Gabriela Melo Rodriguez

James Bowen

David Grossin

Besim Ben-Nissan

Artemis Stamboulis
email: a.stamboulis@bham.ac.uk

Functionalisation of Ti6Al4V and hydroxyapatite surfaces with combined peptides based on KKLPDA and EEEEEEEE peptides

Keywords:

Introduction

ABSTR ACT

Surface modifications are usually performed on titanium alloys to improve osteo-integration and sur face bioactivity. Modifications such as alkaline and acid etching, or coating with bioactive materials such as hydroxyapatite, have previously been demonstrated. The aim of this work is to develop a peptide with combined titanium oxide and hydroxyapatite binders in order to ac hieve a biomimetic hydroxya patite coating on titanium surfaces. The technology would also be applicable for the functionalisation of titanium and hydroxyapatite surfaces for selective protein adsorption, conjugation of antimicrobial peptides, and adsorption of specialised drugs for drug delivery. ln this work. functionalisation of Ti6Al4V and hydroxyapatite surfaces was achieved using combined titanium-hydroxyapatite (Ti-Hap) peptides based on titanium peptide binder (KKLPDA) and hydroxyapatite peptide binder (EEEEEEEE). Homoge neous peptide coatings on Ti6Al4V surfaces were obtained after surface chemical treatments with a 30wt% aqueous solution of H 2 O 2 for 24 and 48 h. The treated titanium surfaces pr esented an average roughness of s. = 197 nm (24 h) and s. = 128 nm ( 48 h); an untreated mirror polished sample exhibited an s. of 13 nm. The advancing water contact angle of the titanium oxide layer after 1 h of exposure to 30wt% aqueous solution of H 2 O 2 was around 65 °, decreasing gradually with time until it reached 35 ° after a 48 h exposure, suggesting that the surface hydrophilicity increased over etching time. The presence of a lysine (L) ami no acid in the sequence of the titanium binder resulted in fluorescence intensity roughly 16% higher compared with the arginine (R) amino acid analogue and therefore the lysine containing titanium peptide binder was used in this work. The Ti-Hap peptide KKLPDAEEEEEEEE (Ti-Hap 1) was not adsorbed by the treated Ti6Al4V surfaces and therefore was modified. The modifications involved the inclusion of a glycine spacer between the binding terminais (Ti-Hap2) and the addition of a second titanium binder (KKLPDA) (Ti-Hap3 and Ti-Hap4). The combined Ti-Hap peptide which exhibited the strongest inten sity after the titanium surface dip coating was KKLPDAKKLPDAEEEEEEEE (Ti-HAp4). On the other hand, hydroxyapatite surfaces, exhibiting an average roughness ors.= 1.42 µm, showed a higher fluorescence for peptides with a higher negative net charge.

Surface modifications on titanium alloys are usually performed to improve osteo-integration and surface bioactivity [START_REF] Liu | Surface modification of titanium, titanium alloys, and related materials for biomedical applications[END_REF]. Bioactive surfaces interact with their biological environment by water and hydrated ions adsorption. inducing bone growth and biochemi cal bonding with proteins [START_REF] Sul | The bone response of oxidized bioactive and non-bioactive titanium implants[END_REF][START_REF] Geetha | Ti based biomaterials, the ultimate choice for orthopaedic implants -a review[END_REF]. Titanium and its alloys are widely used in orthopaedics due to properties such as excellent corro sion resistance, as well as biological-related properties such as the stimulation of bone cell growth [1,3.4). Modifications such as sur face etching with basic (5-7) and acidic solutions [START_REF] Ohtsuki | Bioactivity of titanium treated with hydrogen peroxide solutions containing metal chlorides[END_REF][START_REF] He | Early bone response to sandblasted, dual acid-etched and H2O2/HCl treated titanium implants: an experimental study in the rabbit[END_REF]. or coatings with bioactive materials such as hydroxyapatite, have been shown to increase the bioactivity of titanium and titanium alloy surfaces [START_REF] De Groot | Calcium phosphate coatings for medical implants[END_REF][START_REF] Bigi | Nanocrystalline hydroxyapatite coatings on titanium: a new fast biomimetic method[END_REF][START_REF] Smith | Nanoscale crystallinity modulates cell proliferation on plasma sprayed surfaces[END_REF][START_REF] Wang | Early bone apposition in vivo on plasma-sprayed and electrochemically deposited hydroxyapatite coatings on titanium alloy[END_REF].

A number of techniques used to obtain bioactive coatings include plasma spray [START_REF] Sun | material fundamentals and clinical performance of plasma-sprayed hydroxyapatite coatings: a review[END_REF], electrophoretic deposition [START_REF] Albayrak | Hydroxyapatite coating on titanium substrate by electrophoretic deposition method: effects of titanium dioxide inner layer on adhesion strength and hydroxyapatite decomposition[END_REF], micro arc oxidation [START_REF] Kim | One-step approach for nano-crystalline hydroxyapatite coating on titanium via micro-arc oxidation[END_REF], sol gel [START_REF] Kim | Hydroxyapatite coating on titanium substrate with titania buffer layer processed by sol-gel method[END_REF] and biomimetic methods [START_REF] Habibovic | Biomimetic hydroxyapatite coating on metal implants[END_REF]. The technique most commonly used in the orthopaedic and dental industry is plasma spray [START_REF] Sun | material fundamentals and clinical performance of plasma-sprayed hydroxyapatite coatings: a review[END_REF]. During this method high temperatures are normally used in order to coat metallic surfaces with hydroxyapatite followed by a rapid cooling of the coating [START_REF] Liu | Surface modification of titanium, titanium alloys, and related materials for biomedical applications[END_REF][START_REF] Pfender | Fundamental studies associated with the plasma spray process[END_REF]. As a result, in some cases, heterogeneous hydroxyapatite coatings can be obtained [START_REF] Yan | Characterization of chemical inhomogeneity in plasma-sprayed hydroxyapatite coatings[END_REF][START_REF] Fazan | Dissolution behavior of plasma-sprayed hydroxyapatite coatings[END_REF]. Therefore, research efforts have been focused to develop alternative surface modifications that are cheaper, use less energy and are more biomimetic. One example is surface modification using NaOH or H 2 O 2 to form surface gel layers, sodium titanate gel and titania, respectively, which induce deposition of hydroxyapatite from simulated body fluid (SBF) environments [START_REF] Wang | A comparative study of in vitro apatite deposition on heat-, H2O2-, and NaOH-treated titanium surfaces[END_REF][START_REF] Navarro | Biomaterials in orthopaedics[END_REF]. Apatite deposition onto sodium titanate gel depends strongly on the sodium ion exchange from the gel with H 3 O + ions in SBF [START_REF] Kim | Preparation of bioactive Ti and its alloys via simple chemical surface treatment[END_REF], whereas apatite deposition onto titania gel is associated with the abundance of OH -groups on the surface, presenting a negative charge in SBF [START_REF] Pea | The role of hydrated silica, titania, and alumina in inducing apatite on implants[END_REF].

The titanium oxide layer formed on the titanium surface after treatment with H 2 O 2 presents both acidic (-Ti-O -) and basic (-Ti-OH 2 + ) moieties in aqueous solution at pH 7.5 [START_REF] Sano | A hexapeptide motif that electrostatically binds to the surface of titanium[END_REF]. The amphoteric character of the titanium oxide layer promotes electrostatic interactions with organic molecules such as peptides and proteins. Roddick-Lanzilotta et al. [START_REF] Roddick-Lanzilotta | An in situ infrared spectroscopic study of glutamic acid and of aspartic acid adsorbed on TiO 2: implications for the biocompatibility of titanium[END_REF][START_REF] Roddick-Lanzilotta | An in situ infrared spectroscopic study of the adsorption of lysine to TiO2 from an aqueous solution[END_REF][START_REF] Roddick-Lanzilotta | An in situ infrared spectroscopic investigation of lysine peptide and polylysine adsorption to TiO2 from aqueous solutions[END_REF] have reported electrostatic interactions between modified titanium surfaces and amino acids such as glutamic acid, aspartic acid or lysine; these interactions were highly dependent on solution pH. For example, glutamic acid adsorbs to TiO 2 at higher pH, whereas aspartic acid adsorption is weaker at high pH, but becomes stronger at those pH where electrostatic interactions are favourable [START_REF] Roddick-Lanzilotta | An in situ infrared spectroscopic study of glutamic acid and of aspartic acid adsorbed on TiO 2: implications for the biocompatibility of titanium[END_REF]. It has also been reported that aspartic acid could bridge two titanium ions via a bidentate coordination at pH = 7 [START_REF] Roddick-Lanzilotta | An in situ infrared spectroscopic study of glutamic acid and of aspartic acid adsorbed on TiO 2: implications for the biocompatibility of titanium[END_REF]. Understanding the amphoteric behaviour of the titanium oxide layer when interacting with organic materials led to the synthesis of a titanium peptide binder (RKLPDA) by Sano and Shiba, capable of binding to titanium oxide following treatment with H 2 O 2 [START_REF] Sano | A hexapeptide motif that electrostatically binds to the surface of titanium[END_REF].

