1.52 (1.2)
	PBA3-HST	(C10NH16)0.5H1.5Bi0.1Sr0.85Ta2O71.0H2O	13.3 (13.9)	10.3 (11.2)	3.0 (2.7)	10.01 (9.0)	1.74 (1.7)	1.25 (1.0)

[START_REF] Tan | Recent Advances in Ultrathin Two-Dimensional Nanomaterials[END_REF]

. Finally, the mass losses around 900 °C (1.6%, 1.2% and 1.0% for PBA1-HST, PBA2-HST and PBA3-HST, respectively), associated with an exothermic event, are proposed to be due to partial Bi removal and formation of the tungsten bronze phase -Bi 0.1 Sr 0.85 Ta 2 O 6 . XRD pattern of the intermediate and final phases, as well as phase matching

Table 2 .

 2 Position of the IR absorption bands of the CH

	Product	as (CH2) (cm -1 )	s (CH2) (cm -1 )	δ (CH2) (cm -1 )
	PBA1-HST	2929.6	2860.1	1471.8
	PBA2-HST	2925.5	2853.7	1471.8
	PBA3-HST	2920.5	2851.9	1471.1

2 groups in PBA1-HST, PBA2-HST and PBA3-HST.

  XRD pattern of HST and its reaction products with PBA at 130 °C 0.5 h,1.5 h, 3 h and 4.5 h reaction times. Asterisks indicate diffraction lines attributed to an impurity phase (see text).

		(002)
	u.)	(002) * (004) (004)	* * *			(100) (100)		(110) (110)			(200) (200)	(210) (210)	3 h 4.5 h
	(a. Intensity	(002) (002)	(004) (004) *	(0010) (0010)	(0012) (0012)	(100) (100) (100)		(110) (110) (110)	(0022)	(0024) (0024)	(200) (200) (200)	(210) (210) (210)	0.5 h 1.5 h
				(001)		(002)	(100)	(003)	(110)	(004)		(200)	(210)	HST
				10	15	20	25	30	35		40	45	50	55	60
								2θ (°)
	Figure 8.


Valérie Samouillan

Nofel Merbahi

Mohammed Yousfi

Jean-Pierre Gardou

Florian Delaunay

Jany Dandurand

Colette Lacabanne

Effect of Low-Temperature Plasma Jet on Thermal Stability and Physical Structure of Type I Collagen

Keywords: Denaturation, differential scanning calorimetry (DSC), Fourier transform infra red (FTIR) spectroscopy, lowtemperature plasma jet, type I collagen

This work is devoted to the characterization of type I collagen treated by a low-temperature plasma jet generated in ambient air to determine whether the resulting fibrous material is structurally preserved or reinforced. The physical structure of collagen is checked by differential scanning calorimetry (DSC), which is a well suited technique to analyze thermal transitions in proteins, such as denaturation. DSC is used to evaluate the thermal stability of collagen after the plasma treatments while Fourier transform infra red spectroscopy is used to check the integrity of triple helical domain and to investigate the effects of plasma treatments on the functional groups of collagen. It is more particularly shown that the plasma treatment can stabilize the collagen structure without altering the triple helical structure. This observation is supported by 1) the shift observed toward high-temperature range of the collagen denaturation and 2) the stiffening of the chains by a cross-linking action when compared to the control sample.

I. INTRODUCTION

D EVELOPMENT of biomaterials used as substitutes of the extracellular matrix for the replacement of cardiovascular tissues in associated pathologies or the realization of skin substitutes is a great challenge of repair medicine. Since two decades, detergent and enzymatic protocols have been optimized to obtain non antigenic extracellular matrices, preserving the main fibrillar proteins such as collagen and elastin [START_REF] Lee | Effect of molecular structure of poly(glycidyl ether) reagents on crosslinking and mechanical properties of bovine pericardial xenograft materials[END_REF]- [START_REF] Pasquino | Bovine pericardium for heart valve bioprostheses:in vitro andin vivo characterization of new chemical treatments[END_REF]. The most commonly utilized collagen, collagen type I, is constituted by triple helices units stabilized by covalent intermolecular cross-links [START_REF] Linsenmayer | Cell Biology of Extracellular Matrix[END_REF]- [START_REF] Bateman | Extracellular Matrix: Molecular Components and Interactions[END_REF]. The rich chemistry of collagen allows engineers to alter physicochemical properties such as porosity, crystallinity, and cross-link density, resulting in predictable tissue ingrowth and biodegradable rates [START_REF] Lew | Optimization of UV crosslinking density for durable and nontoxic collagen GAG dermal substitute[END_REF]. Nevertheless, the durability of collagen-based bioprostheses must V. Samouillan, F. Delaunay, J. Dandurand, and C. Lacabanne are with the Université de Toulouse, CIRIMAT-Institut Carnot-UMR 5085, 31062 Toulouse Cedex 9, France (e-mail: valerie.samouillan@iut-tlse3.fr; florian. delaunay@gmail.com; danduran@cict.fr; lacabane@cict.fr).

