Raquel De Oliveira

Mathieu Durand

Lylian Challier

Pierluca Messina

Jean Marie Swiecicki

Margherita Di Pisa

Gérard Chassaing

Solange Lavielle

Olivier Buriez

Eric Labbé

Electrochemical Quenching of the Fluorescence produced by NBD-labelled Cell Penetrating Peptides: a contribution to the study of their internalization in Large Unilamellar Vesicles

Keywords: fluorescence quenching, eElectrochemical, cell penetrating peptides, internalization, Large Unilamellar Vesicles

This work investigates the implementation of a simple and versatile electrochemical setup aimed at achieving a fast and complete fluorescence extinction of cell penetrating peptides contained in 2-5 cm 3 samples containing phosphate buffer + Large Unilamellar Vesicles. The quenching is obtained through a reductive electrolysis in a 2compartment cell homebuilt from disposable plastic labware, which remains inert towards the adsorption of both peptides and lipid vesicles. Considering the micromolar concentration of NBDtagged peptides, the main electrochemical reaction observed is hydrogen evolution, NBD reduction representing a small fraction of the cathodic current/charge engaged. The electrolysis conditions are discussed with respect to the nature of the reduction products formed, the integrity of Large Unilamellar Vesicles and phosphate buffering properties. This electrochemical method is compared to the traditional chemical dithionite quenching of NBD and tested to monitor the internalization of Cell Penetrating Peptides in Large Unilamellar Vesicles.

Introduction

The transport of molecules across biological membranes [START_REF] Swiecicki | How to unveil self-quenched fluorophores and subsequently map the subcellular distribution of exogenous peptides[END_REF] is a crucial issue in drug delivery, signal transduction and peptide/protein internalization. Cell-penetrating peptides (CPPs), namely, act as vectors for a variety of cargoes such as nucleotides, proteins, nanoparticles or therapeutic agents [START_REF] Eguchi | siRNA delivery using peptide transduction domains[END_REF][START_REF] Gupta | Intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides[END_REF][START_REF] Koren | Cell-penetrating peptides: breaking through to the other side[END_REF][START_REF] Torchilin | Cell penetrating peptide-modified pharmaceutical nanocarriers for intracellular drug and gene delivery[END_REF][START_REF] Lindberg | Therapeutic delivery opportunities, obstacles and applications for cell-penetrating peptides[END_REF]. The uptake of these peptides -which are mostly cationic under physiological pH conditions -within cells or liposomes delimited by phospholipid bilayers has risen a number of questions about the postulated energy-

A C C E P T E D

M A N U S C R I P T 2 independent internalization mechanism (also called passive transport) of charged peptides across hydrophobic barriers [START_REF] Koren | Cell-penetrating peptides: breaking through to the other side[END_REF][START_REF] Zorko | Cell-penetrating peptides: mechanism and kinetics of cargo delivery[END_REF].

In this context, fluorescence methods have been intensively used to monitor and quantify the amount of internalized peptides within liposomes during a given incubation period vis-à-vis those located outside [START_REF] Henriques | How to address CPP and AMP translocation? Methods to detect and quantify peptide internalization in vitro and in vivo (Review)[END_REF][START_REF] Mager | Assessing the uptake kinetics and internalization mechanisms of cell-penetrating peptides using a quenched fluorescence assay[END_REF][START_REF] Swiecicki | Accumulation of Cell-Penetrating Peptides in Large Unilamellar Vesicles: A Straightforward Screening Assay for Investigating the Internalization Mechanism[END_REF][START_REF] Miller | Some recent advances in fluorescence spectroscopy[END_REF].

NBD (7-nitrobenz-2-oxa-1,3-diazole) is a small fluorescent probe originally introduced to label proteins through binding to their hydrophobic regions [START_REF] Kenner | New Fluorescent Probe For Protein And Nucleoprotein Conformation -Binding Of 7-(Para-Methoxybenzylamino)-4-Nitrobenzoxadiazole To Bovine Trypsinogen And Bacterial Ribosomes[END_REF]. The poor steric hindrance of NBD, making this fluorescent probe structurally neutral upon covalent grafting has popularized its use in the labelling of phospholipids [START_REF] Mazeres | 7-nitrobenz-2-oxa-1,3-diazole-4-yl-labeled phospholipids in lipid membranes: Differences in fluorescence behavior[END_REF][START_REF] Brown | Self-Quenching Of Nitrobenzoxadiazole Labeled Phospholipids In Lipid-Membranes[END_REF], sugars [START_REF] Funabashi | Utilization of Fluorescent Glucose Analog 2-NBDG as a Metabolic Indicator for FACS Analysis during ES Cell Differentiation[END_REF] or CPPs [START_REF] Swiecicki | Accumulation of Cell-Penetrating Peptides in Large Unilamellar Vesicles: A Straightforward Screening Assay for Investigating the Internalization Mechanism[END_REF][START_REF] Di Pisa | Translocation Mechanism(s) of Cell-Penetrating Peptides: Biophysical Studies Using Artificial Membrane Bilayers[END_REF] .

Chemical structure of NBD

In the study of CPP internalization within cells or vesicles, the quantification of internalised peptides relies on the comparison between fluorescence emitted before incubation and after incubation + chemical quenching of the fluorescence emitted by non-internalised peptides, as depicted in scheme 1:

Scheme 1 Classical DT quenching procedure used to monitor CPP internalization

This procedure is convenient as long as the cell or the vesicle remains hermetic to the agent used to switch fluorescence off after the incubation period. The quenching of NBD fluorescence is generally carried out through the introduction of reducing agents such as hydrogen sulphide [START_REF] Bae | Colorimetric signaling of hydrogen sulfide by reduction of a phenylseleno-nitrobenzoxadiazole derivative[END_REF] and more traditionally dithionite [START_REF] Mcintyre | Fluorescence Assay for Phospholipid Membrane Asymmetry[END_REF][START_REF] Angeletti | Dithionite quenching rate measurement of the inside-outside membrane bilayer distribution of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labeled phospholipids[END_REF], the latter being also described for its fluorescence quenching properties of chlorophyll [START_REF] Neubauer | DITHIONITE-INDUCED FLUORESCENCE QUENCHING DOES NOT REFLECT REDUCTIVE ACTIVATION IN SPINACH-CHLOROPLASTS[END_REF]. However, this cheap, physiologically compatible chemical quencher has recently been found to These considerations prompted us to develop a non-chemical strategy to quench NBD fluorescence.

Considering the redox nature of the reaction between dithionite and NBD, an electrochemical reduction could bring a significant benefit in several aspects [START_REF] Buriez | Selective Electrochemical Bleaching of the outer Leaflet of Fluorescently Labeled Giant Liposomes[END_REF]: i) It avoids adding exogenous chemical agents like dithionite ii) the heterogeneous nature of electron transfers achieved at solid electrodes imposes that the electrochemical reduction only affects species dissolved outside the vesicles or aggregated in the outer phospholipid leaflet of the vesicles iii) the counter-reaction (oxidation) can be separated in another compartment. Electrochemical activation/deactivation of fluorescent probes is not a new concept per se.

Indeed, Miomandre et al. have described the electrochemically controlled switching of chloromethoxytetrazine [START_REF] Miomandre | Fluorescence microscopy coupled to electrochemistry: A powerful tool for the controlled electrochemical switch of fluorescent molecules[END_REF], and other dyes have also been switched on/off electrochemically [START_REF] Mi | AIEE-Active and Electrochromic Bifunctional Polymer and a Device Composed thereof Synchronously Achieve Electrochemical Fluorescence Switching and Electrochromic Switching[END_REF][START_REF] Seo | Electrochemical Fluorescence Switching from a Patternable Poly(1,3,4-oxadiazole) Thin Film[END_REF][START_REF] Kuo | Electrochemical fluorescence switching properties of conjugated polymers composed of triphenylamine, fluorene, and cyclic urea moieties[END_REF][START_REF] Shin | Efficient Fluorescence "Turn-On" Sensing of Dissolved Oxygen by Electrochemical Switching[END_REF].

Herein, we propose an electrochemical method, which allows the quantitative fluorescence extinction of 2-5 mL samples containing NBD-labelled peptides. Our intention was to allow non-electrochemists to both reproduce the electrochemical setup and achieve quantitative electrochemical quenching routinely. Some specifications had to be considered: the setup was dimensioned in view of follow-up fluorescence measurements; the electrolysis therefore concerned 2-5 mL volumes of solution. The surface area of the electrodes had to be large enough compared to the volume of the solution to run reasonably fast electrolyses (< 1 min). Vitreous carbon foams have been found to meet this requirement since high specific surface materials (> 20 cm²/cm 3 ) can be purchased and carved to the desired shape. Once optimized, we have examined the resulting electrochemical setup in terms of buffer capacity, LUV integrity and finally tested on the quenching of fluorescence emitted by non-internalized NBD-labelled peptides.