Hydroxyapatite binding proteins such as dentin phosphophoryn, osteopontin, osteonectin and bone sialoprotein (BSP) contain domains rich in aspartic and/or glutamic acid [START_REF] Fujisawa | Acidic amino acid-rich sequences as binding sites of osteonectin to hydroxyapatite crystals[END_REF]. These two amino acids interact via their carboxyl group with the Ca 2+ ions of the hydroxyapatite crystal surface [START_REF] Koutsopoulos | The effect of acidic amino acids on hydroxyapatite crystallization[END_REF]. Glutamic acid and aspartic acid oligopeptides have been used in bone drug delivery [START_REF] Wang | Evaluation of water-soluble polymeric bone-targeted drug delivery systems[END_REF] due to their ionic interaction between the anionic oligopeptide and the cationic calcium ion of hydroxyapatite at pH = 7.4 [START_REF] Murphy | Synthesis and in vitro hydroxyapatite binding of peptides conjugated to calcium-binding moieties[END_REF]. The aspartic acid oligopeptides with 6 and 20 amino acids had a high affinity with hydroxyapatite but Hunter et al. reported that the interaction had a negative effect on the nucleation of hydroxyapatite during in vitro mineralization [START_REF] Fujisawa | Acidic amino acid-rich sequences as binding sites of osteonectin to hydroxyapatite crystals[END_REF][START_REF] Hunter | Modulation of crystal formation by bone phosphoproteins: structural specificity of the osteopontin-mediated inhibition of hydroxyapatite formation[END_REF]. On the other hand, the glutamic acid oligopeptides with 6 and 20 amino acids had a spontaneous interaction with hydroxyapatite and allowed nucleation of hydroxyapatite within the steady-state gel system [START_REF] Fujisawa | Acidic amino acid-rich sequences as binding sites of osteonectin to hydroxyapatite crystals[END_REF][START_REF] Hunter | Modulation of crystal formation by bone phosphoproteins: structural specificity of the osteopontin-mediated inhibition of hydroxyapatite formation[END_REF][START_REF] Hunter | Modulation of crystal formation by bone phosphoproteins: role of glutamic acid-rich sequences in the nucleation of hydroxyapatite by bone sialoprotein[END_REF]. The main difference between the oligopeptide types was that at neutral pH, the glutamic acid had a left-handed extended helical conformation, whereas the oligopeptide of aspartic acid had a non-helical conformation [START_REF] Hunter | Modulation of crystal formation by bone phosphoproteins: role of glutamic acid-rich sequences in the nucleation of hydroxyapatite by bone sialoprotein[END_REF].

Creating cost effective multifunctional orthopaedic surfaces that combine bioactivity as well as drug delivery capabilities is highly desirable. The aim of this project is to develop a peptide with the capability to bind to both titanium alloys and hydroxyapatite, and can be used to achieve a biomimetic hydroxyapatite coating on titanium surfaces. The peptide can also be used to functionalise titanium and hydroxyapatite surfaces for selective protein adsorption as well as conjugation of antimicrobial peptides and specialised drugs for drug delivery.

Materials and methods

Peptide synthesis, purification and analysis

All the peptide sequences were synthesised according to the principles of solid-phase peptide synthesis (SPPS). HPLC grade fluorenyl-methyloxy-carbonyl chloride (Fmoc-Cl) protected amino acids and Wang resins were supplied by Novabiochem ® . Analytical grade dimethyl-formamide (DMF), dichloromethane (DCM), and acetonitrile were supplied by Fisher Scientific. Analytical grade piperidine, tri-isopropyl-silane, tri-fluoro-acetic acid (TFA), ninhydrin, 5(6)-carboxy-fluorescein dye (5-FAM) and other reagents used in the synthesis of peptides were supplied by Sigma Aldrich.

The different sequences that were synthesized and labelled with 5-FAM are presented in Table 1 together with their isoelectric points that were calculated using the PepCalc online calculator [35].

The purification of the sequences was performed using a preparative high performance liquid chromatographer (Dionex, USA) with a stationary phase of C18-bonded silica and a mobile phase gradient of 0.1 vol% TFA in water and 0.1 vol% TFA in acetonitrile. The peptide aptamer sequences were analysed under the same mobile and stationary phase of the purification technique in an analytical Shimadzu HPLC. The sequence compositions were verified by electrospray mass spectrometry on a Micromass LCT (Waters, UK).

Preparation of surfaces and coatings

Polished to a mirror finish Ti6Al4V plates

Preparation of single-sided polished Ti6Al4V plates of dimensions 10 mm × 10 mm, cut with a guillotine, was carried out by the Struers method of grinding using an MD-Piano disk with water, at 300 rpm and a force of 40 N, until the samples presented with plane surfaces. This was followed by polishing using the MD-Largo with 9 m diamond solution in oil for 5 min, at 150 rpm with a force of 30 N. Final polishing was performed using an MD-Chem disk with 0.04 m colloidal silica solution in 10% v/v aqueous solution of 30% H 2 O 2 , at 150 rpm and a load of 30 N, until mirror finish. Polishing to a mirror finish of Ti6Al4V plates was necessary in order to ensure homogeneity of surface roughness prior to surface modifications by etching and final growth of the titanium oxide layer. The plates were then washed following 15 min consequent ultrasonic baths with deionised water (dH 2 O), acetone, and dH 2 O, before finally being dried at 22 • C. After drying, the plates were etched using a 30 wt% aqueous solution of H 2 O 2 in order to develop a homogeneous TiO 2 layer. After etching, the plates were rinsed three times with deionised water (dH 2 O).

Hydroxyapatite tablets

Hydroxyapatite (HA) powder (Sigma Aldrich, UK, code 21223) was used to make hydroxyapatite tablets; 700 ± 2 mg of HA powder was added to 100 L dH 2 O and the mixture was placed into a home-made stainless steel mould with a 15 mm internal diameter. The tablets were compressed using a screw-driven Instron mechanical testing machine (Model 1195, Instron Corporation, UK), using a maximum compression load of 5 kN and a compression rate of 2 mm/min. The HA tablets were then sintered at 1100 • C for 2 h, followed by controlled cooling at a rate of 20 • C/min, in a furnace (Elite Thermal Systems, UK). After the sintering process three samples were measured with a digital calliper; the mean diameter was 12.30 ± 0.01 mm, and the mean thickness was 2.80 ± 0.04 mm. The tablets were polished with 400 grit carbide paper until the tablets were visibly flat and smooth; residual materials was removed by rinsing with acetone. After each tablet preparation, the mould was cleaned with acetone until the visible residue of hydroxyapatite disappeared, before drying at 20 • C for 3 min.

Peptide coating

The titanium plates and hydroxyapatite tablets were dip coated for 17 h in 0, 1, 5 and 10 M of 5-FAM labelled peptide solution in phosphate buffer saline (PBS) at pH 7.4 and at a temperature of 20 • C. After the coating procedure, the solution was removed using a micro-pipette (Gilson, USA). The coated titanium plates and hydroxyapatite tablets were soaked in water for 1 min, three consecutive times, and the samples were left at 20 • C in a plastic covered container with silica gel until dried. They were then kept in the fridge and covered with aluminium foil to protect them from exposure to ambient light.

Surface characterisation

The titanium surfaces were imaged with a JEOL 7000 scanning electron microscope (SEM) using an electron voltage of 20 kV and a magnification of X3000. For the fluorescence imaging, a DM6000 Leica microscope was used to track the 5-FAM labelled peptides on the Ti6Al4V and HA surfaces, using a filter according to the requirements for the fluorescent dye used and with an excitation wave length of 495 nm and an emission of 519 nm. The fluorescence intensity is presented in arbitrary units and was calculated using ImageJ (NIH, USA) for three non-overlapping areas, for each sample that exhibited a homogeneous green colour on the surface; the dimensions of each area was 1280 m × 957 m. The fluorescence intensity was not measured for surfaces that did not exhibit a homogeneous colour in the fluorescence microscopy study. The surface roughness was measured using a MicroXAM interferometer (Omniscan, UK) and white light in the visible wavelength range was used. Regions of dimensions 432 m × 321 m were analysed using a 20X lens. A minimum of three regions were recorded per sample in non-overlapping areas. The water contact angle was measured three times per sample in a home-built apparatus using a static drop of deionised water (dH 2 O); the captured images were analysed using ImageJ.

Results

The titanium surfaces obtained after etching using an aqueous solution of 30 wt% H 2 O 2 for 0, 1, 3, 6, 13, 24 and 48 h were analysed by SEM. Micrographs of (a) the untreated surface and (b) surfaces etched for 24 h are presented in Fig. 1, showing the topography change caused by the etching treatment. The polished titanium surface was not completely free of surface features, indicating some titanium oxide layer may be present already on the surface. The surface of the etched sample was clearly different with evidence of crystal growth on the surface. Values of surface roughness obtained using white light interferometry are presented in Table 2. The average roughness (S a ) increased with increasing etching time, and was significantly higher for the treated samples, compared with the polished to mirror finish Ti6Al4V sample.