N. Merbahi and J.-P. Gardou are with the Université de Toulouse, LAPLACE, UMR CNRS 5213, 31062 Toulouse Cedex 9, France (e-mail: nofel.merbahi@Laplace.univ-tlse.fr; gardou@Laplace.univ-tlse.fr).

M. Yousfi is with the Université de Toulouse, LAPLACE, UMR CNRS 5213, Université Paul Sabatier, 31062 Toulouse Cedex 9, France (e-mail: yousfi@Laplace.univ-tlse.fr).

Digital Object Identifier 10.1109/TPS.2012.2190303 be improved by the mechanical stabilization of proteins, and their preserving against proteolytic degradation is necessary to a complete recellularization [START_REF] Hønge | Recellularization of decellularized mitral heart valves in juvenile pigs[END_REF].

One of the best methods to preserve collagen is the crosslinking of collagenic fibers in order to restitute the tridimensional network of the protein generally impaired by the cellular purification of tissues [START_REF] Neethling | Mitigation of calcification and cytotoxicity of a glutaraldehyde-preserved bovine pericardial matrix: improved biocompatibility after extended implantation in the subcutaneous rat model[END_REF], [START_REF] Madaghiele | Collagen-and gelatine-based films sealing vascular prostheses: evaluation of the degree of crosslinking for optimal blood impermeability[END_REF]. This cross-linking can be achieved by chemical methods (glutaraldehyde, widely used in surgery or carboimides) or by physical methods, such as dehydrothermal treatment or gamma rays or UV irradiation. The main disadvantage of chemical treatment using aldehydes for collagen cross-linking is the cytotoxicity and the calcification of fibers, decreasing the durability of the bioprosthesis [START_REF] Hønge | Recellularization of decellularized mitral heart valves in juvenile pigs[END_REF], [START_REF] Bottio | The changing hydrodynamic performance of the decellularized intact porcine aortic root: considerations on in-vitro testing[END_REF]. The physical treatments could have the advantage to lead the formation of chemical bonds between collagen macromolecules without the incorporation of any exogenous molecules. For instance, UV treatments have been investigated to cross-link collagen [START_REF] Sionkowska | Thermal stability of UV-irradiated collagen in bovine lens capsules and in bovine cornea[END_REF], although irradiation can cause both stabilization and destabilization of the collagen structure [START_REF] Sionkowska | Thermal stability of UV-irradiated collagen in bovine lens capsules and in bovine cornea[END_REF]- [START_REF] Ohan | Synergistic effects of glucose and ultraviolet irradiation on the physical properties of collagen[END_REF]. Under such a context, it seems interesting to investigate the effects of nonthermal atmospheric pressure plasmas as they are not yet used for the treatment of collagen fibers, but already successfully used in various other biomedical applications. This concerns plasma sterilization and decontamination of surfaces [START_REF] Sarrette | Observation of antibacterial effects obtained at atmospheric and reduced pressures in afterglow conditions[END_REF], [START_REF] Villeger | Synergy between N and O atom action and substrate surface temperature in a sterilization process using a flowing N 2 -O 2 microwave post discharge[END_REF], tissues engineering and biomaterial treatment for their functionalization [START_REF] Sardella | Nano-structured celladhesive and cell-repulsive plasma-deposited coatings: Chemical and topographical effects on keratinocyte adhesion[END_REF], [START_REF] Desmet | Nonthermal plasma technology as a versatile strategy for polymeric biomaterials surface modification: a review[END_REF] and more recently plasma medicine for blood coagulation, disinfection of living tissues involving wound healing, and more generally the interaction of plasmas with eukaryotic cells [START_REF] Morfill | Nosocomial infections-A new approach towards preventive medicine using plasmas[END_REF]- [START_REF] Dobrynin | Physical and biological mechanisms of direct plasma interaction with living tissue[END_REF].

Therefore, the aim of this work is to analyze some effects of atmospheric pressure nonthermal plasmas on collagen fibers. We used more particularly a low-temperature plasma jet generated in ambient air [START_REF] Merbahi | Device for emitting a plasma jet from the atmospheric pressure air at ambient temperature and pressure, and use of said device[END_REF] and producing various active species (excited species, free radicals, charged particles, and photons (covering a large spectrum from UV up to visible) that are in contact with type I collagen fibers under both freeze-dried and hydrated states.