Experimental section

Preparation of symmetric LUVs

DPPG (1,2-Dipalmitoyl-sn-glycero-3-phosphorylglycerol sodium salt) was purchased from Avanti Polar Lipids, as a solution in chloroform. Depending on the desired LUV composition, the appropriate amount of phospholipids (PLs) was introduced in a round-bottomed flask and chloroform was slowly evaporated under vacuum at 40 °C using a rotating evaporator and then under high-vacuum during at least 2 h. The lipids were hydrated with phosphate-buffered saline solution (50 mM) at pH=7.4 to a concentration of 1 mM. The turbid suspension of multilamellar vesicles was subsequently extruded 7 times through a 200 nm polycarbonate track-etch membrane and 10 times through a 100 nm polycarbonate track-etch membrane (Whatman) using a 10 mL Thermobarrel extruder (LipexBiomembranes). Since DPPG has a high transition

A C C E P T E D M A N U S C R I P T 4
temperature (41°C), the extruder was heated using a thermocontrolLed circulation bath. The LUV solutions were used from the day and within two weeks after their preparation.

Fluorescence experiments

Fluorescence emission spectra were recorded with a Jasco Fluorescence Spectrophotometer at 20 °C controlled by a JASCO MCB-100 mini circulation bath. A xenon lamp was used as the excitation source and both excitation and emission slit widths were set at 5 nm. To avoid peptide adsorption and subsequent fluorescence quenching, polystyrene cuvettes were used instead of glass. During measurements the solutions were stirred at 800 rpm.

For the internalization experiments, unlabelled LUVs were used with NBD-labelled CPPs introduced at a concentration of 0.1 µM. The excitation wavelength was fixed at 460 nm while the emission was monitored at 555 nm.

Quantification of the internalization into LUVs

For experiences in the gel phase, peptides (0.1 µM) were incubated at 20 ºC for 10 min with the LUVs (DPPG, 10 µM) in 50 mM PBS + 0.1mM HCl. The reductions by adding dithionite or by electrochemical methods were performed at the same temperature as well as the quantification of fluorescence.

For experiences in the fluid phase, peptides were incubated at 41 °C for 10 min with the LUVs in 50 mM PBS + 0.1mM HCl. The chemical (dithionite) or electrochemical reduction was performed at 20 ºC as well as the quantification of fluorescence.

Electrochemical measurements

Voltammetric measurements and both galvanostatic and potentiostatic electrolyses were performed at room temperature using a potentiostat (Autolab type PGSTAT100N, Ecochemie) controlled by GPES 4.9 software.

Cyclic voltammetry

Cyclic voltammetry was performed using glassy carbon as working electrode. Electrodes (1 mm diameter)

were polished to a mirror finish with emery paper of decreasing grain size and alumina powder (Minitech The sequences were synthesized manually on a MBHA Resin Iris BR 1120 (0.54 mmol g -1 , 0.2 mmol) using SPPS Boc-strategy. For the different couplings, activation was accomplished with DCC/HOBt in NMP.

Coupling of NBD-Cl on the N-terminus amine was achieved by addition of NBD-Cl (0.4 mmol) and DIEA (1 mmol) in DMF (2 mL) to the peptidyl-resin (0.1 mmol). The solution was stirred overnight and then rinsed with DMF, CH 2 Cl 2 and MeOH (5 mL) and the peptidyl-resin was dried under vacuum. Then, the peptide was cleaved from the resin by treatment with HF (2 h, 0°C) using anisole (1.5 mL/g peptidyl-resin) and methylsulfide (0.25 mL/g peptidyl-resin) as scavengers. The peptide was precipitated in cold diethylether and the scavengers were removed by filtration. The crude peptide was dissolved in a 10% solution of acetic acid in water and lyophilized before purification.

General purification and peptide characterization

The peptides were analysed and purified by RP-HPLC using the following solvents: A = (0.1 % TFA in H 2 O) and B (0.1% TFA in acetonitrile) on a Waters System with detection at 220, 280 nm and/or 340 nm for peptides having an NBD-Tag. Analytical RP-HPLC was performed using an Ace 5 C8-300 column (4.6 x 250 mm) at a flow rate of 1.0 mL.min -1 . Preparative RP-HPLC was performed using a SymmetryPrep C8 column (7.8 x 300 mm) at a flow rate of 5.0 mL.min -1 using optimized gradients. Purified peptides, (over 95% purity), were characterised by Maldi-Tof MS using -cyano-4-hydroxycinnamic acid (HCCA) as the matrix [START_REF] Swiecicki | How to unveil self-quenched fluorophores and subsequently map the subcellular distribution of exogenous peptides[END_REF].

Typical electrolysis experiments 2.95 mL of PBS 50 mM were introduced in each compartment of the electrochemical cell. In the cathodic compartment, the following solutions were successively added : 1) 20µL of aqueous HCl 1M 2) 30 µL of a solution of DPPG LUVs (DPPG at 1 mM) 3) 3 µL of a solution of NBD-labelled CPP at 0.1 mM was introduced.

The solution was homogenised by pumping up and down the resulting solution 4 times. Then, the electrodes were introduced in each compartment (GC cylinder of 1.2 cm diameter and ca. 3.5 cm height immersed). The electrolysis was performed ( -150 mA for 20 s). After electrolysis, the electrodes were removed from the cell and the solution contained in the cathodic compartment transferred in a fluorescence cuve.

A C C E P T E D M A N U S C R I P T 7 3 Results and discussion

Electrochemical behaviour of NBD-Glycine (NBD-G)

We first used NBD-glycine (NBD-G) as a test-compound to collect information on the electrochemical behaviour of the NBD probe covalently bonded to an amino acid. The cyclic voltammogram recorded in PBS is presented in Figure 1. The residual cathodic current observed at potential values more negative than -1

V/SCE is ascribed to the slow electrochemical reduction of water according to:

H 2 O + e -→ ½ H 2 + OH - (1)
In Figure 1, the sluggish evolution of the cathodic residual current (dashed line) recorded at a glassy carbon electrode (electrode material known for its large proton/water reduction overpotential) is consistent with a slow reduction process. In the presence of NBD-G, a broad, partially reversible reduction wave is observed, overlapping the background current at around -0.65 V. Since glycine alone does not show any faradaic process in the potential window explored, this wave accounts for the electrochemical reduction of the NBD probe. The electrochemical reduction of aliphatic and aromatic nitro compounds has been extensively reviewed [START_REF] Lund | Cathodic Reduction of Nitro and related Compounds, Organic Electrochemistry -an introduction and a guide[END_REF][START_REF] Jaworski | Aromatic Nitrogen-Containing Compounds[END_REF]. The reduction may engage up to 6 electrons to yield the parent amino compound. Actually, in water and more generally in protic solvents the cyclic voltammograms display two successive reduction steps, the first one producing an hydroxylamino intermediate:

Ar-NO 2 + 4e -+ 4 H + → Ar-NHOH + H 2 O (2)
This reduction step is observed at potential values, which strongly depend on several factors, namely the pH of the medium. At neutral pH values like in Figure 1, reaction (2) is observed at -0.7 V vs. SCE [START_REF] Squella | Recent developments in the electrochemistry of some nitro compounds of biological significance[END_REF], i.e.

similar to the reduction potential value of NBD-G in Figure 1. Therefore, the wave at -0.65 V observed in

Optimization of the electrochemical setup used for electrolyses

We have tested glass electrochemical cells to perform the electrolyses of PBS solutions containing both LUVs and cationic peptides. Using such "classical" cells for electrochemists, a noticeable loss of fluorescence (typically 20-50% of relative fluorescence) was observed upon introducing the NBD-tagged CPPs in the cell, in the absence of electrolysis. Actually, the adsorption of cationic peptides on glass has been reported [START_REF] Kristensen | Adsorption of Cationic Peptides to Solid Surfaces of Glass and Plastic[END_REF] and the glass surface area represented by the cell walls and the frit separator may contribute significantly to the adsorption of CPPs. In order to minimize the adsorption of CPPs during the electrolyses, we decided to build an electrochemical cell essentially made of plastic containers. A special focus was made on the accessibility/availability of these cells to non-electrochemists as well as their volume compatibility with the follow-up fluorescence measurements in spectrometric cells of 2-5mL. The cell finally used is described in the experimental section and features 5 mL plastic syringes (anodic and cathodic compartments) connected to each other through the smallest commercially available glass frit.

Such a cell configuration also facilitated the introduction of cylindrical vitreous carbon foams as electrodes, which allowed the electrolysis of the NBD-labelled peptides to proceed in 20s. Additionally, we have carried out successive electrolyses on LUVs solutions, which had been previously incubated at 41°C with RW9-NBD.

The fluorescence appeared unchanged by these electrolyses, showing that electrolyses did not affect the integrity of LUVs (considering that most of the fluorescence is emitted by NBD located in the hydrophobic region of the LUV lipidic bilayer).