The wettability study using water contact angle on mirror polished and etched Ti6Al4V samples is presented in Fig. 2. The study demonstrated that the hydrophilicity of Ti6Al4V surfaces increased with increasing etching time of up to 6 h. The hydrophilicity of the samples etched for 6-48 h was similar for all samples, with a small decrease for the sample etched for 24 h. Fig. 3 shows the fluorescence microscopy images of etched titanium surfaces (etching times of 1 h and 24 h) coated with Ti1 peptide binder. The titanium surface that was not etched prior to coating with the peptide, did not present any fluorescence emission, whereas all etched surfaces showed some peptide agglomerations/clusters. The coating shown in Fig. 3a was not homogeneous (according to Materials and Methods, the fluorescence intensity was measured only for the surfaces presenting a homogeneous green colour), but the fluorescence emission increased as peptide agglomerations were present when the time of etching was longer, suggesting a greater interaction with the peptides. On the other hand, Ti6Al4V etched for 24 h presented a homogeneous green colour, suggesting a homogeneous peptide coating (Fig. 3b) The fluorescence intensity for Ti1 (RKLPDA) was 157 ± 1 a.u. and for Ti2 (KKLPDA) it was 188 ± 8 a.u.. Ti2 exhibited 16% higher fluorescence intensity when adsorbed onto Ti6Al4V surface, compared with Ti1. Therefore, the titanium peptide binder used in this work was Ti2 (KKLPDA).

The first combined Ti-Hap peptide binder used in this study was the (5-FAM) labelled KKLPDA EEEEEEEE (Ti-Hap1). Fluorescence microscopy of the Ti6Al4V surface etched for 24 h and dip coated in a solution of 1 M Ti-Hap1 in PBS at pH 7.4 exhibited no emission, indicating that the peptide did not adsorb onto the surface. For this reason, three other peptides were designed; Ti-Hap2, Ti-Hap3 and Ti-Hap4. The coatings were prepared following exactly the same dip coating conditions, using Ti6Al4V etched for 24 h. Homogeneous coatings were obtained for all three combined Ti-Hap peptides and the fluorescence intensities were measured. The Ti-Hap4 coated sample exhibited higher fluorescence intensity compared with Ti-Hap2 and Ti-Hap3 (Fig. 4). For this reason, Ti-Hap4 was used in solutions of concentration 1, 5 and 10 M to coat titanium surfaces etched for 24 h and 48 h, in order to study

Table 3

Fluorescence intensity of Ti6Al4V after etching with 30 wt% H2O2 aqueous solution for 24 and 48 h, followed by immersion in 1, 5 and 10 M concentration of Ti-Hap4 in PBS solution. The errors correspond to standard deviation, with number of samples n = 3.

Ti-Hap4 peptide concentration (M)

Fluorescence intensity (a.u.) on Ti6Al4V surface after [START_REF] Pea | The role of hydrated silica, titania, and alumina in inducing apatite on implants[END_REF] the effect of peptide concentration, as well as the effect of etching time, on the quality of the obtained coatings.

From the fluorescence intensities measured for the 24 h and 48 h etched Ti6Al4V samples (Table 3), it was noticed that the 48 h samples coated with 10 M Ti-Hap4 exhibited 40% higher fluorescence intensity compared with the rest of the samples, suggesting an enhanced interaction of the peptide with the Ti6Al4V surface. Also from Table 3, the fluorescence intensity increased with increasing the peptide concentration in the coating solution, as well as with the etching time of the Ti6Al4V surface.

The average roughness (S a ) of the hydroxyapatite surface after polishing, obtained by white light interferometry and shown in Fig. 5, was S a = 1.42 m, much higher compared with all titanium surfaces. The hydroxyapatite tablets were dip coated with the hydroxyapatite binder (Hap1) as well as the combined peptides Ti-Hap1-4. The hydroxyapatite samples coated with the hydroxyapatite binder Hap1 had the highest fluorescence intensity compared with the combined peptides, showing strong interaction with HA surfaces as expected (Fig. 4). The samples coated with the combined Ti-Hap peptides showed also some adsorption. The Ti-Hap1 peptide showed higher fluorescence intensity compared with the other combined Ti-Hap peptides. Among the modified combined Ti-Hap peptides, the sequence which presented higher fluorescence intensity was the peptide with the glycine spacer (Ti-Hap2). Ti-Hap3 and 4 exhibited similar fluorescence intensities, both lower than Ti-Hap2 (Fig. 4). It should be noted, that the setup of the fluorescence microscope was different between Ti6Al4V and HA surface measurements, and therefore of the measured intensity should not be made between the two different materials in Fig. 4.

Discussion

The roughness of Ti6Al4V surfaces increased with etching time (24 h and 48 h in 30 wt% H 2 O 2 ) compared with the untreated sample. This is in agreement with a similar study, where a commercial pure titanium grade I was etched with 30% v/v H 2 O 2 ; the surface exhibited nanoscale topographical changes when etched for 1-6 h, but exhibited microscale topographical changes when etched for 24 h or more [START_REF] Nagassa | Optimisation of the hydrogen peroxide pre-treatment of titanium: surface characterisation and protein adsorption[END_REF].

The Ti6Al4V samples etched with H 2 O 2 were more hydrophilic than the polished to mirror finish control Ti6Al4V sample, whereas samples etched between 6 h and 48 h were more hydrophilic compared with those etched for up to 3 h (Fig. 2). Other studies also showed that the hydrophilicity of titanium oxide and Ti6Al4V increased in samples treated with H 2 O 2 . For example, titanium (IV) dioxide particles after treatment with peroxide became more hydrophilic [START_REF] Macdonald | Adsorption and dissolution behaviour of human plasma fibronectin on thermally and chemically modified titanium dioxide particles[END_REF]. Similarly, Ti6Al4V surfaces became more hydrophilic after treatment with aqueous 30 wt% H 2 O 2 [START_REF] Macdonald | Thermal and chemical modification of titanium-aluminum-vanadium implant materials: effects on surface properties, glycoprotein adsorption, and MG63 cell attachment[END_REF]. It was concluded that the use of H 2 O 2 resulted in topographical changes, an increase in the oxide layer thickness, and the formation of peroxide complexes such as Ti(IV)O 2 1-, Ti(IV)O 2 2-and Ti(III)O 2 [START_REF] Macdonald | Thermal and chemical modification of titanium-aluminum-vanadium implant materials: effects on surface properties, glycoprotein adsorption, and MG63 cell attachment[END_REF]. Generally, it has been reported that hydrophilic surfaces exhibited improved adsorption of peptides and proteins, as well as enhancing cell attachment [START_REF] Pea | The role of hydrated silica, titania, and alumina in inducing apatite on implants[END_REF][START_REF] Macdonald | Thermal and chemical modification of titanium-aluminum-vanadium implant materials: effects on surface properties, glycoprotein adsorption, and MG63 cell attachment[END_REF][START_REF] Feng | Characterization of surface oxide films on titanium and adhesion of osteoblast[END_REF][START_REF] Ponsonnet | Relationship between surface properties (roughness, wettability) of titanium and titanium alloys and cell behaviour[END_REF].

Surfaces etched for 24 h and 48 h exhibited homogeneous coatings with Ti-Hap2-4 peptides, indicating improved interaction. Samples treated with peroxide for up to 13 h displayed visibly heterogeneous coatings (Fig. 3). A recent fibronectin adsorption study showed that samples of Ti6Al4V pre-treated with peroxide for short periods, e.g., 5 min, did not present increased fibronectin coverage compared with the untreated control sample [START_REF] Macdonald | Thermal and chemical modification of titanium-aluminum-vanadium implant materials: effects on surface properties, glycoprotein adsorption, and MG63 cell attachment[END_REF]. A separate study, where the etching time ranged from 1 h to 4 weeks, showed that a significant increase in the adsorption of fluorescein isothiocyanate (FITC) conjugated albumin serum was observed, compared with the untreated control sample [START_REF] Nagassa | Optimisation of the hydrogen peroxide pre-treatment of titanium: surface characterisation and protein adsorption[END_REF]. The study suggested that the OH - ions or peroxide complexes on the etched surfaces interacted with the charged protein. Hence, the ionic composition of surfaces plays an important role for the adsorption of proteins onto the etched surfaces.

Sano and Shiba [START_REF] Sano | A hexapeptide motif that electrostatically binds to the surface of titanium[END_REF] suggested that the Ti1 (RKLPDA) peptide binder can bind to the titanium surface by the cis-folding of P causing an orientation of R and D amino acids to the surface. An electrostatic interaction between the Ti-O -and R, a Lewis base, and an electrostatic interaction between -Ti-OH 2 + and D, a Lewis acid, summarises the attractive interactions between Ti1 and the titanium surface. This suggests that any other cationic amino acid in the first position in the peptide sequence, and/or anionic amino acid in the fifth position in the peptide sequence, can be used to bind to titanium surfaces.

In this study, Ti1 and Ti2 differ only in their first amino acid, which is either R or K. The coatings formed at pH 7.4 on Ti6Al4V with RKLPDA and KKLPDA were both homogeneous, and the fluorescence intensity in Fig. 4 was slightly higher for KKLPDA. It should be noted that the isoelectric points of both sequences are relatively close to 8.8 (Table 1). It is likely that the amphoteric behaviour of the etched titanium surfaces at pH 7.4 made the interaction with both peptides possible. The homogeneity of the coating for both cases suggests that R or K can be used in the first position of the sequence. In this study, Ti2, with K in the first position of the peptide sequence, was preferred because it resulted in greater surface adsorption compared with Ti1.