To quantify the effect of the plasma treatment on the properties of type I collagen, we chose to combine two suitable protein characterization methods: differential scanning calorimetry (DSC) and Fourier transform infra red (FTIR) spectroscopy. DSC is a powerful technique providing direct measurements of thermodynamic characteristics and, unlike the other methods, is efficiently applicable to collagen solutions, fibers, and tissues. It is widely used in collagen research to infer the level of cross-linking within a given sample through elucidation of its denaturation temperature. Furthermore, FTIR spectroscopy is a useful method to monitor changes in secondary structures of a protein, in particular variation in the amide A, amide B, amide I, II, and III regions.

II. EXPERIMENTAL SETUPS AND METHOD OF ANALYSIS

A. Overview on Low-Temperature Plasma Jet

There are in the literature many devices developed to produce a low-temperature plasma jet more particularly for biomedical applications as emphasized in the review of Laroussi [START_REF] Laroussi | Arc-free atmospheric pressure cold plasma jets: A review[END_REF]. A plasma jet allows a remote treatment very practical for in vivo treatment where it is dangerous to put the living tissue inside the zone of plasma generation. A plasma jet is also very interesting for biomaterial treatment more particularly when it is needed for instance to immerse the biomaterial in water for a treatment under hydrated mode. There are many examples of plasma jet devices for instance plasma needle [START_REF] Kieft | Plasma treatment of mammalian vascular cells: a quantitative description[END_REF], plasma pencil [START_REF] Laroussi | Room-temperature atmospheric pressure plasma plume for biomedical applications[END_REF], plasma brush [START_REF] Duan | Low-temperature direct current glow discharges at atmospheric pressure[END_REF], and other setups of plasma jets driven by various power supplies (dc, ac, or RF sources). Such plasma devices generally produce low-temperature plasmas using various gas compositions and designs of the electrode configuration. This allows the launching of the plasma outside the generation zone with the help of a continuous gas flow. The low-temperature plasma jet used in the present work for the collagen treatment has been the subject of a patent [START_REF] Merbahi | Device for emitting a plasma jet from the atmospheric pressure air at ambient temperature and pressure, and use of said device[END_REF]. The measured plasma temperature on the top of the jet, that has a length of about 1 cm, does not exceed 27 • C. The plasma jet is generated directly in the ambient air at atmospheric pressure and launched by itself without any system of gas inlet feed. This means that it is easily transportable because there is neither gas bottle nor gas pumping. It is a low-temperature plasma generated by a specific corona discharge design giving a natural repetitive discharge current with a frequency of about 20 kHz under a high-voltage dc power supply. Fig. 1 shows an overview on the experimental setup involving schematically the plasma jet power supply, the tools of measurements (monochromator for spectroscopy and oscilloscope for electric data). Fig. 1 also displays a schematic view of the plasma jet and the treated collagen placed at a distance d from the plasma. Fig. 2(a) shows the instantaneous discharge current with a peak of about 11 mA that corresponds to a dissipated power of about 100 mW. The UV-visible spectrum corresponding to the light emission of the top of the air plasma jet is shown in Fig. 2(b) and(c). This shows the classical emission bands of nitrogen such as the second positive system of N

2 (C 3 π u ) v → N 2 (B 3 π g ) ′
v from about 290 nm up to 440 nm corresponding to the different vibration states v and v ′ (for instance at the peaks at 315.93 nm, 337.13 nm, 357.69 nm, 375.54 nm, and 380.49 nm). The first positive system N 2 (B 3 g ) → N 2 (A 3 Σ + u ) which is apparent in the visible-near infrared (between about 600 nm up to 900 nm) range indicates the formation of the metastable N 2 (A 3 Σ + u ) states. The detection of the first negative system (FNS) of N + 2 (B 2 Σ + u ) → N + 2 (X 2 Σ + g) around 390 nm is synonymous of high electron energies leading to the ion formation. However, the presence of positive nitrogen ion is probably very low due to the very small intensity of the FNS emission. There are also the oxygen emissions at for instance 759 nm coming from the band

O 2 (b 1 Σ + g v = 0) → O 2 (X 3 Σ - g , v = 0
) and the atomic line of the triplet state of O at 777.47 nm. An atomic line of nitrogen for N 4 P -4 S 0 is also observable. However, the NO γ bands due to the emission of NO(A 2 Σ + ) v → NO(X 2 ) ′ v synonymous of dissociation of molecular nitrogen and oxygen leading to NO formation, are not observable between about 200 nm up to 290 nm due to the quenching of NO γ bands by the molecular oxygen following the reaction:

NO(A 2 Σ + ) + O 2 → NO(X 2 ) + O 2
with a rate coefficient that is 1000 times higher than the NO(A 2 Σ + ) quenching by N 2 .