A C C E P T E D M A N U S C R I P T 9

We have carried out several electrolyses in single compartment cells, using different electrode materials (platinum, gold, carbon). Unfortunately, those electrolyses led to anode passivation and sometimes to turbid solutions in the presence of LUVs. Moreover, the possible generation of hydroxyl radicals at the anode, which are known intermediates in the electrochemical oxidation of water [START_REF] Chaplin | Critical review of electrochemical advanced oxidation processes for water treatment applications[END_REF], may oxidize lipids in the LUVs [START_REF] Waterfall | The measurement of lipid peroxidation in vivo[END_REF], making single compartment electrolyses too aggressive with respect to the integrity of LUVs.

In this context, we decided to perform the electrolyses in compartmented electrochemical cells from 50 mM PBS solutions.

Electrochemical quenching of the fluorescence emitted by NBD-G

With this respect, the electrochemical reduction of NBD-G was carried out using the homemade 2compartement cells described in the experimental section. Stepwise galvanostatic electrolyses of 0.3C were performed and the relative fluorescence of the solution reported as a function of the total charge passed.

The results are reported in Figure 2.

Figure 2

White circles : relative fluorescence intensity recorded as a function of the charge during the 150 mA galvanostatic reductive electrolysis of a 3 mL solution of NBD-G (0.5 µmol.L -1 ) in aqueous PBS (50 mmol.L -1 ).

Black circle : relative fluorescence after addition of excess dithionite (DT) to the solution obtained after the ultimate electrolysis (3C).

The evolution presented in Figure 2 A final addition of a large excess of dithionite (black circle in Fig. 2), introduced to fully quench the remaining fluorescence, revealed that all NBD-G fluorophores had already been reduced after a 3C electrolysis. Therefore, the quenching of NBD-labelled molecules can be quantitatively achieved through electrolysis, which proceeds fast enough (compared to incubation periods, i.e. 20s at -150 mA with respect to few minutes) to be envisioned in CPP transport studies based on the quenching of non-internalized fluorophores.

Buffering capacity of PBS under electrolysis conditions

The effect of a fast pH jump on the fluorescence quenching of NBD has been recently described and used to evaluate the intravesicular accumulation of NBD-labelled peptides [START_REF] Swiecicki | Accumulation of Cell-Penetrating Peptides in Large Unilamellar Vesicles: A Straightforward Screening Assay for Investigating the Internalization Mechanism[END_REF]. A full fluorescence quenching was observed in a 7-11 pH range upon introduction of hydroxyl anions for a NBD labelled cell penetrating peptide. With this respect, the reductive electrolysis carried out in dilute NBD-G solutions feature the coulometric generation of hydroxyl anions according to (1) as the main electrochemical reaction. Such a quantitative production of HO -during the electrolysis could alter the buffer capacity of PBS and subsequently cause a significant pH increase. In order to discard a quenching of fluorescence only due to the pH jump during our electrolyses, we decided to focus on the buffer capacity of PBS solutions under electroreductive conditions.

A priori, PBS solutions are expected to buffer the pH evolution for a limited release of H + or OH -. In order to evaluate the impact of the electrolysis on the pH value within the cathodic compartment, we have monitored the pH during both chemical (with NaOH) and coulometric titrations of PBS. The chemical (NaOH) titration was performed to establish the actual H 2 PO 4 -/ HPO 4 2-buffer capacity before running the electrolysis, since the PBS tablets used to make the solutions did not explicitly mention these concentrations. The corresponding titration curves are presented in Figure 3. If one assumes that only the electrochemical reaction (1) is taking place in the cathodic compartment, the pH variation observed in Figure 3B(1) fits the calculated "faradaic" titration of the PBS buffer. In our conditions, the quenching of NBD-G appeared complete after 3C (Fig. 2) Therefore, the addition of a small amount (20µL) of a strong acid as in Figure 3B (2) should prevent significant pH evolution during any electrolysis of less than 3C, and would also allow recovering the pH value of PBS (7.2). In unbuffered solutions, the electrochemical release of hydroxyl anions would dramatically increase the pH value in the vicinity of the electrode, then possibly triggering an alkaline NBD quenching as discussed earlier in this paragraph [START_REF] Swiecicki | Accumulation of Cell-Penetrating Peptides in Large Unilamellar Vesicles: A Straightforward Screening Assay for Investigating the Internalization Mechanism[END_REF]. In our conditions, the phosphate buffer and the continuous homogenization caused by the discharge of H 2 bubbles make the occurrence of local pH gradients unlikely. In order to verify that the addition of small amounts of a strong acid had no impact on the electrochemical quenching of NBD-G fluorescence, we have monitored the relative fluorescence of an acidified solution of NBD-G during stepwise electrolyses (Figure 4). As can be observed in Figure 4, the addition of a strong acid preserves the quenching capacity of the electrochemical procedure towards NBD fluorescence. The addition of a small amount of a relatively strong acid (hydrochloric or phosphoric) finally provides adequate pH conditions for the electrolysis without significantly affecting neither the composition of the buffer nor its osmolarity (mostly fixed by sodium chloride). We have systematically applied these optimized experimental conditions in the following experiments featuring NBD-labelled cel penetrating peptides incubated with LUVs.

A C C E P T E D M A N U S C R I P T

Electrochemical quenching of NBD labelled CPPs in the presence of LUVs

We have implemented the electrochemical reduction procedure to quench the fluorescence of NBD-tagged CPPs previously incubated with LUVs. This methodology was aimed at providing a more reliable quenching method to estimate the proportion of peptides internalized in large unilamellar vesicles. As already underlined, NBD fluorescence is routinely quenched with dithionite S 2 O 4 2- [START_REF] Swiecicki | Accumulation of Cell-Penetrating Peptides in Large Unilamellar Vesicles: A Straightforward Screening Assay for Investigating the Internalization Mechanism[END_REF][START_REF] Mcintyre | Fluorescence Assay for Phospholipid Membrane Asymmetry[END_REF], a chemical reducer, which may partially and slowly penetrate in the lumen of LUVs [START_REF] Moreno | Translocation of phospholipids and dithionite permeability in liquid-ordered and liquid-disordered membranes[END_REF] and therefore lead to underestimate The proportion Q of quenched peptides is estimated as the ratio between the fluorescence lost during incubation (F° -F) and the fluorescence at the origin of the incubation period F°, i.e. Q = (F°-F)/F°.

Accordingly, the percentage of extinction reported in Table 1 is defined as (1-Q)x100. The relative uncertainty of this estimation increases for incubations carried out at 41°C which corresponds to the fluid phase of DPPG and therefore to faster dynamics for lipid re-organization and this may explain the variability in fluorescent peptide distribution.

Table 1 shows that the electrochemical procedure at 20°C (T<Tm, liquid ordered phase) gives results similar to the DT method, whatever the peptide considered. This good correlation between electrochemical-and DT-quenching discards the occurrence of LUV destabilization namely through a possible electroporation

A C C E P T E D M A N U S C R I P T 14 generated at the electrode. Even if this cannot be totally ruled out for long timescales, electroporation is unlikely or negligible in our electrolysis conditions since it would have allowed CPPs to undergo trafficking through the pores and therefore total fluorescence extinction would have been observed. At 20°C, when the LUVs are in a liquid ordered phase, the extinction of NBD fluorescence is nearly quantitative, indicating that the peptides have not been internalized. At 41°C, a fraction of R9-NBD was internalized (nearly 12-14%) while secondary amphipathic peptides RW9-and Pen-NBD exhibited a higher proportion of internalization, as expected and described using only DT as quencher [START_REF] Swiecicki | Accumulation of Cell-Penetrating Peptides in Large Unilamellar Vesicles: A Straightforward Screening Assay for Investigating the Internalization Mechanism[END_REF].

These results show that the electrochemical extinction of NBD fluorescence affects a similar proportion of CPPs than the one quenched with DT, whatever the nature of the CPP. These observations fully validate the electrochemical procedure as a method efficient for the quenching of non-internalized NBD-labelled peptides.

Conclusion

We have developed a quantitative electrochemical methodology aimed at quenching the fluorescence of NBD-labelled peptides contained in milliliter-range samples. Once optimized, this procedure does not require expert electrochemical setup/apparatus. It appears reliable for the monitoring of peptide internalization in large unilamellar vesicles and favourably compares with the classical dithionite reductive method, since the quenching proceeds without any additional chemical agent. Accordingly, several drawbacks observed with dithionite (slow internalization in LUVs, degradation with time, reaction with O 2 ) are discarded, making this electrochemical method more versatile to perform large screening assays on various peptide/phospholipid combinations and at different temperatures. Accordingly, a more reliable information on the passage of peptides across lipid membranes can be expected from the implementation of this electrochemical procedure. This methodology also opens new perspectives in the quenching of fluorescence emitted by other fluorophores and other molecules. Indeed, NBD-tagged phospholipids have been found to undergo "leaflet-specific" electrochemical reduction in Giant Unilamellar Vesicles, i.e. only the external leaflet is electroactive, as confirmed with unsymmetrical vesicles [START_REF] Buriez | Selective Electrochemical Bleaching of the outer Leaflet of Fluorescently Labeled Giant Liposomes[END_REF].