The peptide sequences Ti-Hap2-4 were modified from Ti-Hap1 by adding amino acids between the titanium and the hydroxyapatite peptide binders. TiHap2-4 were found to adsorb on the Ti6Al4V surfaces, whereas the Ti-Hap1 did not. This may be a consequence of the additional negative charge from the glutamic acid coil that resulted in a lower isoelectric point of the peptide (3.65) compared with Ti2 (8.72); furthermore at pH = 7.4, the Ti-Hap1 peptide presented a negative charge that would interact weakly with the amphoteric titanium surface. Although the isoelectric point of Ti-Hap2, modified to include a 4-glycine spacer, was the same as Ti-Hap1, only Ti-Hap2 coating was found to adsorb onto the Ti6Al4V surface. This can be explained by the increase in conformational freedom conferred by the glycine spacer in the sequence, which improves the exposure of the titanium binder to the surface, resulting in improved peptide/surface interactions. The role of glycine spacers between a self-assembly sequence RADA 16-I and the functional biological motif PFSSTKT was reported by Taraballi et al. [START_REF] Taraballi | Glycine-spacers influence functional motifs exposure and self-assembling propensity of functionalized substrates tailored for neural stem cell cultures[END_REF], who concluded that a glycine spacer of four residues or more can lead to a more stable self-assembling 3D nanostructure and improved exposure of the functional motif to neural stem cells, resulting in improved cell adhesion, viability and differentiation ability. The peptides Ti-Hap3-4 presented an extra sequence of titanium peptide binder KKLPDA, and hence the isoelectric point increased to 3.94 with the addition of two lysines. These sequences had increased net charge and greater distance between each peptide terminal, but the charge of both sequences at pH 7.4 remained negative. The difference between these two sequences was the position of hydroxyapatite and titanium peptide binders (Table 1). The Ti-Hap4 coating at pH 7.4 showed almost double the fluorescence intensity compared with the other sequences. This observation suggests that the order of the charged amino acids in the sequence affects the interaction with the surface.

The coatings on the hydroxyapatite surface were performed with all peptides containing the glutamic acid motif. It was noted that for peptides with higher negative net charge, the adsorption was higher (Fig. 4). Previous studies reported that the adsorption of proteins on hydroxyapatite was generally better than on titanium. For example Zeng et al. [START_REF] Zeng | Analysis of bovine serum albumin adsorption on calcium phosphate and titanium surfaces[END_REF] reported that bovine serum albumin was adsorbed twice as much on hydroxyapatite than on pure titanium, whereas Kilpadi et al. [START_REF] Kilpadi | Hydroxylapatite binds more serum proteins, purified integrins, and osteoblast precursor cells than titanium or steel[END_REF] reported that both fibronectin and vitronectin were adsorbed on both materials, hydroxyapatite exhibiting higher adsorption than pure titanium once again. Shen et al. [START_REF] Shen | Molecular simulation of protein adsorption and desorption on hydroxyapatite surfaces[END_REF] reported that the electrostatic energy plays a key role in the interactions between proteins and hydroxyapatite surface, whereas COO -and NH 3 + are the main chemical groups present in model proteins interacting with calcium phosphate surfaces. Based on the above findings in the literature, one would expect that the adsorption of peptides onto hydroxyapatite and titanium surfaces in our case should follow a similar trend. However, direct comparison of the fluorescence intensity between the titanium and hydroxyapatite surfaces was not possible as mentioned earlier and therefore such an argument would not be supported by the data presented here.

Conclusions

The ability of a number of peptide sequences to be adsorbed by Ti6Al4V and hydroxyapatite surfaces was studied. It was shown that longer periods of Ti6Al4V surface treatment with peroxide resulted in increased titanium peptide binder (RKLPDA or KKLPDA) adsorption which may be associated with changes in the topography and hydrophilicity of the surface. Further work needs to be conducted to establish the relationship between roughness, hydrophilicity and adsorption of peptides. A small modification of RKLPDA by replacing one arginine by one lysine, both amino acids exhibiting a cationic charge at physiological pH, resulted in a small improvement of the adsorption intensity for the KKLPDA peptide. Four combined Ti-Hap peptides were synthesised by combining the hydroxyapatite peptide binder EEEEEEEE with the titanium peptide binder KKLPDA in different ways using in one case a 4-glycine spacer. The combined Ti-Hap peptide with the highest adsorption to Ti6Al4V was the Ti-Hap4 at pH 7.4, whereas the highest adsorption ability for hydroxyapatite at the same pH was exhibited by the more negatively charged Ti-Hap1 peptide. Both Ti-Hap2, containing the 4-glycine spacer, and Ti-Hap3 were successfully adsorbed onto Ti6Al4V and hydroxyapatite surfaces. The peptide Ti-Hap4 also bound to both surfaces successfully, suggesting that it could be used for further experiments in an arrangement that contains both materials.

Fig. 1 .

 1 Fig. 1. SEM micrographs X3000 of (a) polished to mirror finish Ti6Al4V surface; (b) polished to mirror finish Ti6Al4V surface after etching for 24 h.

Fig. 2 .

 2 Fig. 2. Water contact angle of etched Ti6Al4V using 30 wt% H2O2 aqueous solution for 0, 1, 3, 6, 13, 24 and 48 h; error bars correspond to standard deviation, with number of samples n = 3.

Fig. 4 .

 4 Fig. 4. Fluorescence Intensity on Ti6Al4V surfaces etched for 24 h and HA surfaces. Samples were immersed in a 1 M concentration of peptide in PBS for 17 h; The error bars correspond to standard deviation, with number of samples n = 3. Control surfaces were uncoated; the Ti-Hap1 coating on the Ti6Al4V surface was visibly heterogeneous, and therefore the fluorescence intensity was not calculated. The set-up of the fluorescence microscope varied between the Ti6Al4V and HA surface measurements, and therefore intensity comparisons are not possible between the two different materials.

Fig. 3 .

 3 Fig. 3. Fluorescence microscopy images with 10X magnification (100 m scale bar) of Ti6Al4V samples coated with Ti1 peptide, using a 1 M concentration in PBS solution. Prior to coating, samples were polished to mirror finish and then etched using 30 wt% H2O2 aqueous solution for (a) 1 h and (b) 24 h.

Fig. 5 .

 5 Fig. 5. White light interferometry of the surface of a hydroxyapatite tablet prior to coating with peptides. The average roughness was Sa = 1.42 m.

Table 1

 1 Peptide aptamer sequences, nomenclature, target material for binding, and isoelectric point of each sequence calculated using PepCalc, available at http://pepcalc.com/.

	Peptide aptamer sequence a	Name of peptide	Target material	Isoelectric point
	RKLPDA	Ti1	Ti6Al4V	8.86
	KKLPDA	Ti2	Ti6Al4V	8.72
	EEEEEEEE	Hap1	HA	3.11
	KKLPDA EEEEEEEE	Ti-Hap1	Ti6Al4V and HA	3.65
	EEEEEEEE GGGG KKLPDA	Ti-Hap2	Ti6Al4V and HA	3.65
	EEEEEEEE KKLPDA KKLPDA	Ti-Hap3	Ti6Al4V and HA	3.94
	KKLPDA KKLPDA EEEEEEEE	Ti-Hap4	Ti6Al4V and HA	3.94

a All peptide sequences were labelled with 5-FAM.

Table 2

 2 Average roughness, Sa, of Ti6Al4V after etching with 30 wt% H2O2 aqueous solution for 0, 1, 3, 6, 13, 24 and 48 h, measured by white light interferometry. The errors correspond to standard deviation, with number of samples n = 3.

	Etching time (h)	Sa (nm)
	0	13 ± 9
	1	37 ± 3
	3	29 ± 1
	6	78 ± 4
	13	33 ± 3
	24	197 ± 7
	48	128 ± 2

Acknowledgements

GMR would like to thank the School of Metallurgy and Materials/University of Birmingham for providing a PhD EPSRC studentship. This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 645749.


Yasuhiro Kiyota

Ie-Rang Jeon
email: ie-rang.jeon@univ-rennes1.fr

Olivier Jeannin

Maxime Beau

Tadashi Kawamoto

Pere Alemany

Enric Canadell
email: canadell@icmab.es

Takehiko Mori

Marc Fourmigué
email: marc.fourmigue@univ-rennes1.fr

Electronic engineering of a tetrathiafulvalene charge-transfer salt via reduced symmetry induced by combined substituents

A 1:1 metallic charge-transfer salt is obtained by cosublimation of (Z,E)-(SMe) 2 Me 2 TTF and TCNQ. Xray diffraction studies confirm that the formation of segregated stacks comprising donor and acceptor molecules in [(E)-(SMe) 2 Me 2 TTF](TCNQ). The crystal packing features lateral S•••S interactions between TTF stacks, which is in sharp contrast to that in (TTF)(TCNQ). Structural analysis and theoretical studies afford a partial charge-transfer (r » 0.52), leading to a system with the electronic structure close to quarter-filled. Resistivity measurements reveal that this material behaves as a metal down to 56 K and 22 K at 1 bar and 14.9 kbar, respectively. The thermopower is negative in the metallic regime, indicating the dominant role of the acceptor stacks for the observed conducting behavior. Analysis of a single-crystal EPR spectra shows the remaining spin susceptibility at 4.3 K, suggesting the importance of the Hubbard U correction. These results highlight the judicious engineering of electronic and geometrical effects on the TTF core; the combined use of methyl and thiomethy groups has decreased the TCNQ bandwidth while keeping the segregated stacks, converting the metal to insulator (M-I) transition more 4k F like. In addition, the enhanced S•••S contacts between the TTF stacks lead to more rapidly decreasing M-I transition temperature under pressures.