The same remark is true for the OH(A 2 Σ + ) → OH(X 2 3/2 ) emission bands between about 300 nm up to 320 nm coming from the dissociation of water vapor impurities present in ambient air and not observed due to the quenching by molecular oxygen following the reaction:

OH(A 2 Σ + ) + O 2 → OH(X 2 3/2 ) + O 2
with a rate coefficient that is roughly 10 times higher than the OH(A 2 Σ + ) quenching by N 2 .

This overview on plasma jet spectrum means that the excited species present at the top of jet are at least those involved by the emission bands such as N2(C

3 π u ) v , N 2 (B 3 π g ) ′ v , O 2 (b 1 Σ + g v = 0), O 2 (X 3 Σ - g , v = 0), N 2 (A 3 Σ + u )
, O and N. Due to the kinetics of formation of such excited species, it is obvious that we have also another metastable states of molecular nitrogen and oxygen, and also dissociation products of air such as atomic nitrogen N and even atomic hydrogen. There are also other species in the present ambient air plasma jet that are active during the collagen treatment but they are not always detectable in the optical emission spectrum of Fig. 2 due to mainly the quenching processes or to emission outside the present wavelength range (for instance specific atomic lines of nitrogen are in the VUV range below 200 nm).

B. Treated Collagens

Commercial insoluble type I collagen (Fluka Chemie AG, Switzerland) was extracted from bovine Achilles tendon and available in the form of air-dried fibers. The type I collagen is considered under two states:

-Freeze-dried state: Collagen fibers were compressed into pellets (thickness 0.5 mm, diameter 10 mm), and each face of the pellet was exposed to the plasma jet during 5, 10, and 60 min at ambient temperature (20 • C). These times were found after preliminary tests to be representative of short and long exposure times. -Hydrated state: 80 mg of collagen fibers was placed in 7 mL of deionized water, equilibrated for 1 h so that the fibers are swollen and subsequently exposed to the plasma jet during 10, 20, 45, and 90 min under stirring at ambient temperature (20 • C). As in the dehydrated state, these times were found to be representative of short and long exposure times.

A set of samples were then carefully put on absorbent paper to remove excess water before thermal analysis. Another set of samples were freeze-dried again before further thermal analysis.

C. Setups of DSC and FTIR Spectroscopy

The DSC phase transition thermograms were recorded with a Pyris Diamond differential scanning calorimeter from Perkin Elmer. The temperature and energy scales were calibrated using the manufacturer's instructions with mercury, indium, and tin as standards. Samples of 5 mg of weight were sealed in aluminum pans. Empty pans were used as references. Thermal analysis is performed to get insight into the denaturation phenomenon of collagen, occurring in the [40 • C; 80 • C] temperature range in the hydrated state and in the [180 • C; 230 • C] in the dehydrated state [START_REF] Bateman | Extracellular Matrix: Molecular Components and Interactions[END_REF]. That is why investigations in the hydrated state were performed between 10 • C and 90 • C with 10 • C/min heating rates, in hermetic pans. Investigations of the dehydrated state were performed between 30 • C and 250 • C with 10 • C/min heating rates, in nonhermetic pans. Determination of transition parameters was performed with Origin software.

The FTIR spectra were performed in the attenuated total reflection infrared spectroscopy using a Nicolet 5700 FTIR (Thermo Electron Corporation) equipped in ATR device with diamond crystal. All spectra were recorded in absorption mode between 4000 cm -1 and 450 cm -1 at 4 cm -1 interval and 64 accumulations. Spectra were performed in duplicate on two series of treated samples to check the reproducibility in the position of absorption bands (inferior to a deviation of 1 cm -1 ). The background spectrum was subtracted and a baseline correction was performed. Fourier-self-deconvolution (FSD) of the infrared spectra that allows resolution of several overlapping bands [START_REF] Hu | Determining beta sheet crystallinity in fibrous proteins by thermal analysis and infrared spectroscopy[END_REF] was performed in the amid I-II regions to find the position of the different bands using Omnic software.

III. RESULTS AND DISCUSSION

The freeze-dried and hydrated collagens are treated by the low-temperature plasma jet for increasing exposures times. The main electric operating parameter of the plasma setup (magnitude of dc voltage of the power supply) is varied within a voltage range between about 8 kV up to 13 kV that allows maintaining the corona regime generating our low-temperature plasma jet. Under these conditions, no change has been observed in the DSC thermograms of the treated collagen. Such results was expected because in this voltage range, only the length of plasma jet undergoes a small variation (10%), but the plasma remains at the same low temperature (because the spark regime, which can heat the plasma, appears only at higher voltages).