A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T

3 phase [ 21 ]

 321 permeate POPC/cholesterol vesicles (POPC = 1-Palmitoyl-2-oleoylphosphatidylcholine) , the permeation relative rate of dithionite vs. a model NBD-labelled phospholipid (NBD-DMPE, DMPE = 1,2-Dimyristoyl-snglycero-3-phosphoethanolamine) being significantly higher in liquid ordered phase than in liquid disordered . This permeation property could therefore severely affect translocation studies based on middle-to long-term fluorescence monitoring after dithionite quenching, since this questions the postulate that internalized fluorescent probes remain unaffected by quenching agents introduced in the external medium.

AFluorescent peptides used in the study 1 ) 4 . 6

 146 C C E P T E D M A N U S C R I P T 5 233 by PRESI), sonicated for 10 min in ultrapure water, and dried before use. The reference electrode was a commercial saturated calomel electrode (SCE -Radiometer Analytical). Electrolysis Galvanostatic electrolyses were carried out at -150 mA. All electrolyses were performed in twocompartment homemade cells separated by a porosity 4 glass frit membrane. The cell compartments were made of two 5 mL syringe barrels capped at the bottom of the tip to avoid leakage. A hole was cut in the extremity of each barrel to introduce a plastic tube with the glass frit membrane of 3 mm diameter. The region around the plastic tube was externally sealed with araldite (Araldite 2011, Huntsman). The glass frit separator was purchased from Ellipse® at the lowest available diameter to minimize the adsorption of CPPs. The working and counter electrodes were made from a vitreous carbon foam (VC003830, Goodfellow). The carbon foam was melded in a shape of a cylinder having almost the same size as the syringe barrel allowing the galvanostatic electrolysis to proceed at current values high enough for fast quenching without imposing too negative potential values. In this way the high electrode surface area allowed performing fast and exhaustive electrolyses. The volume of PBS solutions introduced in each compartment was 3.0 mL. NBD-Pen (sequence NBD-RQIKIWFQNRRMKWKK-NH 2 ) 2) NBD-RW9 (sequence NBD-RRWWRRWRR-NH 2 ), NBD-R9 (sequence NBD-RRRRRRRRR-NH 2 ) Plastic cylindrical bridge sealed with agar-agar , filled with aqueous PBS buffer hosting the Ag/AgCl wire reference electrode A C C E P T E D M A N U S C R I P T General procedure for Boc solid-phase peptide synthesis Standard Boc-amino acids, MBHA resin (0.54 mmol.g -1 ), 1-hydroxybenzotriazole (HOBt), dicyclohexylcarbodiimide (DCC) were purchased from Iris Biotech, GmbH or Novabiochem. 4-Chloro-7nitrobenz-2-oxa-1,3-diazole, (NBD-Cl), was obtained from Sigma-Aldrich.

Figure 1 CV

 1 Figure 1CV of NBD-G at a 1 mm diameter GC electrode in PBS aqueous buffer (50 mmol.L -1 ). Dashed line:background CV before addition of NBD-G. Solid line: NBD-G 4 mmol.L -1 . Scan rate: 0.1 V.s-1

Figure 1 Figure 1 (

 11 Figure 1 is coherent with the reduction of the nitro group. Considering the CV of aqueous PBS presented in Figure 1 (dashed line), any electrolysis at potential values more negative than -0.7 V would simultaneously reduce both water and NBD-G, featuring the overall (1) + (2) reductions.

10 escape

 10 unambiguously demonstrates the quenching effect of the electrochemical reduction on the fluorescence emitted by NBD. The cathode potential stabilized at -1.7 V vs. SCE, a value more negative than the reduction wave of NBD-G in Figure1. Moreover, gas bubbles from the cathode all along the electrolyses. These observations are consistent with the simultaneous reduction of water and NBD from (1) + (2) reactions, as anticipated from the results of cyclic voltammetry.

Figure 3 A:B: 1 : 2 :

 312 Figure 3A: Titration of 10 mL PBS (50 mmol.L -1 ) by NaOH 50 mmol.L -1 -Circles : experimental points. Line: calculated evolution.B: Intentiostatic electrolysis of 3 mL of PBS (50 mmol.L -1 ) at a GC foam cathode (150 mA)-1: experimental (circles) and calculated (solid line) pH evolution for a quantitative faradaic conversion of H 2 O to OH -from reaction (1) based on the H 2 PO 4-/ HPO 4 2-concentrations estimated from Figure3A.2: experimental (triangles) and calculated (dashed line) pH evolution during the electrolysis of 3 mL of a solution composed of PBS (50 mmol.L -1 ) + 20 µL of HCl (1 mol.L -1 ).

12 Figure 4

 124 Figure 4 White circles : relative fluorescence intensity recorded as a function of the charge during the 150 mA galvanostatic reductive electrolysis of a 3 mL solution of NBD-G (0.5 µmol.L -1 ) in aqueous PBS (50 mmol.L -1 ) -same data as Figure 2-. Black squares : relative fluorescence intensity recorded as a function of the charge during the 150 mA galvanostatic reductive electrolysis of a 3 mL acidified solution of NBD-G (0.5 µmol.L -1 ) in aqueous PBS (50 mmol.L -1 )+ 20 µL phosphoric acid at 1M.

  U S C R I P T the proportion of internalized peptides. In this context, the heterogeneous character of the reduction of NBD using electrochemical techniques should overcome this drawback. We have tested the electrochemical quenching method in the quantification of peptide internalization. We have selected three representative NBD-labelled cell penetrating peptides (R9-NBD, RW9-NBD and penetratin-NBD) and compared the results with the classical dithionite (DT) quenching procedure below the phase transition temperature (Tm) and at T = Tm (Tm = 41°C for DPPG). Actually, the drawbacks of DT highlighted in the introduction (slow internalization in vesicles) should not interfere in this fast quenching procedure, allowing an evaluation of the electrochemical extinction method vis à vis DT quenching, used as reference (considering that fluorescence quenching is carried out at 20°C, NBD internalization should be negligible at short timescales). The electrochemical reduction was systematically carried out at a constant current electrolysis of 150 mA during 20 s, therefore engaging 3 coulombs. The percentage of fluorescence extinction obtained for each peptide after an incubation of 5 minutes in the presence of DPPG LUVs is reported in

Table

Table 1

 1 1: Electrochemical and chemical (DT) quenching of the fluorescence of NBD-labelled peptides (0.1 µM) incubated with DPPG LUVs (DPPG 10 µM) (each experiment was performed in triplicate)

		5 min incubation with LUVs at 20°C	5 min incubation with LUVs at 41°C
	Cationic CPP	Electrochemical extinction at 20°C	DT extinction at 20°C	Electrochemical extinction at 20°C	DT extinction at 20°C
	NBD-R9
	(non	97 ± 1 %	99  1 %	88 ± 2 %	84  7 %
	amphipathic)
	NBD-RW9
	(secondary	98 ± 2 %	99  1 %	46 ± 7 %	32  5 %
	amphipathic)
	NBD-Pen
	(secondary	97 ± 2 %	99  1 %	70 ± 3 %	74  5 %
	amphipathic)

Acknowledgements

The authors warmly acknowledge Christian Amatore for his kind interest in this work and for the enriching discussions. This work was supported by the Agence Nationale de la Recherche (grant number: ANR-12-BS08-0002-01 "ELIPTIC").

Graphical abstract

ACCEPTED MANUSCRIPT

A C C E P T E D M A N U S C R I P T 18

Research highlights ► A quantitative electrochemical fluorescence quenching method is proposed for the study of CPP internalization into LUVs.

►The electrochemical quenching proceeds in 20s without affecting neither the buffer capacity nor the integrity of LUVs.

►The method favorably compares to the classical dithionite quenching and has been validated on selected cell penetrating peptides.


N Doudin

S Pomp

M Blatnik

R Resel

M Vorokhta

J Goniakowski

C Noguera

F P Netzer

S Surnev

Epitaxial NiWO4 films on Ni(110): experimental and theoretical study of surface stability

Keywords: Nickel tungstate, polar surfaces, faceting, scanning tunneling microscopy, X-ray photoelectron spectroscopy, X-ray diffraction, density functional theory

Despite the application potential of nickel tungstate (NiWO4) in heterogeneous catalysis, humidity and gas sensing, etc, its surfaces have essentially remained unexplored. In this work, NiWO4 nanoparticles and films with the wolframite structure have been grown via a solidstate reaction of (WO3)3 clusters and a NiO(100) film on a Ni(110) crystal surface and characterized by a variety of experimental techniques, including x-ray photoelectron spectroscopy (XPS), scanning tunneling microscopy (STM) and x-ray diffraction (XRD), combined with ab-initio density functional theory (DFT) calculations. NiWO4 grows initially as three-dimensional (3D) crystalline nanoparticles displaying mainly two crystalline facets vicinal to the (100) surface, which merge with increasing the (WO3)3 coverage into a quasicontinuous epitaxial film. The DFT results provide an account of the energetics of NiWO4 low index surfaces and highlight the role of faceting in the stabilization of extended polar (100) terraces. These combined experimental and theoretical results show that interaction with a metal substrate and vertical confinement may stabilize oxide nano-objects with high energy facets, able to enhance their reactivity.