Introduction

The discovery of metallic behaviour in crystalline organic molecular materials has stimulated a plethora of different research areas covering chemistry and physics. 1 The most notable example is the charge transfer donor-acceptor compound, (TTF)(TCNQ) (TTF = tetrathiafulvalene; TCNQ = 7,7,8,8tetracyanoquinodimethane), which was the first organic conductor to present a metallic conductivity in a wide temperature range down to 59 K. 2 This compound features an anisotropic crystal structure where donor and acceptor molecules form segregated stacks which facilitate a good overlap of respectively HOMOs and LUMOs π-type frontier orbitals along the stack direction to render a 1D electronic structure. 3 The accumulated knowledge on such a system emphasizes the critical role of structural chemistry and crystal engineering. [START_REF] Pouget | Semiconductors and Semimetals[END_REF] In these molecular systems, a subtle change in chemical structure is known to drastically modify geometrical and electronic structures, which in turn enable to discover new exotic physical phenomena and to underpin physical understanding. 5 It is particularly interesting that the intriguing interplay of 2k F and 4k F instabilities have been extensively investigated in (TTF)(TCNQ). [START_REF] Pouget | Semiconductors and Semimetals[END_REF] Scheme 1. Tetrathiafulvalene derivatives with different substituents.

It has been known that tetramethyl-TTF (TMTTF) as well as hexamethylene-TTF (HMTTF) stabilize the metallic conduction, 12 whereas tetrathiomethyl-TTF (TTMTTF) makes insulating TCNQ complexes due to the mixed-stack structures in DDA and DDAA sequences. 13a This comes from the strong dimerization of TTMTTF associated with the steric hindrance of the out-of-plane thiomethyl groups. Methyl substitution increases the donor ability, but thiomethyl substitution reduces the donor ability (Figure S5). Therefore, in order to finely control the electronic structure without destroying the segregated stacks, we have brought our attention to a TTF donor molecule with two thiomethyl-and two methyl-substituents (Z,E)-(SMe) 2 Me 2 TTF, (Z,E)-1 (Scheme 1).

At variance with classical TTF, TMTTF, TTMTTF and BEDT-TTF (bis(ethylenedithio)-tetrathiafulvalene) molecules with D 2h symmetry, such systems can exist into Z (C 2v ) and E (C 2h ) forms, which easily interconvert in solution upon oxidation, making often difficult their (electro)crystallization 6 despite their readily accessible synthesis. Such a difficulty is well demonstrated by the scarcity of the structurally characterized donor-acceptor two-chain charge transfer salts comprising such Z-or E-TTF isomers. 7 Our specific interest for 1 also comes from its first oxidation potential (E 1/2 = +0.41 V vs. SCE, Figure S1) close to that of TTF itself (+0.33 V), a consequence of the electron-rich methyl groups which compensate in part the electron withdrawing effect of the thiomethyl groups. We postulate that such a close proximity of electrochemical potentials likely triggers a non-zero charge transfer if this donor is associated with TCNQ, 8 as demonstrated below.

Results and discussion

Co-crystallization and structural analyses

The neutral (Z,E)-1 was prepared according to the modified literature procedure, by successive deprotection of (Z,E)-(SCH 2 CH 2 CN) 2 (Me) 2 TTF with CsOH and further alkylation with MeI. 9 Cosublimation of (Z,E)-1 and TCNQ in a two-zones furnace 10 under vacuum afforded black, flake-shaped crystals of [(E)-1](TCNQ) in 3 weeks. In addition to [(E)-1](TCNQ), we have also observed the deposition of orange needle-shaped crystals of the donor molecules and yellow flake-shaped crystals of the acceptor molecules within the cosublimation tube (see Experimental section). NMR analysis on the orange crystals that remained after the sublimation indicated that it only contains E-isomer (Figure S2). This observation strongly suggests that the Z-isomer converts to E-isomer during the vaporization in the presence of the TCNQ vapor, and therefore, exclusively affords [(E)-1](TCNQ) as a co-crystallized product. Please note that the sublimation of (Z,E)-1 without TCNQ afforded the mixture of Z-and E-isomers (Figure S2). To the best of our knowledge, such a discrimination of isomers using co-sublimation has never been reported. It is worh to mention that the solution diffusion method for co-crystallization was not successful to obtain single crystals for further analysis, although it provided microcrystalline powders consisting of [(E)-1](TCNQ), as indicated by the powder X-ray diffraction (figure S3). The compound crystallizes in the triclinic system, P-1 space group, with both (E)-1 and TCNQ molecules on an inversion centre. The structure consists of segregated stacks of donor and acceptor molecules running both along the crystallographic a axis, and alternating along the c axis, as shown in Figure 1a.

The intermolecular overlaps along the stacks adopt the bond-over-ring motif commonly observed in highly conducting compounds, with plane-to-plane distances of 3.474 and 3.227 Å in the stacks of (E)-1 and TCNQ respectively. Note that these values are larger than those reported in the RT structure of (TTF)(TCNQ) (3.451 and 3.188 Å respectively) (Table S1), 11 an indication of possibly weaker intermolecular interactions along the stacks in [(E)-1](TCNQ). In the crystallographic ac plane (Figure S4), all molecules are tilted in the same direction with respect to the (100) plane, with an angle of 22.23 and 31.65° for (E)-1 and TCNQ respectively, leading to an interplanar dihedral angle of 9.42°. A remarkable feature is the relative arrangement of the stacks which allows direct S•••S inter-stack contacts along the b axis (Figures 1a andS4) while such lateral interactions are absent in (TTF)(TCNQ) (Figure 1b). Taken together, the arrangement and the packing of donor and acceptor molecules in [(E)-1](TCNQ) strongly differs from that of the unsubstituted (TTF)(TCNQ). 11 Analogous 1:1 salts were also reported with the D 2h -symmetry TTFs such as TMTTF and TTMTTF. The (TMTTF)(TCNQ) metallic salt crystallizes in the P2/c space group with stacks of TMTTF molecules interacting also sideways, but with large and opposite tilt angles vs. the stacking axis. 12 The (TTMTTF)(TCNQ) charge transfer complex is an insulating compound with mixed stacks, as explained in Introduction. 13 Although tetraethyl-TTF (TETTF) has a similar chemical potential located near TCNQ (Figure S5), it also makes mixed stacks with TCNQ, (TETTF)(TCNQ) 2 , owing to the out-of-plane ethyl groups. 13b However, DEDMTTF forms segregated stacks in the TCNQ complex, 13c where the terminal methyl moieties are within the same plane as the TTF part but extend to side of the TTF. The resulting molecular packing of (DEDMTTF)(TCNQ) is very similar to the present complex (Figure 1a), and has direct contacts between the TTF stacks. These comparisons indicate that the balanced substitution between -SMe and -Me groups in the present donor 1 indeed allows for an original structure which enhances the structural dimensionality of the conducting sheets while avoiding DADA mixed stacks.

To estimate the degree of charge transfer, the bond distances within TCNQ were carefully examined using Kistenmacher's relationship (Table S2 and Figure S6). 14 The calculated charge of TCNQ is 0.52, implying a nearly quarter-filled band system. Note that this value is also coherent with r = 0.59 estimated from the C≡N stretch of the FT-IR spectrum 15 and the theoretical calculations (vide-infra). The variable-temperature electrical resistivity was measured under different pressures on single crystals along the a stacking axis (Figure 2). At 300 K, the conductivity is 118 S/cm under ambient pressure, which is comparable to the known organic charge transfer conducting compounds. 3 As temperature decreases, the resistivity decreases smoothly to reach a minimum value at 56 K before an abrupt increase, indicating the occurrence of a metal to insulator (M-I) transition. The activation energy in the insulating region is estimated at E a = 31 meV. Before the M-I transition, the resistivity drop shows a deviation around 130 K, which is confirmed by the maximum of the derivative (Figure S7), likely related to the electronic instability in either the donor or the acceptor stacks (vide-infra).