A. Freeze-Dried Collagen Treated by Plasma Jet

Fig. 3 shows the DSC thermograms of control collagen and collagen treated by the plasma jet for several time exposures. Table I displays the corresponding denaturation temperatures T d, defined as the maximum of the peak and the corresponding enthalpies of denaturation ∆Hd computed from a series of measurements. Calorimetric measurements of control collagen show a broad endothermic phenomenon occurring between 50 • C and 150 • C due to the evaporation and vaporization of residual bound water in collagen [START_REF] Samouillan | Thermal analysis characterization of aortic tissues for cardiac valve bioprostheses[END_REF]. Another endothermic event is observed above 200 • C attributed to the denaturation of collagen. It is addressed to the rupture of hydrogen bonds that maintain the secondary and tertiary structure of collagen inducing the uncoiling of the triple helix in α chains of random conformation, individually or covalently linked depending on the degree of heating [START_REF] Privalov | Stability and mobility of the collagen structure[END_REF], [START_REF] Balian | The Structure and Properties of Collagen[END_REF]. The denaturation of the dry protein occurs in the [180 • C; 250 • C] temperature range as an endothermic peak [START_REF] Samouillan | On the molecular basis of fouling resistance[END_REF], [START_REF] Samouillan | Characterisation of elastin and collagen in aortic bioprotheses[END_REF]. The denaturation parameters of the collagen control (T d = 225 • C and ∆Hd = 7.05 J • g -1 ) are similar to thermal parameters of pure freeze-dried collagen generally observed in literature [START_REF] Miles | Polymer-in-a-box mechanism for the thermal stabilization of collagen molecules in fibers[END_REF]. In this case, the value of the denaturation enthalpy of collagen for hydration < 6% (corresponding to less than one mole of water per tripeptide) is assigned mainly to the breaking of the direct hydrogen bonds between alpha chains [START_REF] Miles | Polymer-in-a-box mechanism for the thermal stabilization of collagen molecules in fibers[END_REF].

In the case of treated collagen, the DSC thermograms (Fig. 3) show several changes. The denaturation becomes a complex event, implying denaturing zones at lower and higher temperature when compared to the control collagen (Fig. 3 andTable I). The destabilized zones could be attributed to the reduction of collagen into polypeptides of different molecular weight [START_REF] Sionkowska | Thermal stability of UV-irradiated collagen in bovine lens capsules and in bovine cornea[END_REF].

Nevertheless, the high enthalpy associated with destabilized zones for a 5-min exposure is a feature of a triple helical domain. The stabilized zones can be attributed to a crosslinking of collagen. For a 10-min exposure, the total enthalpy is slightly greater than the enthalpy corresponding to the control collagen, meaning that the stabilization is mainly entropic, such as induced by an increase in the packing density of collagen molecules in the fibers [START_REF] Sarrette | Observation of antibacterial effects obtained at atmospheric and reduced pressures in afterglow conditions[END_REF]. For a 60-min exposure time, the shape of denaturation peaks and the low value of the associated enthalpy suggest an important degradation of collagen triple helical structure. The optimum time seems to be comprised between 10 min and 60 min. By increasing exposure time to the low-temperature plasma jet, peptide bond cleavage becomes predominant. The relative proportion of these two competing reactions (cross-linking and bond cleavage) is unknown but was shown to depend on the water content and the oxygen tension [START_REF] Sionkowska | Thermal stability of UV-irradiated collagen in bovine lens capsules and in bovine cornea[END_REF]. That is why similar experiments were also performed on hydrated collagen.

B. Hydrated Collagen Treated by Plasma Jet

Figs. 4 and5 display the DSC thermograms (first and second scans) of control collagen (i.e., without plasma treatment) and collagen treated by the plasma jet for gradual exposure times (up to 90 min). The second scan, performed between -40 • C and 20 • C, was used to evaluate free water quantity in collagens. This experiment, that will be discussed in first, was posterior to the analysis of the denaturation zone in order to minimize the influence of calorimetric measures (here a cooling to -40 • C) on the shape of denaturation.