INTRODUCTION

The first row transition metal tungstates MWO4 (M = Mn, Fe, Co, Ni) belong to a technologically important family of materials, which crystallize with the monoclinic wolframite structure, space group P2/c. Among them, nickel tungstate (NiWO4) has a great application potential in the heterogeneous catalysis, [START_REF] Gil-Llambias | Hydrodesulfurization Catalysts: Electrophoretic Study of Mo (or W)-Co, Mo (or W)-Ni, and Mo (or W)-Ca Sulfided Phases[END_REF][START_REF] Scheffer | Temperature-Programmed Reduction of NiO-WO3/A12O3 Hydrodesulphurization Catalysts[END_REF][START_REF] Diaz-Real | Hydrotreatment of Athabasca Bitumen Derived Gas Oil over Nickel-Molybdenum, Nickel-Tungsten, and Cobalt-Molybdenum Catalysts[END_REF][START_REF] Stern | Propane Oxydehydrogenation over Metal Tungstates[END_REF][START_REF] Bi | NiWO4 Nanoparticles: A Promising Catalyst for Hydrodesulfurization[END_REF][START_REF] Pourmortazavi | Structure Characterization and Catalytic Activity of Nickel Tungstate Nanoparticles[END_REF][START_REF] Mohamed | Unprecedented High Photocatalytic Activity of Nanocrystalline WO3/NiWO4 Hetero-junction towards Dye Degradation: Effect of Template and Synthesis Conditions[END_REF] humidity and gas sensors, [START_REF] Bhattacharya | Environment Sensitive Impedance Spectroscopy and DC Conductivity Measurements on NiWO4[END_REF][START_REF] Dusastre | Symmetrical Alkyl-substituted Oligothiophenes as Ligands: Complexation of the [(η-C5H5)Ru] + Moiety by Hexyl-substituted Ter-, Quater-and Quinque-thiophenes[END_REF][START_REF] Sundaram | Electrical and Humidity Sensing Properties of Lead(II) Tungstate-Tungsten(VI) Oxide and Zinc(II) Tungstate-Tungsten(VI) Oxide Composites[END_REF] microwave devices, [START_REF] Pullar | MgWO4, ZnWO4, NiWO4 and CoWO4 Microwave Dielectric Ceramics[END_REF] and photoanodes. [START_REF] Pandey | Optical, Electrical and Photovoltaic Electrochemical Characterization of Spray Deposited NiWO4 Thin Films[END_REF][START_REF] Zhu | Photoelectrochemical Activity of NiWO4/WO3 Heterojunction Photoanode under Visible Light Irradiation[END_REF] Also, NiWO4 has been demonstrated recently to exhibit electrochromic properties, [START_REF] Kuzmin | AFM and Raman Studies of Nickel Tungstate Electrochromic Thin Films[END_REF][START_REF] Green | Electrochromism in Sputter Deposited Nickel-containing Tungsten Oxide Films[END_REF][START_REF] Green | Structure and Optical Properties of Electrochromic Tungsten-containing Nickel Oxide Films[END_REF] which are superior to those of its binary NiO and WO3 constituents. With the advance of nanotechnologies there is a growing interest in preparing NiWO4 structures at the nanoscale, where surfaces and interfaces are playing a key role in determining novel physical and chemical properties of these materials. Whereas the bulk crystal structure of NiWO4 has been well established by x-ray [START_REF] Keeling | Structure of NiWO4[END_REF] and neutron [START_REF] Weitzel | Kristallstrukturverfeinerung von Wolframiten[END_REF] diffraction, the NiWO4 surfaces are essentially unexplored. This is due to the fact that nickel tungstate compounds have been mostly synthesized in a polycrystalline form, while only few studies report on the growth of well-shaped NiWO4 single crystals. [START_REF] Oishi | Growth of NiWO4 Crystals from a Na2W2O7 Flux[END_REF] Fundamental studies of wolframite surfaces are scarce, in general, and even more so when polar orientations are considered. Growing epitaxial oxide thin films on single crystal metal supports allows one to prepare well-defined surfaces, which can be investigated with atomic precision using modern surface analytic tools. [START_REF] Netzer | Ordered Oxide Nanostructures on Metal Surfaces, Oxide Ultrathin Films[END_REF] Recently, we have fabricated two-dimensional (2-D) Cu 21and Fe [START_REF] Pomp | Two-dimensional Iron Tungstate: a Ternary Oxide Layer with Honeycomb Geometry[END_REF] tungstate nanolayers on Cu(110) and Pt(111) surfaces, respectively, via an ultrahigh vacuum (UHV) surface epitaxial growth route using an on-surface solid-state chemical reaction in two dimensions. This preparation approach differs drastically from the conventional methods for bulk NiWO4 production, where typically chemical reaction of molten powders, mechanical grinding in a vibrating mill, or high-temperature decomposition of complex precursors are used.

Here, instead, the reacting components are brought together in the form of structurally welldefined oxide phases, a NiO(100) film and (WO3)3 clusters, on a single crystal Ni(110) support; their reaction to form an epitaxial stoichiometric NiWO4(100) film has been initiated by oxidative annealing at elevated temperature and has been investigated in situ by a combination of x-ray photoelectron spectroscopy (XPS) with synchrotron radiation and scanning tunneling microscopy (STM) with atomic resolution, and ex situ by x-ray diffraction (XRD). In contrast to any previous work on NiWO4 films, this study is the first one showing the growth of well-ordered epitaxial NiWO4 films, whose surface structure was characterized at the atomic level. The stability of various low index surfaces of NiWO4 has been explored via DFT+U calculations, with a specific focus on the observed polar (100) orientation. We conclude that the observed morphology of the NiWO4 film is likely driven by the interaction with the Ni substrate and by the vertical confinement in the ultrathin oxide film. Two structural phases are highlighted which show that the NiWO4(100) surface is faceted into two vicinal surfaces, (1611) and (811), with low energy (211) ledges. Models of edge and terrace configurations are proposed.

METHODS

The STM experiments were performed in an UHV system (base pressure 1 x 10 -10 mbar) containing a VT-STM/AFM (Omicron), a LEED optics and a cylindrical mirror analyzer (CMA) for Auger electron spectroscopy (AES), as well as the usual facilities for sample manipulation, surface cleaning and thin film evaporation. [START_REF] Steurer | Metamorphosis of Ultrathin Ni-oxide Nanostructures on Ag(100)[END_REF] STM images were recorded at room temperature in constant current mode with electrochemically etched W tips; the bias values cited are referred with respect to the sample.

High-resolution x-ray photoelectron spectroscopy (XPS) experiments were conducted at the Materials Science beamline, at the Elettra synchrotron light source in Trieste, Italy. The UHV chamber was equipped with a spherical electron energy analyzer (mean radius of 150 mm), rear view LEED optics and facilities for sample cleaning and thin film deposition. The photoemission spectra were taken at photon energies of 120 eV, 130 eV, 640 eV and 950 eV for the valence band and the W 4f, O 1s and Ni 2p core levels, respectively, with a total energy resolution of ~ 200 meV. The binding energy scale was calibrated with respect to the Fermi energy of the metal substrate, and all spectra were normalized to the secondary electron background at a few eV lower binding energy than the respective core level peak. A Shirley background was subtracted from the core level spectra prior to the peak decomposition analysis. The latter has been performed with the help of free-ware least-squares minimization software, FitXPS. 24 The core-level spectra have been decomposed into several components, whose individual shape consists of a Donjach-Šunjić profile convoluted with a Gaussian distribution. [START_REF] Doniach | Many-electron Singularity in X-ray Photoemission and X-ray Line Spectra from Metals[END_REF] X-ray diffraction measurements were performed at atmospheric conditions with a Philips X'Pert diffractometer equipped with an ATC3 texture cradle using chromium radiation from a sealed tube. At the secondary side a flat graphite single crystal was used to select monochromatic Kα radiation ( = 2.291 Å), but the monochromator was transmissive also for the wavelengths /2, /3, …. . The experimental data are presented in terms of scattering vector q instead of scattering angles 2θ obtained by the following transformation: q = (4/) sinθ. Specular scans were performed to detect the orientation of the single crystalline substrate, of the NiO and NiWO4 crystallites relative to the substrate surface. The results are evaluated by comparing the experimentally observed peak positions with peak positions calculated by using the single crystal structure of the materials. An fcc structure of nickel with a lattice constant of 3.5228 Å, [START_REF] Swanson | Standard X-ray Diffraction Powder Patterns[END_REF] an NaCl type structure of NiO with a lattice constant of 4.168 Å 27 and a monoclinic structure of NiWO4 with a = 4.5992 Å, b = 5.6606 Å, c = 4.9068 Å and ß = 90.03° were used. [START_REF] Keeling | Structure of NiWO4[END_REF][START_REF] Weitzel | Kristallstrukturverfeinerung von Wolframiten[END_REF] The peak positions were computed by the software POWDERCELL 2.3. [START_REF] Kraus | POWDER CELL -A Program for the Representation and Manipulation of Crystal Structures and Calculation of the Resulting X-ray Powder Patterns[END_REF] The in-plane alignments of the different types of crystallites relative to each other were determined by pole figure measurements. [START_REF] Shultz | A Direct Method of Determining Preferred Orientation of a Flat Reflection Sample Using a Geiger Counter X-Ray Spectrometer[END_REF] The experimentally observed pole directions (net plane normals) were compared with stereograms using the software STEREOPOLE. [START_REF] Salzmann | Software for the Analysis of X-ray Diffraction Pole Figures with IDL[END_REF] The Ni(110) surface was cleaned by cycles of Ar ion sputtering (1.5 keV) and annealing to 1000 K. In order to remove residual carbon contaminations the Ni crystal was exposed to oxygen (pressure of up to 1x10 -7 mbar) at 570 K, followed by a brief flash to 1000 K in UHV.