Transport properties

Upon increasing pressure, the room-temperature conductivity increases and the M-I transition temperature significantly shifts to lower temperatures, to reach 470 S/cm and 22 K, respectively, at 14.9 kbar (Figures 2b and2c). At this highest available pressure, the resistivity rise shows no saturation and E a decreases by half (E a = 16 meV). This is in stark contrast with (TTF)(TCNQ), where the conductivity increases less than twice and the M-I transition temperature hardly varies under similar pressures. 3a,16 The comparatively large pressure-sensitivity of the present compound is likely attributable to the enhanced structural dimensionality coming from the side-by-side S•••S contacts. The applied pressure may indeed reinforce the inter-stack contacts which might ultimately lead to the suppression of the electronic Peierls instability expected for a purely 1D electronic system. As shown in Figure 3, the thermopower exhibits small negative values over the metallic region, indicating that the transport is dominated by the acceptor stacks. 17 However, in contrast to (TTF)(TCNQ) where it amounts to -40 µV/K at 300 K, the small absolute value for [(E)-1](TCNQ), -5 µV/K, indicates an increased contribution of the donor stacks to the conductivity. Upon lowering temperature, the thermopower shows a small peak around 150 K then a gradual decrease. Given the negative carriers become more dominant below 150 K, the anomaly at 150 K is probably to be associated with the setting of 4kF instability in the donor stack. Note that such a peak was observed also for (TMTTF)(TCNQ). 17 Below 70 K, the strong upturn of the thermopower is observed, which may be due to the setting of modulation in the TCNQ stacks that finally condense and open a gap around 50 K. S8). [START_REF]In general, charge transfer donor-accepter complexes show a single Lorentzian due to the sufficiently rapid movement[END_REF] Given that the two Lorentzians manifest at the same angles in all three samples investigated, this is likely an inherent property of the compound and the splitting stems from the existence of inversely tilted domains, which have borders in the ab plane (Figure S9). In view of the extremely small deviation from the monoclinic cell (b = 90.292 o ), this seems to be inherently associated with the present crystal. The electronic structure of [(E)-1](TCNQ) was determined from first-principles DFT+U (U being an effective Hubbard correction term 20 ) calculations based on the 150 K crystal structure. Although DFT calculations usually provide a good description of the electronic structure of molecular conductors, twochain molecular conductors like the present system still face us with some noticeable problems. For instance, the calculated charge transfer in TTF-TCNQ is ~ 0.72-0.74 irrespective of the particular functional and basis set used 21,22 whereas the experimental estimations are 0.55-0.59. [START_REF] Pouget | Semiconductors and Semimetals[END_REF] As we have recently discussed, 22 this problem mostly concerns two-chain conductors containing TTF or TTF-based donors because of a deficient description of their HOMO bands.

ESR analyses

These bands are usually found to be wider than the experimental estimations in two-chain systems for which 4k F fluctuations associated with the TTF-based component have been observed. These observations mean that electronic repulsions in the TTF-based chains are important and that they are underestimated by the DFT approach. The simplest way to remedy for this drawback is by using an effective Hubbard correction term for S 3p orbitals which contracts them and leads to narrower HOMO bands and smaller charge transfers. 22 Since the present system contains TTF-based chains we have considered the U dependence of the electronic parameters of TTF-TCNQ, HMTTF-TCNQ and TMTTF-TCNQ (see for instance Figures S10 to S11) and found that a value U= 6 eV leads to reasonable results for band widths and charge transfers.

For instance, using their room temperature crystal structure the calculated vs. experimental charge transfers (determined at 25 K) [START_REF] Pouget | Semiconductors and Semimetals[END_REF] for TTF-TCNQ, TMTTF-TCNQ and HMTTF-TCNQ are 0.57/0.59, 0.56/0.57 and 0.68/0.72. Thus, we have adopted this value in our study.

The calculated band structure and density of states are reported in Figure 6. The upper band at G is based on the (E)-1 HOMO and the lower band is based on the TCNQ LUMO. The two bands cross along the intrachain G-X line, at the Fermi energy. However, in these two-chain conductors the avoided crossing between the HOMO and LUMO bands is usually slightly overestimated by DFT so that we believe that the gap along G-X is smaller and probably irrelevant for most temperatures. The small but non nil dispersion The avoided band crossing occurs for a k F value of 0.13a* and consequently, the charge transfer (r = 4´k F /a*) is 0.52. Integration of the DOS curves (see Figure 6b where the total DOS as well as the separate contributions of the donor and acceptor bands are also shown) leads to r = 0.53. These values are very similar to the r = 0.52 one estimated above from the bond lengths of the acceptor thus confirming that the charge transfer must be very close to 0.5. This situation is clearly different from that of the archetypal (TTF)(TCNQ) for which the charge transfer (r = 0.59) is definitely far from the quarterfilled/empty case and thus a different physical behavior may be expected. Note that whatever the value of U used in the calculations, the difference with (TTF)(TCNQ) does not practically change (Figure S10) so that the result is a robust one.

Comparison with (TTF)(TCNQ)

What is the origin of the charge transfer difference with (TTF)(TCNQ)? It may be argued that it is due to the decrease in the HOMO energy induced by the -SMe substituents, illustrated by the increase of first oxidation potential when compared with TTF. However, the charge transfer in these two-chain molecular conductors subtly depends on both electronic and structural factors. A decrease of the charge transfer can also occur if one or both of the HOMO and LUMO bands become narrower. The dispersion of the TCNQ band depends essentially of the C•••C contacts along the intrachain a direction. As mentioned above, these contacts in [(E)-1](TCNQ) (Table S1) are clearly longer than even those for the RT structure of (TTF)(TCNQ). Hence we can conclude that the TCNQ band in the present salt must be narrower than in (TTF)(TCNQ).

The dispersion of the donor band depends essentially from the six intra-stack S•••S contacts. In the RT structure of TTF-TCNQ, the same contacts are again found to be shorter (Table S1), as evidenced also by the cell parameter along the chains direction (3.842 Å for [(E)-1](TCNQ) at 150 K vs 3.819 Å for (TTF)(TCNQ) at RT). However, the (E)-1 donor has two additional sulfur atoms involved in the HOMO.

The extra S SMe •••S TTF intra-stack contacts increase the HOMO•••HOMO interaction with respect to (TTF)(TCNQ) and it is not clear if, in the present compound, the dispersion of the HOMO band should slightly decrease or even increase.

Assuming a linear relationship for the thermal evolution of the DFT band widths, we estimate that the widths of the HOMO and LUMO bands for (TTF)(TCNQ) at 150 K should be 0.59 and 0.83 eV, respectively. The calculated band widths for the present compound are 0.70 and 0.68 eV, respectively. As expected, the band width for the TCNQ band decreases in [(E)-1](TCNQ). In contrast, the decrease in the strength of the intrachain S•••S interactions of the TTF core is more than compensated by the extra S SMe •••S TTF contacts, leading ultimately to an increase of the HOMO band width. However, we note that the variation for the TCNQ band is somewhat larger. In addition, the increase in the donor band width (and thus, of charge transfer) will mostly compensate the intrinsic lowering in the HOMO energy (and thus, the decrease of the charge transfer) due to the -SMe substituents. We thus suggest that the substantial decrease of the C•••C contacts in the TCNQ stacks is the main reason behind the small but significant charge transfer decrease, now very close to the quarter-filled/empty value.

Low temperature behaviour scenario

What are the possible scenario for the low temperature behavior of this compound? An important factor to consider is the filling of the LUMO/HOMO bands which at low temperatures may become almost exactly quarter-filled/empty (r = 0.5) or can keep a slightly different value. In view of the transport measurements reported above and relying on analogies with other TTF-based two-chain salts [START_REF] Pouget | Semiconductors and Semimetals[END_REF]17 we suggest the following scenario. The small anomaly occurring around 150 K clearly seen in the thermopower measurements is probably to be associated with the setting of 4k F fluctuations in the donor stacks since the negative carriers become more dominant below 150 K. The strong upturn around 70 K may be due to the setting of a fluctuations regime in the TCNQ stacks which finally condense and open a gap at around 50 K. However, if a charge transfer very near to the quarter-filled/empty case remains at low temperatures the modulation on the TCNQ stacks could be a 2k F tetramerization and the regime change in the (E)-1 stacks could then be of the 4k F charge-ordering or Mott-dimer types, explaining that the magnetic susceptibility remains nonzero until very low temperatures. 23 X-ray diffuse scattering studies are needed in order to shed light on these issues.

Conclusions

In this study, we have successfully grown a 1:1 co-crystal of (E)-1 and TCNQ and have investigated its physical properties in detail. The obtained crystal of [(E)-1](TCNQ) features the segregated D and A stacks with the DA packing that is notably different when compared with the analogous charge transfer TCNQ salts comprising the classical TTF donors. In particular, the lateral S•••S contacts between donor stacks give rise to the structural two-dimensionality in [(E)-1](TCNQ).

The band structure of the compound features close to a quarter-filled system with r » 0.52, leading to a metallic behaviour down to 56 K. Moreover, both the conductivity and the metal-to-insulator transition temperature are significantly influenced by the applied pressure, likely due to the enhanced structural dimensionality.

In particular, slight delocalization in the HOMO of the donor by means of the two thiomethyl-and two methyl-substituents leads to a situation where the donor and acceptor band widths are very similar and the charge transfer is very near the commensurate value 0.5. To the best of our knowledge the only other twochain salts of this family exhibiting both features are (DEDMTTF)(TCNQ) and (TMTTF)(DMTCNQ) (DMTCNQ = 2,5-dimethyl-7,7,8,8-tetracyanoquinodimethane). 4,13c These salts lie just at the borderline between those where the dominant structural instability changes from 2k F to 4k F . [START_REF] Pouget | Semiconductors and Semimetals[END_REF] Thus, it would be extremely interesting to study if this is also the case for the present salt. If it is indeed the case, small structural or electronic modifications could bring substantially different physical behaviors and provide invaluable information to settle the difficult issue of the competition between structural instabilities in twochain conductors.