Fig. 4 shows the large endotherm between 0 • C and 20 • C of ice melting. In hydrated collagen, freezable water can be quantified by dividing the area of the measured endotherm by 330 J • g -1 corresponding to the melting enthalpy of pure ice. The total amount of water in the collagen sample was evaluated by gravimetry. The quantification of freezable and total water is computed on Table II. Since denaturation parameters are very sensitive to the level of hydration of collagen fibers, we had to check that all the collagens have been analyzed under similar conditions. Some authors [START_REF] Miles | Polymer-in-a-box mechanism for the thermal stabilization of collagen molecules in fibers[END_REF] showed that the denaturation enthalpy of collagen was constant below 6 moles of water per tripeptide of collagen (corresponding to 0.4 g of water/g collagen), and that the denaturation enthalpy was constant above 30 moles of water per tripeptide (corresponding to 1.9 g of water per g of collagen). Since the levels of hydration in the present study are largely superior to this last limit, we can compare the denaturation parameters versus the exposure time without hydration influence.

In Fig. 5, a complex endothermic peak addressed to the denaturation phenomenon of hydrated collagen is detected for all samples, splitting into two components or more for 10 min, 45 min, and 90 min of plasma exposure. sponding denaturation enthalpies ∆Hd computed from a series of measurements.

The denaturation parameters of control hydrated collagen (T d = 78.3 • C and ∆Hd = 47.8 J • g -1 ) are close to thermal parameters of pure freeze-dried collagen generally observed in the literature [START_REF] Miles | Polymer-in-a-box mechanism for the thermal stabilization of collagen molecules in fibers[END_REF]. The high enthalpy of unfolding collagen immersed in water-when compared with the values found in the dehydrated state-is thought to derive mainly from the breaking of hydrogen bonds forming the hydration network around the collagen molecules. These hydrogen bonds comprise intrachain and interchain as in the dehydrated state but also intermolecular water bridges. More precisely, hydroxyproline and hydroxyl groups are exposed to the solvent and can support hydrogenbonds water bridges [START_REF] Sionkowska | Thermal stability of UV-irradiated collagen in bovine lens capsules and in bovine cornea[END_REF], [START_REF] Miles | Polymer-in-a-box mechanism for the thermal stabilization of collagen molecules in fibers[END_REF], [START_REF] Miles | Identification of an intermediate state in the helix-coil degradation of collagen by ultraviolet light[END_REF].

For 10 min of exposure time, the split of the denaturation peak can be attributed to destabilized zones and stabilized ones, showing in this case as in the dehydrated one the competition between two antagonist processes: reduction into polypeptides of various molecular weights and cross-linking of collagen. The value of enthalpy indicates a conservation of the triple helical structure. Nevertheless, a qualitative comparison between the two peaks shows that the destabilization mechanism is dominant.

For 20 min of exposure time, stabilized zones and destabilized zones must be roughly formed in equal parts, giving rise to a broad and complex denaturation peak.

For 45 min of exposure time, the cross-linking mechanism appears to be the main phenomenon according to peak height and area.

Finally, a special feature is noticed for 90 min of exposure time. Contrary to the evolution at long exposure time reported in the dehydrated case, a sharp and intense peak is detected at 231 • C, corresponding to a highly stabilized collagen domain. The peak previously attributed to destabilized zones is also shifted toward higher temperature, the temperature of the peak maximum being analogous to the control collagen one. Thereofore, the cross-linking mechanism is rather distinct from that observed in the dehydrated state, and seems better to obtain highly stabilized samples.

C. FTIR Spectroscopy Analysis of the Treated Freeze-Dried Collagen

The FTIR spectra of protein molecules can be correlated directly to their backbone conformation. The identification of the different absorption bands of the control sample was performed using bibliographic data on collagen type I [START_REF] He | Modification of collagen with a natural cross-linker, procyanidin[END_REF]- [START_REF] Sionkowska | Chemical and thermal cross-linking of collagen and elastin hydrolysates[END_REF] and general data on protein FTIR absorption bands [START_REF] Barth | The infrared absorption of amino acid side chains[END_REF].

Fig. 6(a) and (b) show the FTIR spectra of the freeze-dried collagen samples decomposed in two wavenumber ranges for a better clarity (4000 cm -1 to 2000 cm -1 and 2500 cm -1 to 400 cm -1 ). The amide A and B bands at 3300 cm -1 and 3075 cm -1 [Fig. 6 ] is assigned to the C-N stretching and N-H bending vibrations from amide linkages, as well as wagging vibrations of CH 2 groups in the glycine backbone and proline side chains [START_REF] Madhan | Stabilization of collagen using plant polyphenol: role of catechin[END_REF]. In addition to the amide bands largely exploited for the characterization of proteins, the 3000-2800 cm -1 region [Fig. 6 important absorptions due to the high level of aliphatic side chains in collagen.