Surface order and cleanliness were monitored by LEED, STM and AES and XPS. The NiWO4(100) films have been prepared by a three-step procedure. First, the Ni(110) surface has been oxidized in 5x10 -6 mbar O2 at 773 K for 10 min to produce a NiO(100) layer [START_REF] Eierdal | Interaction of Oxygen with Ni(110) Studied by Scanning Tunneling Microscopy[END_REF] with a thickness of ~ 10 Å, as established by XPS (see below). In a second step, (WO3)3 clusters from a molecular beam 21 have been deposited in UHV onto the NiO film at 300 K. Finally, the mixed oxide film has been subjected to oxidative annealing in 5x10 -6 mbar O2 at 973 K for 15 min. The last step was crucial to initiate the solid-state reaction between the binary NiO and (WO3)3 phases, forming the ternary NiWO4 phase. The latter was found to exhibit a moderate structural order in STM, but annealing in UHV to 823 K resulted in better ordered films. The (WO3)3 clusters beam was generated by thermal sublimation of WO3 powder at 1300 K in a thermal or e-beam evaporator, with the evaporation flux monitored by a quartz microbalance. The NiWO4 coverage, cited in the text, is set equal to the (WO3)3 coverage measured in the quartz microbalance, with 1 monolayer equivalent (MLE) (WO3)3 corresponding to the density of the surface atoms of Ni(110), i.e. 1.14x10 15 atoms cm -2 .

DFT calculations were performed with the Vienna Ab-initio Simulation Package (VASP) [START_REF] Kresse | Ab initio Molecular-Dynamics Simulation of the Liquid-Metal-Amorphous-Semiconductor Transition in Germanium[END_REF] using the Projector Augmented Wave (PAW) method [START_REF] Blöchl | Projector Augmented-wave Method[END_REF] to represent the electron-core interaction. Standard Ni and W, and soft oxygen (energy cutoff of 270 eV) pseudopotentials provided by VASP were used. The typical imprecision with respect to standard oxygen pseudopotential (energy cutoff of 400 eV) is of 0.01 Å (bulk NiWO4 lattice parameters) and 0.01 J/m 2 [(1x1)-NiWO4(100) surface energies]. The Perdew-Wang 91 (PW91) [START_REF] Perdew | Molecules, Solids, and Surfaces: Applications of the Generalized Gradient Approximation for Exchange and Correlation[END_REF] gradientcorrected exchange-correlation functional was employed, within the DFT+U approach as proposed by Dudarev, [START_REF] Dudarev | Electron-Energy-Loss Spectra and the Structural Stability of Nickel Oxide: An LSDA+U Study[END_REF] with U -J = 6 eV for both Ni and W, in order to improve the description of the bulk band gap Eg. The calculated bulk lattice parameters a = 4.65 Å, b = 5.72 Å and c = 5.02 Å are in good agreement with the experimental ones. [START_REF] Keeling | Structure of NiWO4[END_REF] Considering the very small distortion of the monoclinic cell with respect to orthorhombicity, in all the following, we have assumed that  = 90°.

NiWO4 has an antiferromagnetic insulating ground state, associated to a doubling of the unit cell along the a axis. The present DFT+U settings allow to well reproduce this ground state and produce a density of states in agreement with hybrid DFT simulation of bulk NiWO4. [START_REF] Kuzmin | First-principles LCAO Study of Phonons in NiWO4[END_REF] The calculated band gap width Eg (Eg = 3 eV) is significantly larger than the GGA value of 0.9 eV and close to the experimental Eg = 3.6 eV. [START_REF] Ejima | Microscopic Optical and Photoelectron Measurements of MWO4 (M=Mn, Fe, and Ni)[END_REF] Reproducing a correct band gap is required to correctly describe the iono-covalent character of the cation-oxygen bonds, i.e. the electron distribution in the system, [START_REF] Noguera | Physics and Chemistry at Oxide Surfaces[END_REF] as well as a correct ordering of polar versus non-polar surface energies. [START_REF] Nilius | Incorrect DFT-GGA Predictions of the Stability of Non-stoichiometric/Polar Dielectric Surfaces: the Case of Cu2O(111)[END_REF] A sketch of the wolframite structure is shown in Figure 1, using VESTA. [START_REF] Momma | VESTA 3 for Three-dimensional Visualization of Crystal, Volumetric and Morphology Data[END_REF]