Work is underway to prove the ground state of [(E)-1](TCNQ) and to explore the promising role of (Z,E)-1 as electron donor to grow co-crystals with intriguing electronic structures.

Experimental General methods

Chemicals and materials from commercial sources were used without further purification. Solvents were dried using a commercial solvent purification system from Inert. The (Z,E)-(SCH 2 CH 2 CN) 2 (Me) 2 TTF was prepared according to the literature procedure. 9 Melting points were measured on a Kofler hot-stage apparatus. NMR spectra were recorded on a Bruker AV III 300 MHz NMR spectrometer at room temperature using CDCl 3 . Column chromatography was performed using Teledyne Isco CombiFlash® Rf400. Cyclic voltammetry measurements were carried out in a one-compartment cell under argon, equipped with a platinum wire, a glassy carbon, and a Saturated Calomel Electrode (SCE) as working, counter, and reference electrodes, respectively, using a Bio-Logic SP-50 potentionstat. Analyte solutions (10 -3 M solution of complex) were prepared in 0.1 M solutions of (NBu 4 )(PF 6 ) in CH 2 Cl 2 .

Synthesis of (Z,E)-1

To a solution of the (Z,E)-(SCH 2 CH 2 CN) 2 (Me) 2 TTF (300 mg, 0.75 mmol) in 75 mL of THF was added under argon a solution of CsOH•H 2 O (372 mg, 2.22 mmol) in 10 mL of MeOH dropwise at 0 °C. The resulting solution was stirred at 25 °C for 90 minutes to afford a dark mixture. MeI (0.3 mL, 4.82 mmol) was then added to the flask, and the resulting mixture was stirred for 12 h to give a yellow solution. After quenching the reaction with 60 mL of distilled H 2 O, the organic solvent mixture was removed from the reaction under vacuum, and the resulting residue was extracted into CH 2 Cl 2 (4 x 60 mL). The organic extracts were dried over magnesium sulfate, filtered, and then concentrated under reduced pressure to give a red oil. The crude oil was purified by flash column chromatography on a silica gel to afford (Z,E)-1 as an orange solid (198 mg, 82 %). The product was recrystallized by slowly evaporating the saturated EtOAc solution. Column conditions: silica cartridge 12 g; solid deposit on Celite®; λ detection : (254 nm, 280 nm); CH 2 Cl 2 in light petroleum with a gradient 0 -30%. mp = 133 °C. 1 H NMR (300 MHz, Chloroform-d) δ = 2.31-2.32 (s 3H), 2.12-2. 13 (s, 3H).

Crystallization of [(E)-1](TCNQ)

Single crystals were prepared by the co-sublimation method. To opposite ends of a 30 cm thin-necked glass tube, (Z,E)-1 and TCNQ were placed. The glass tube was then sealed in vacuo with a pressure of 1 × 10 -5 mbar. In a two-zone furnace, the segments of the tube were heated to 130 and 250°C for the donor and the acceptor, respectively, to which the temperature was gradually raised from room temperature at a rate of 0.04 °C/min. The temperature of the central zone was approximately 180°C. After three weeks, black crystals grow near the center of the tube. Since a single central zone of crystals composed of a charge transfer salt was obtained, other complexes with (Z)-1 or different compositions were not obtained by this method. Note that our co-sublimation condition did not allow all vaporized donor and acceptor molecules flew to the central zone to react. Accordingly, there was also the deposition of each starting material along the tube, orange needle-shaped crystals of the donor molecules and yellow flake-shaped crystals of the acceptor molecules.

X-ray structure determination

Single crystals of (E-SMe 2 Me 2 TTF)(TCNQ) that are suitable for X-ray analysis were coated with Paratone-N oil and mounted on a MicroMounts TM rod. The crystallographic data were collected on a Bruker D8 venture diffractometer equipped with MoKα microsource and PHOTON100 detector at the Centre de Diffractométrie (CDIFX, Université de Rennes 1, France) at 150 K. Raw data were integrated and corrected for Lorentz and polarization effects using Bruker APEX3 v. 2015. [START_REF]Bruker Analytical X-Ray Systems[END_REF] The structure was solved by direct methods using the SIR92 program, [START_REF] Altomare | [END_REF] and then refined with full-matrix least-square methods based on F 2 (SHELXL-2014) 26 with the aid of the WINGX program. 27 All non-hydrogen atoms were refined with anisotropic atomic displacement parameters.

Electrical resistivity and thermopower

Electrical resistivity was measured by the four-probe method down to 2.7 K. Four gold wires (15 µm diameter) were attached to a crystal using carbon paste. Low-frequency AC current was applied to the outer wires and the resistance was estimated from the voltage measured at the inside wires. Pressure was applied to a crystal by a conventional clamp cell with an oil Daphne #7373 as a pressure medium. 28 The absolute value of the pressure was estimated from the resistance change of a manganin wire at room temperature.

Results for 9.1 kbar to 14.9 kbar are taken using a different sample from that for 1 bar to 7.4 kbar. For thermopower measurement, a crystal was attached to two copper blocks via gold foils using carbon paste.

The copper blocks were alternately heated by glued tip resistors to generate DT less than 1K. ΔT was measured by an AuFe-chromel thermocouple. Thermopower was estimated from the slope of the generated voltage DV vs. the temperature difference DT. 29 All measurements were conducted along the a-axis.

ESR measurement

ESR measurements were carried out for a single crystal using X-band spectrometer (JEOL JES-TE100).

The g-values were calibrated by the 3 rd (g = 2.034) and 4 th (g = 1.981) signals of a Mn 2+ marker. The crystal was located so as to make the long axis parallel to the magnetic field, and rotated by 15 o steps up to 180 o for the angle dependence measurements (Figure S5). Although the crystal was chosen carefully, the observed signal was composed of two Lorentzians when the crystal long axis was not parallel to the magnetic field. The signal was analyzed by a sum of two Lorentzians, where H was the magnetic field, I was the intensity of the signal, H 0 was a center of the signal, and DH pp was the linewidth of the signal. Temperature dependence measurements were carried out down to 4.3 K for the single Lorentzian and double Lorentzians.

Computational details

The density functional theory (DFT) calculations were performed using a numerical atomic orbitals DFT 30 approach developed for efficient calculations in large systems and implemented in the SIESTA code. 31 The generalized gradient approximation to DFT and, in particular, the functional of Perdew, Burke, and Ernzerhof (PBE) 32 has been used. Recently 22 we have found that inclusion of a Hubbard correction term 20 U for S 3p orbitals is needed for accurately describing the electronic structure of TTF-TCNQ and related two-chain molecular solids for which accurate experimental information on the bandwidth and charge transfer is available. In the present work, we have considered the dependence of the experimentally observed charge transfer for several sulfur-containing two-chain salts and found that a good agreement is found for U= 6 eV. Thus, except otherwise stated we have used this value in our calculations. The contribution of core electrons has been described by norm-conserving Troullier-Martins relativistic pseudo-potentials, 33 factorized in the Kleinman-Bylander form. 34 Valence electrons were treated explicitly using a split-valence basis set of double-ξ plus polarization functions obtained with an energy shift of 10 meV. 35 The energy cutoff for the real space integration mesh was set to 300 Ry and the Brillouin zone was sampled using a mesh with 9×25×3 k-points obtained using the method of Monkhorst and Pack. 36 All calculations were performed using the experimental crystal structure.

(a) T. J. Kistenmacher, T. J. Emge, A. N. Bloch and D. O. Cowan, Acta Cryst., 1982, B38, 1193; (b) K. Bechgaard, T. J. Kistenmacher, A. N. Bloch, Acta Cryst., 1977, B33, 417.

Figure 1 .

 1 Figure 1. Projection views of the crystal packing in [(E)-1](TCNQ) (a) and (TTF)(TCNQ) (b). Yellow, blue, gray, and pink spheres represent S, N, C, and H atoms, receptively.

Figure 2 .

 2 Figure 2. (a) Temperature dependence of the resistivity under pressure. The inset is a logarithmic plot for clearly at low temperatures. Pressure dependence of (b) the room-temperature conductivity and (c) T M-I determined by the resistivity minimum.

Figure 3 .

 3 Figure 3. Variable-temperature thermopower of [(E) -1](TCNQ).

Figure 4 .

 4 Figure 4. Angle dependence of the g-value and the linewidth at (a)(b) 294 K and (c)(d) 4.3 K, highlighting two independent Lorentzians indicated with red (open) and blue (filled) symbols.

Figures 4

 4 Figures 4 shows the angle dependence of the extracted g-values and linewidths at room temperature and at 4.3 K. Although single crystals were chosen carefully, the observed ESR signal was systematically composed of two Lorentzians (FigureS8).[START_REF]In general, charge transfer donor-accepter complexes show a single Lorentzian due to the sufficiently rapid movement[END_REF] Given that the two Lorentzians manifest at the same angles in

Figure 5

 5 Figure 5 shows temperature dependence of the spin susceptibility and the linewidth estimated from a single Lorentzian at 0 o and double Lorenzian at 30 o . The spin susceptibility is almost constant with a slight decrease down to 65 K, indicating the Pauli-paramagnetic like behavior. Below 65 K, the spin susceptibility gradually increases. The linewidth also takes a minimum at 65 K and then increases. The observed similar tendency for both Lorentzians supports the same origin of the two signals at 30 o . Broadening of the ESR signal is likely related to the nesting of the TCNQ Fermi surface as observed in related compounds,(TTF)(TCNQ) and (TMTTF)(TCNQ).[START_REF]In general, charge transfer donor-accepter complexes show a single Lorentzian due to the sufficiently rapid movement[END_REF]19 At low temperature, the spin susceptibility remains non-zero

  along the interchain b*-direction (G-Y) of the donor band, which is associated with an S•••S contact of 3.767 Å, slightly lowers the energy of the upper band and consequently overlaps with the top of the lower band, definitely leading to a band overlap inside the Brillouin zone and thus, a metallic-type behavior.