The superimposition of the spectra of collagen control and plasma-treated collagen indicates that the global positions of the major amide bands do not change with the plasma treatment, particularly the amide I band related to collagen triple helix [START_REF] Payne | Fourier transform ir spectroscopy of collagen and gelatin solutions: Deconvolution of the amide I band for conformational studies[END_REF]. Moreover, the IR absorption ratio of amide III to 1454 cm -1 (AIII/A1454 cm -1 ) is also considered to be a measure of the preservation of integrity of collagen triple helices [START_REF] He | Modification of collagen with a natural cross-linker, procyanidin[END_REF], [START_REF] Andrews | Interaction of collagen with corilagin (a hydrolysable tannin)[END_REF] when it is equal or higher than one; it falls to 0.6 for denatured collagen [START_REF] Silvester | Collagen banded fibril structure and the collagen-platelet reaction[END_REF]. Table IV shows that the ratios for plasma-treated collagens slightly increase from 1 for control collagen, indicating that the triple helix conformation is not destroyed by the plasma treatment.

Since the amide I-II region consists of several bands strongly dependent on secondary conformations, it was subjected to a FSD in order to resolve the various underlying and overlapping spectral features that contribute to this complex region. The renormalized amide I-II regions and the corresponding FSD traces of control collagen and 60-min treated collagen are presented in Figs. 7(a) and(b).

The amide I region of control collagen results into five components that are the signatures of the different conformations of the protein at 1693 cm -1 (antiparallel β-sheets), 1679 cm -1 (β-turns), 1660 cm -1 (native triple helical conformation), 1645 cm -1 (random coil), and 1632 cm -1 (β-sheets) [START_REF] Rabotyagova | Collagen structural hierarchy and susceptibility to degradation by ultraviolet radiation[END_REF], [START_REF] Walton | Influence of telopeptides, fibrils and crosslinking on physicochemical properties of type I collagen films[END_REF]- [START_REF] Tamburro | Supramolecular amyloid-like assembly of the polypeptide sequence coded by Exon 30 of Human Tropoelastin[END_REF]. For 60 min of plasma exposure, the FSD trace confirms the integrity of triple helical conformation (1660 cm -1 ), and none increase of the random coil conformation (1645 cm -1 ) as observed in denatured collagen [START_REF] Rabotyagova | Collagen structural hierarchy and susceptibility to degradation by ultraviolet radiation[END_REF]. The main difference concerns the decrease of the absorption in the 1700 cm -1 -1680 cm -1 region (that gradually decreases upon the time of treatment, results not shown), suggesting a slight modification of the secondary structure of treated collagen in this case, with a diminution of the associated conformations (antiparallel β-sheets and β-turns). Nevertheless, we must take into account that the side chain C = 0 stretching of acid residues (like aspartic acid and glutamic acid, as well as the side chain C = N stretching of basic residue arginine) absorb in the 1700 cm -1 -1680 cm -1 region, and their contribution is not negligible since their proportion is high in collagen. Also, another possible hypothesis for the diminution of the absorbance in this zone is the modification of these three kinds of residues upon plasma treatment.

Another slight modification concerns the diminution and disappearance of the small absorption band at 3004 cm -1 , addressed to the stretching of aromatic and vinylic C-H bonds. Plasma treatment could have an effect of the C = C bond of collagen residues such as tyrosine and phenylalanine. This assertion is corroborated by the modification of the amide II in the 1520-1505 cm -1 region, corresponding to the ring mode of tyrosine and phenylalanine. It must be recalled that tyrosine and phenylalanine are the main absorber of UV light that are shown to decrease with UV radiation due to a possible aggregation of the fibers [START_REF] Kato | Aggregation of collagen exposed to UVA in the presence of riboflavin: A plausible role of tyrosine modification[END_REF].

As the low-temperature plasma jet generates radicals, exited and ionized species and also UV emissions, cross-linking of collagen in our experiments could be partly attributed to the UV emissions of the plasma jet. However, this affirmation must be considered with care because it has to be confirmed by a plasma jet treatment of the collagen done with and without an UV filter. Such a phenomenon was reported both with UVB [START_REF] Sionkowska | Thermal stability of UV-irradiated collagen in bovine lens capsules and in bovine cornea[END_REF] and UVC [START_REF] Caruso | Functional evaluation of collagen fiber scaffolds for ACL reconstruction: cyclic loading in proteolytic enzyme solutions[END_REF] irradiation or with the presence of a photosensitizing agent and laser irradiation in the visible spectra [START_REF] Chan | Photochemical crosslinking improves the physicochemical properties of collagen scaffolds[END_REF].