EXPERIMENTAL RESULTS

A. X-ray photoelectron spectroscopy characterization

The chemical composition and electronic structure changes occuring during the reaction of 5 MLE (WO3)3 clusters with the NiO(100) film, supported on the Ni(110) surface, have been followed by recording XPS spectra in the Ni 2p, O 1s and W 4f core level and valence band regions, the results are presented in Fig. 2. The Ni 2p3/2 spectrum of the clean Ni(110) surface (bottom spectrum in Fig. 2a) contains a main emission line with a binding energy (BE) of 852.6 eV (marked Ni 0 ) and a satellite peak (marked S) shifted by ~ +6 eV, which is due to a two hole c -1 3d 9 4s 2 (c -1 is a core hole) final state effect. [START_REF] Hüfner | Photoelectron spectroscopy[END_REF] The NiO(100) film is spectroscopically well distinguished from the Ni(110) substrate and displays a characteristic Ni 2p3/2 line shape (Fig. 2a, middle spectrum), which can been fitted with four core-level components with BEs of 854.4 eV (marked A), 856.1 eV (B), 861.0 eV (C) and 862.9 eV (D), typical for Ni 2+ species in bulk NiO. [START_REF] Van Veenendaal | Nonlocal Screening Effects in 2p X-ray Photoemission Spectroscopy Core-level Line Shapes of Transition Metal Compounds[END_REF] The lowest BE main line A has been assigned to a c -1 3d 9 L -1 final state, where c -1 and L -1 refer to a hole in the 2p core level and the ligand band, respectively, whereas the component B has been ascribed to a non-local screening process from neighbouring NiO6 units [START_REF] Alders | Nonlocal Screening Effects in 2p X-ray Photoemission Spectroscopy of NiO (100)[END_REF] and/or surface effects. [START_REF] Preda | Interface Effects in the Ni 2p X-ray Photoelectron Spectra of NiO Thin Films Grown on Oxide Substrates[END_REF] The emission structures at the higher binding energy side, C and D, correspond to a c -1 3d 10 L -2 and c -1 3d 8 final states, respectively. [START_REF] Van Veenendaal | Nonlocal Screening Effects in 2p X-ray Photoemission Spectroscopy Core-level Line Shapes of Transition Metal Compounds[END_REF] Some remnant intensity of the underlying Ni substrate can be also seen at the low BE side of the Ni 2p3/2 spectrum of the NiO film, which proofs useful in determining the thickness of the latter. From the attenuation of the substrate Ni 0 signal by the NiO film and using the electron inelastic mean free length of 4.9 Å for NiO at a kinetic energy of 100 eV, [START_REF] Powell | NIST Electron Inelastic-Mean-Free-Path Database[END_REF] we estimate a thickness of 10.5 Å, which correspond to ~ 5 NiO layers. The O 1s spectrum of the NiO film (Fig. 1b, bottom curve) contains a single peak with a maximum at a binding energy of 529.9 eV, in agreement with similar values reported in the literature. [START_REF] Kuhlenbeck | Molecular Adsorption on Oxide Surfaces: Electronic Structure and Orientation of NO on NiO(100)/Ni(100) and on NiO(100) as Determined from Spectroscopies and Ab initio Cluster Calculations[END_REF][START_REF] Caffio | Early Stages of NiO Growth on Ag(001): A Study by LEIS, XPS, and LEED[END_REF] The corresponding valence band spectrum (Fig. 2d, middle curve) is also typical for NiO(100) surfaces of bulk crystals [START_REF] Wulser | Valence-band Density of States During Hydrogen Reduction[END_REF] or thin films, [START_REF] Agnoli | Core and Valence Band Photoemission Study of Highly Strained Ultrathin NiO Films on Pd(100)[END_REF] containing a doublet emission structure at 2.5 eV and 4.1 eV due to a multiplet of hybridized 3d 8 L -1 final states, a broad band between 5 eV and After the thermal treatment of the mixed (WO3)3-NiO layer at 973 K in 5x10 -6 mbar O2 distinct changes are observed in all analyzed XPS spectra (top curves in Fig. 2), reflecting the generation of a new oxide phase, which has been unambiguously identified as NiWO4 in the XRD measurements, discussed below. The W 4f spectrum contains a single W 4f7/2 -W 4f5/2 doublet peak, with the W 4f7/2 peak located at 35.6 eV, in good agreement with binding energy values reported for NiWO4 compounds. [START_REF] Green | Structure and Optical Properties of Electrochromic Tungsten-containing Nickel Oxide Films[END_REF][START_REF] Mancheva | Direct Mechanochemical Synthesis of Nanocrystalline NiWO4[END_REF][START_REF] Green | Structure and Composition of Sputter-deposited Nickel-Tungsten Oxide Films[END_REF] The corresponding O 1s spectrum contains a single component with a BE of 530.3 eV, which is also compatible with NiWO4 literature data. [START_REF] Green | Structure and Optical Properties of Electrochromic Tungsten-containing Nickel Oxide Films[END_REF] The absence of NiOand (WO3)3related core-level components in the O 1s spectrum suggests that the original binary oxide phases have completely reacted together to form the ternary NiWO4 phase. Note, that in the latter both W 4f7/2 and O 1s peaks display markedly narrowed line shapes with respect to corresponding spectra of the (WO3)3 clusters, with FWHM of 0.75 eV and 1.25 eV, respectively, which is fully in line with the crystalline nature of the NiWO4 phase, as reported below. The Ni 2+ fine structure is generally maintained in the Ni 2p3/2 spectrum of the NiWO4 film (Fig. 2a, top spectrum), but the peaks A and B are now shifted by +0.6 eV, the peak C by ~ +1 eV. These shifts are well documented in the literature [START_REF] Green | Structure and Optical Properties of Electrochromic Tungsten-containing Nickel Oxide Films[END_REF][START_REF] Green | Structure and Composition of Sputter-deposited Nickel-Tungsten Oxide Films[END_REF] and reflect the different local chemical environment of the Ni 2+ species in the NiWO4 phase, as compared to NiO. The substrate Ni 0 signal in the Ni 2p spectrum of the NiWO4 covered Ni(110) surface can again assist to estimate the average thickness of the NiWO4 layer. Using the electron inelastic mean free length of 4.6 Å for NiWO4 at kinetic energy of 100 eV, [START_REF] Powell | NIST Electron Inelastic-Mean-Free-Path Database[END_REF] the NiWO4 thickness is determined to be ~ 9.8 Å. Note that the intensity decrease of the W 4f and Ni 2p peaks in the NiWO4 spectra, with respect to the corresponding spectra of the (WO3)3 and NiO layers, respectively, is mostly due to the three-dimensional character of the NiWO4 film growth, as established by STM (see e.g. Fig. 5), but some sublimation of (WO3)3 and/or reduction of the NiO layer at the high reaction temperature (923 K) cannot be excluded. Finally, the valence band spectrum (top curve in Fig. 2d) reflects the changes in the electronic band structure accompanying the formation of the NiWO4 layer. Here, mainly hybridized Ni 3d and O 2p emissions are present. The 3d 8 L -1 multiplet structure at the valence band maximum is shifted in NiWO4 by +0.3 eV and partially overlaps with the O 2p band, which can be ascribed to the modified ligand field in the NiWO4 layer with respect to NiO. The broad satellite structure around 10 eV is strongly attenuated, indicating a smaller weight of the unscreened 3d 7 emission channel in NiWO4. On annealing in UHV to 823 K, which was employed to improve the structural order of the NiWO4 layer, no significant changes in all XPS spectra were observed, besides some decrease of the W 4f and Ni 2+ intensities, accompanied by an increase of the substrate Ni 0 signal, which we attribute to morphological changes in the oxide overlayer.

B. X-ray diffraction analysis

We have performed ex-situ XRD measurements of the 5 MLE NiWO4 film on Ni(110), which has been prepared in UHV and characterized in-situ with STM (see Fig. 5b). The sample has then been taken out and left for one night in air, and afterwards inserted back in the UHV chamber. No differences in the film morphology and structure were found in STM and LEED, proving that the NiWO4 film is robust and stable in air.

A specular x-ray diffraction pattern of the sample is depicted in Fig. 3a. Three different types of crystal structures are observed. The Ni(110) single crystal substrate generates a peak at qz = 2.52 Å -1 , which originates from the (110) plane and corresponds to the 220 reflection from radiation with the wavelength /2. The NiWO4 crystallites contribute with the 100 and 200 diffraction peaks and small traces of the 011 peak; from the NiO phase the 200 peak is observed. These results reveal that the NiWO4(100) plane (and/or (100) plane), as well as the NiO(100) plane, are parallel to the Ni(110) surface. We note parenthetically that the NiO phase has been formed during the exposure of the sample in air as a result of oxidation of the Ni(110) substrate. This is due to the fact that the NiWO4 film, although thick enough to generate x-ray diffraction reflections, is not completely closed (see e.

C. Surface morphology and structure

The large-scale STM images in Fig. 5(a,b) reveal that the NiWO4 film grows in a threedimensional (3D) manner on the Ni(110) substrate. At a (WO3)3 coverage of 2 MLE wellfaceted crystallites form (Fig. 5a), whose height varies between 2 and 5 nm. With increasing (WO3)3 coverage the density of the NiWO4 crystallites increases and eventually at a coverage above 5 MLE they merge forming more planar, but discontinuous NiWO4 film with a RMS roughness of 0.5 nm (Fig. 5b). This film morphology is possibly the reason for the oxidation of the underlying Ni(110) substrate to NiO upon exposing the NiWO4 film to air, as observed in the XRD. The surface of the NiWO4 film is terminated exclusively by the A-type facets, which are separated by straight step edges including similar characteristic angles as in Fig. 5a. The analysis of these angles (see the sketch in Fig. 5d The high-resolution STM images of the A-and B-facets, presented in Fig. 7a and7b, respectively, assist identifying their geometrical structure and orientation. The Afacet displays single stripes oriented along the Ni[110] direction with a periodicity of 15.8 Å ± 0.2 Å across the stripes (see line profile 2-2' in Fig. 7a and7c). The stripes consist of a periodic array of bright protrusions, elongated along the stripe direction, with a separation of 7.6 Å ± 0.2 Å (line profile 1-1' in Fig. 7a and7c). These two periodicities define an oblique unit cell for the Afacet (indicated with a black solid line in Fig. 6a) with an obtuse angle of 100° ± 2°. Between two neighboring stripes a row of elongated maxima with a darker contrast can be also recognized in both the STM image (Fig. 7a) and the line profile 2-2' (Fig. 7c). The Bfacet shows a more complex appearance in the STM images (Fig. 6b): it consists of double zig-zag stripes running also parallel to the Ni[110] direction, with the distance between the maxima along the stripe direction being the same as in the Afacet, i.e. 7.6 Å, but the periodicity across the stripes is twice larger, i.e. 31.5 Å. The corresponding unit cell is drawn with the solid line on the STM image in Fig. 7b, with the two unit cell vectors including the same angle of 100°, as in the facet A. We note in passing that the STM images of the A-and B-facets show no significant sample bias and polarity dependence in the range (-2.0 to +2.0 V), with the best contrast achieved for the A-facet at positive sample bias, and for the B-facet at negative sample bias.

COMPUTATIONAL RESULTS

To the best of our knowledge, no NiWO4 surface has been theoretically studied in the past.

We have thus made a preliminary assessment of the stability of several NiWO4 low index surfaces, prior to focusing on the polar (100) orientation.

A. NiWO4 low energy surfaces

We have considered a series of low Miller index surfaces, restricting ourselves to configurations with (1x1) surface unit cells. In Table 1 we report results on the most stable of them, in the order of increasing surface energy. The corresponding atomic surface are given in Figure S1 (Supplementary material). bound to an oxo-ligand (e.g. the (210) surface) with a very short W-O bond (~1.74 Å). We note that a simple picture with a constant ratio between the Ni-O and W-O bond strengths cannot account for the complex hierarchy of the surface energies in Table 1, which is also influenced by fine details of surface bond relaxation and long range electrostatic effects. As a peculiarity of the wolframite structure, the surfaces of lowest Miller indexes have not necessarily the lowest density of broken bonds, and, as a corollary, not the lowest surface energy (e.g. the (100) and the (001) surfaces), at variance with simpler crystallographic structures.