Figure 6 .

 6 Figure 6. Calculated band structure (a) and Density of states (b) for Me 2 (SMe) 2 TTF-TCNQ at 150 K. The Fermi levelis the energy zero. G = (0, 0, 0), X = (1/2, 0, 0), Y = (0, 1/2, 0), M = (1/2, 1/2, 0) and Z = (0, 0, 1/2) in units of the triclinic reciprocal lattice vectors. The DOS is given in units of electrons per eV and per formula unit.

J. P. Perdew, K.Burke and M. Ernzerhof, Phys. Rev. Lett., 1996, 77, 3865.

Acknowledgements

This research was supported by the French National Research Agency Grants, ANR 16-ACHN-0007 and ANR 17-ERC3-0003, and a PhD grant (to M. Beau) from the Bretagne region. Y. Kiyota is grateful to JSPS for a research fellowship (17J05098). We thank Dr. A. Vacher (Rennes) for the preparation of (Z,E)-(SCH 2 CH 2 CN) 2 (Me) 2 TTF and CDIFX-Rennes for access to X-ray diffractometer. Work in Spain was supported by MICIU (PGC2018-096955-B-C44 and PGC2018-093863-B-C22), MINECO through the Severo Ochoa (SEV-2015-0496) and Maria de Maeztu (MDM-2017-0767) Programs and Generalitat de Catalunya (2017SGR1506 and 2017SGR1289). Work in Japan was supported by JPSJ KAKENHI (Grant Number 16K13974 and 18H02044) and Takahashi Industrial and Economic Research Foundation.

Notes and references


Patrick Bardsley

Maxence Cassier
email: maxence.cassier@fresnel.fr

Fernando Guevara Vasquez

Imaging small dielectric inclusions with polarization data

Keywords: Polarization imaging, Kirchhoff migration, phaseless imaging, coherency matrix

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction and mathematical model

We consider the problem of imaging the position and polarizability tensor of N small dielectric polarizable inclusions in an homogeneous medium (with permeability µ) by using polarization data only. This is a common assumption in optics, where it is easier to measure polarization (a statistical property of light) than the full electrical field. We assume that the medium is probed by a single electric source whose position x s ∈ R 3 is known and dipole moment is j s (ω) ∈ C 3 at the frequency ω. Furthermore, the source and the two dimensional array A of receivers (within the plane z = 0) are supposed to be far from the inclusions. To model "white light" we assume its "rescaled dipole moment" j s (ω) = µ ω 2 j s (ω) ∈ C 3 is given at each frequency by a circular symmetric Gaussian random vector with zero mean j s (ω) = 0. The covariance of this vector is assumed known and determines the polarization state of the probing wave. It is given as U s J s (ω)U * s , where J s (ω) is a 2 × 2 complex positive definite matrix and U s is 3 × 2 unitary matrix whose range Ran(U s ) is the cross-range of the source. This corresponds to losing the range component of the source's polarization in the far field, where the probing wave is close to a plane wave near the inclusions [START_REF] Bardsley | Imaging small polarizable scatterers with polarization data[END_REF].

At each realization, the source emits an incident wave: E i (x; ω) = G(x, x s ; ω) j s (ω), where G(x, x s ; ω) denotes the standard Dyadic Green tensor [START_REF] Novotny | Principles of nano-optics[END_REF]. As the inclusions are assumed to be small with respect to the wavelength, they can be modelled as a point-like inclusion localized at y n ∈ R 3 whose scattering properties are determined by a polarizability tensor α (y n ; ω) for n = 1, . . . , N that is a 3 × 3 complex symmetric matrix. We denote by α(y n ) = µ ω 2 α (y n ; ω) "the rescaled polarizability tensor", assumed here to be frequency independent. The incident wave is then reflected and the resulting scattered field (see [START_REF] Novotny | Principles of nano-optics[END_REF]) is given under the first Born approximation by: E s (x; ω) = Π(x; ω)j s (ω) with

Π(x; ω) = N n=1 G(x, y n ; ω) α(y n ) G(y n , x s ; ω).
Finally, one records at each receiver x r ∈ A the 2 × 2 coherency matrix:

ψ(x r ; ω) = ( E(x r ; ω)E(x r ; ω) 1:2,1:2 , (1)
that encodes the polarization of the total field E = E i +E s on A (where • is the expectation).

Kirchhoff imaging with ideal data

We consider now the case of ideal data, that is when one can measure Π(x; ω) for x ∈ A or equivalently the scattered field for three independent source's dipole moments. We use a matrix-valued electromagnetic version of the Kirchhoff imaging function [START_REF] Cassier | Imaging polarizable dipoles[END_REF]:

I[Π](y; ω) = A dx r G(x r , y; ω)Π(x r ; ω)G(x s , y; ω).
We denote by U r = [e 1 , e 2 ] (where (e 1 , e 2 , e 3 ) is the canonical basis of R 3 ) the 3 × 2 unitary matrix whose range is the cross-range of A. We extract from I a stable reconstruction [START_REF] Bardsley | Imaging small polarizable scatterers with polarization data[END_REF][START_REF] Cassier | Imaging polarizable dipoles[END_REF] of the position y n and of the 2 × 2 block α n = U * r α(y n )U s of the polarizability tensor α(y n ) (up to a complex phase). The other components of α(y n ) are lost in this regime of propagation. In the Fraunhofer regime [START_REF] Bardsley | Imaging small polarizable scatterers with polarization data[END_REF], resolution estimates for the reconstruction of these two quantities are given (as in acoustics) by the Rayleigh criterion in cross-range and by c/B in range (where the constant c is the wave propagation speed and B the frequency bandwidth of the data Π(•, ω)). Moreover, one shows that

I[Π](y; ω) ≈ I[U r ΠU * s ](y; ω) with Π = U * r ΠU s .
Thus, instead of using Π, one can image as well with U r ΠU * s , i.e. the ideal data Π projected on the left on the cross-range of the array and on the right on the cross-range of the source.

The Phase-less imaging method

The strategy we use for imaging generalizes the approach for scalar waves of [START_REF] Bardsley | Kirchhoff migration without phases[END_REF]. One first preprocesses the data ψ(x r ; ω) to calculate the matrix p(ψ) = U r ΠU * s + error terms. It consists in particular to partially recover the data U r ΠU * s by eliminating the contribution of the incident field E i in [START_REF] Bardsley | Imaging small polarizable scatterers with polarization data[END_REF]. The remaining error terms are antilinear and sesquilinear in Π. The key is that these terms do not affect the Kirchhoff images for high frequencies. Indeed, one shows [START_REF] Bardsley | Imaging small polarizable scatterers with polarization data[END_REF] via a stationary phase argument and under mild assumptions on the geometry of the problem that the imaging functions with either p(ψ) or Π data are similar, i.e. as ω → ∞ we have

I[p(ψ)](y; ω) = I[U r ΠU * s ](y; ω) + o(1).
Our method is illustrated in figure 1 where the medium contains two dipoles located at y 1 = (-6, 4, 105)λ 0 and y 2 = (7, 4, 105)λ 0 with λ 0 the central wavelength of B. To image the position, we use the Frobenius norm α(•) F of the recovered polarizability tensor. To visualize the polarization tensor (up to a complex phase) we visualize Re α = 1/2( α + α) and Im α = (1/2i)( αα), which are real non-symmetric matrices. We visualize 2 × 2 real matrices A with the ellipse E(A) = {Av | v 2 = 1}. To emphasize that the matrices are not symmetric, we also display the vectors σ 1 v 1 and σ 2 v 2 as axes, where the σ j are the singular values and v j the right singular vectors of A. Here (up to a reference phase [START_REF] Bardsley | Imaging small polarizable scatterers with polarization data[END_REF]) the true tensor Re α (resp. Im α) is depicted by the white (resp. yellow) ellipses/axes, whereas the recovered tensor is depicted using black (real part) and magenta (imaginary part) ellipses/axes.

Figure 1 :

 1 Figure 1: Cross-range (top) and range (bottom) images of α(•) F . The columns show reconstructions with Π (left) and with p(ψ) (right).Here (up to a reference phase[START_REF] Bardsley | Imaging small polarizable scatterers with polarization data[END_REF]) the true tensor Re α (resp. Im α) is depicted by the white (resp. yellow) ellipses/axes, whereas the recovered tensor is depicted using black (real part) and magenta (imaginary part) ellipses/axes.

This work was partially supported by the National Science Foundation grant DMS-1411577. The work of M. C. was supported by Simons Foundation grant #376319 (M. I. Weinstein).