IV. CONCLUSION

The present work on the thermal denaturation of freeze-dried and hydrated collagens brings us interesting information on the effects of the low-temperature plasma jet treatment on the triple helical structure and the stability of this protein. There is a competition between reduction of collagen in polypeptides and cross-linking mechanisms. In the case of the freeze-dried state, the destabilization of the triple helical structure is the main event for the longest exposure times. The feature is distinct when collagen I fibers are exposed to the plasma jet in the hydrated state. In this case, the cross-linking phenomenon becomes predominant for the longest exposure times (between 45 min and 90 min). Therefore, it is clear that the plasma jet treatment of freeze-dried fibers must be avoided. In addition, it is shown from FTIR analysis slight modifications in absorption bands synonymous to the preservation of the integrity of the triple helical structure of collagen. This however underlines a possible effect of plasma treatment on aromatic side chain and polar side chain of collagen, and could constitute a first step to elucidate the thermal stability enhancement of triple helical structure with the plasma exposure.

The present exploratory work is a first step to determine the effects of low-temperature plasmas generated in ambient air on the preservation of the collagen structure and the enhancement of triple helical stabilization. However, in the light of the present work, it is clear that further investigations are needed to investigate the effects of specific active species on the collagen using other kinds low-temperature plasmas as for instance rare gas-oxygen or rare gas-nitrogen mixtures. Last, it will be very interesting to study in collaboration with biochemists, the biocompatibility and the cellular adhesion and proliferation of the collagenous biomaterials exposed to low-temperature plasma jets.

Fig. 1 .

 1 Fig. 1. Overview on the right side on the experimental setup up for plasma jet in ambient air with the spectroscopic coupled ICCD camera and electric (oscilloscope) tools for measurements NB. (a) The treated collagen is schematically displayed at distance d from the plasma jet). (b) The left side picture shows a finger exposed to the low-temperature plasma jet (the temperature of the top of the jet does not exceed 27 • C).

Fig. 2 .

 2 Fig. 2. (a) Instantaneous current of the corona discharge generating the plasma jet in ambient atmospheric pressure air. (b) Spectrum in the 200 nm-450 nm range collected in the top of the plasma jet. (c) Spectrum in the 600 nm-1000 nm range collected in the top of the plasma jet.

Fig. 3 .

 3 Fig. 3. DSC first scans (from 30 • C to 150 • C, 20 • C/min) of freeze-dried collagens (control and plasma jet-treated collagens).

Fig. 4 .

 4 Fig. 4. DSC second scans (from -40 • C to 20 • C, 10 • C/min) of hydrated collagens (control and plasma jet-treated collagens).

Fig. 5 .

 5 Fig. 5. DSC first scans (from 10 • C to 90 • C, 10 • C/min) of hydrated collagens (control and plasma jet-treated collagens).

Fig. 6 .

 6 Fig. 6. (a) FTIR spectra of control and plasma-treated collagens in the wavenumber range 4000-2000 cm -1 . (b) FTIR spectra of control and plasmatreated collagen in the wavenumber range lower than about 2500 cm -1 .

  (a)] are mainly associated with the stretching vibrations of N-H groups. The amide I band at 1630 cm -1 [Fig. 6(b)] is dominantly attributed to the stretching vibrations of peptide C = O groups. The amide II absorbance at 1540 cm -1 [Fig. 6(b)] arises from the N-H bending vibrations coupled to C-N stretching vibrations. The amide III centered at 1237 cm -1 [Fig. 6(b)

  (a)] addressed to symmetric and asymmetric stretching modes of CH, CH 2 and CH 3 shows

Fig. 7 .

 7 Fig. 7. (a) Renormalized absorbance for amide I-II regions versus wavenumber in the case of control and treated collagen during 2 × 60 min (60 mn for each side). (b) Fourier-self-deconvolution (FSD) traces for amide I-II regions versus wavenumber in the case of control and treated collagen during 2 × 60 min (60 min for each side).

TABLE I DENATURATION

 I PARAMETERS OF FREEZE DRIED COLLAGENS (CONTROL

AND PLASMA JET TREATED)

  Table III displays the corresponding denaturation temperatures T d and the corre-

TABLE III DENATURATION

 III PARAMETERS OF HYDRATED COLLAGENS (CONTROL

AND PLASMA JET TREATED)

TABLE IV IR

 IV ABSORPTION RATIO OF AMIDE III (A III ) AND 1454 cm-1 WAVENUMBER (A 1454 ) IN THE CASE OF COLLAGEN TREATED BY PLASMA FOR SEVERAL EXPOSURE TIMES AND COMPARED TO CONTROL COLLAGEN (0 min).

ACKNOWLEDGMENT

Authors would like to acknowledge UFR PCA of University Paul Sabatier for their financial support.