The calculated relatively low stability of the (100) surfaces (to be discussed below) suggests that the large facets vicinal to the (100) orientation observed experimentally are either due to a kinetic hindrance or, more likely, to a limited thickness of the oxide film and to its substantial interaction with the metallic Ni substrate.

B. NiWO4(100) surface

The NiWO4(100) surface is polar, according to Tasker's classification, [START_REF] Tasker | The Stability of Ionic Crystal Surfaces[END_REF] because the repeat unit perpendicular to the surface consists of successive charged layers 2Ni/4O/2W/4O, and thus bears a non-zero dipole moment. As a consequence, electrostatic stabilization requires compensating charges in the surface layers. Considering the absence of experimental evidence of changes of cation oxidation states, we will focus here on stabilization by nonstoichiometric surface reconstructions, which have proven to efficiently heal polarity. [START_REF] Goniakowski | Polarity of Oxide Surfaces and Nanostructures[END_REF][START_REF] Noguera | Polarity in Oxide Nano-objects[END_REF] For the Ni and W terminations, the required compensating surface charge is equal to half the layer charge, so that, e.g., one half of surface cations have to be removed. Conversely, at the two nonequivalent oxygen terminations, either one or three out of four oxygen atoms per unit cell have to be removed, depending if the underlying layer is composed of W or of Ni. We have analyzed these alternative surface terminations with stoichiometric bulk-cut slabs. They are symmetric when terminated by Ni or W (e.g., W/4O/2Ni/4O/…/2W/4O/2Ni/4O/W) and asymmetric for the oxygen terminations (3O/2W/4O/2Ni/4O/…/2W/4O/2Ni/O). As a consequence, only the average surface energy of the oxygen terminations is obtained.

The most stable surface is Ni-terminated (Es = 1.01 J/m 2 ), with only three broken Ni-O bonds per surface unit cell (Fig. 8, left panel). The surface Ni atoms undergo a noticeable inward relaxation which strongly shortens the three remaining Ni-O bonds (1.85 Å compared to 2.12 Å in the bulk) and leads to an energy lowering of nearly 80% (from 1.82 J/m 2 to 1.01 J/m 2 ).

Much stronger atomic rearrangements take place at the W termination, where an oxygen atom bound to two subsurface Ni moves outwards and binds on-top the outermost W (Fig. 8, right panel). With respect to the unrelaxed termination, two Ni-O bonds per unit cell are broken and one strong W=O bond is formed with a length (1.74 Å) typical of a terminal oxo-ligand. [START_REF] Nugent | Metal-ligand multiple bonds[END_REF] Upon such rearrangement, the surface energy is divided by roughly a factor of 5 (from 5.64 J/m 2 to 1.10 J/m 2 ), which brings it close to, but slightly higher than, that of the Nitermination. At this point it is worth noting that the comparison between the Ni-and W=Oterminated unrelaxed surface energies shows that the ratio between W-O and Ni-O bond strengths is roughly equal to 3, consistently with the previous consideration of the relative stability of the different surface orientations (Table 1). As far as the oxygen terminations are concerned, due to the asymmetry of the two sides of the slab, only the average surface energy can be obtained without relying on grand potential analysis, which would be beyond the scope of the present work. Since their average surface energies are much higher than those of the Ni-or W-terminated surfaces (Table S1, Supplementary Material), the oxygen terminations will not be further discussed. To elucidate the effect of misorientation on the stability of the (100) surface, we have considered a series of vicinal terminations, with (100) terraces separated by steps running along NiWO4 <011> direction. Since such steps require (n11) step ledges, we have chosen the (211) ones due to their low surface energy (Table 1). Construction of such vicinal surfaces, with different proportions of polar (100) terraces and non-polar (211) ledges, rises a general question of polarity compensation condition at facetted surfaces. Indeed, the standard evaluation of compensating charge density from the dipole density of the repeat unit cell is possible, but becomes cumbersome in such complex case. A method in which the polarity of ledges and terraces is separately compensated raises the question of how to treat edge and counter-edge atoms. The safer method, when possible, consists in representing the vicinal surfaces by stoichiometric symmetric slabs. Since, in such case, a stoichiometric bulk repeat unit without dipole moment can always be found [START_REF] Goniakowski | Polarity of Oxide Surfaces and Nanostructures[END_REF] and since both the bulk region and each surface region are stoichiometric, the two surfaces are necessarily charge-neutral and thus polar compensated.

Table 2 summarizes results on the series of surfaces with an increasing proportion of (211) ledges. They all display surface energies smaller than the planar (100) one. The energy reduction is driven by the contribution of the (211) ledges, whose energy is lower than that of (100) terraces. As a consequence, surface energies strongly decrease as the proportion of (211) facets increases, thus pointing towards a thermodynamic preference for a strong faceting of the (100) surface. These results suggest that the limited faceting found in the experiment is likely due to the thinness of the NiWO4 film and to its non-negligible interaction with the Ni substrate.

Surface Es We note that all structural characteristics of the (811) surface (the lengths of its lattice parameters A and B, the angle between them, and its misorientation angle with respect to the (100) surface) match very closely the experimental structure of the A facet. The B facet corresponds most likely to a (1611) vicinal orientation: A = 7.61 Å, B = 30.78 Å, 97.58.7°. This surface has not been explicitly included in the present series of ab initio calculations, but from Table 2 its energy can be estimated in the range 0.96-1.0 J/m 2 .

(J/m 2 ) A (Å) B (Å)     (

  It is built from distorted NiO6 and WO6 octahedra, sharing edges, and forming zig-zag chains along the c axis. Structurally inequivalent oxygen atoms exist in the structure, half of them being bound to 1 W and 2 Ni, and the other half to 2 W and 1 Ni. This results in inequivalent Ni-O and W-O distances. The Ni-O distances are not very different from each other (2.09 Å, 2.08 Å and 2.08 Å, two of each type), while the WO6 octahedra are much more distorted (2.12 Å, 1.95 Å and 1.84 Å).

Figure 1 .

 1 Figure 1. The NiWO4 bulk structure (W-blue, Ni-green, O-red) seen perpendicular to the a (left) and c (right) axes

7

  eV, related to O 2p-derived states, and a satellite peak around 10 eV, which has been attributed to emission from unscreened 3d 7 final states.The deposition of 5 MLE (WO3)3 onto the NiO(100) film on Ni(110) at room temperature results in the formation of a layer of intact (WO3)3 clusters, as confirmed by the W 4f (Fig.2c, bottom curve) and O 1s (Fig.1b, middle curve) spectra. The W 4f spectrum of the (WO3)3 clusters has been fitted with a single W 4f7/2 -W 4f5/2 doublet peak, with BEs of 35.9 eV and 38.1 eV, respectively, which are compatible with the oxidation state 6+ of the W atoms in the intact (WO3)3 clusters.21 The (WO3)3 layer exhibits no long-range order (as confirmed by LEED), therefore the W 4f spectrum displays a broad line shape with a full width at half maximum (FWHM) of the W 4f7/2 peak of 1.2 eV. The same holds for the (WO3)3 related component in the corresponding O 1s spectrum, which is characterized by broad (FWHM = 2.2 eV) line shape with a maximum at 530.8 eV, and is energetically well separated and distinguished from the attenuated O 1s peak of the underlying NiO film at 529.9 eV.

Figure 2 .

 2 Figure 2. Ni 2p (a), O 1s (b), W 4f (c) core level and valence band (d) spectra of the different surfaces investigated: the Ni(110) surface, the NiO(100) film on Ni(110), the (WO3)3 clusters on NiO, and the NiWO4 film on Ni(110). The core level spectra have been decomposed into different components, described in the text.

  g. STM image in Fig 5b). In a next step, the x-ray diffraction pole figures were investigated. From each crystalline phase a set of two individual pole figures was measured. The 111 pole figure of the nickel substrate is shown in Fig. 3b. The experimentally observed pole densities are explained by the peaks 111 and 111. In a subsequent step the in-plane direction [110] of the Ni(110) surface has been determined. A single pole figure of the NiWO4 crystallites is displayed in Fig. 3c. Two different alignments of the NiWO4 crystallites (marked by triangles up and triangles down, respectively) are required to explain the observed pole densities. In both cases either the [011] or [011] direction of NiWO4 phase is parallel to the [110] direction of the Ni(110) substrate, while either the (100) plane is parallel to the substrate surface (triangles down) or the (100) plane (triangles up). In summary, two different epitaxial alignments of the NiWO4 crystallites relative to the Ni(110) surface are observed by the x-ray diffraction: either NiWO4(100) or NiWO4(100) || Ni(110) and either NiWO4[011] or NiWO4[011] || Ni[110].

Figure 3 .

 3 Figure 3. (a) Specular x-ray diffraction scan of the 5 MLE NiWO4 film on Ni(110); (b) and (c) are xray diffraction pole figures of the Ni substrate and NiWO4 film, respectively.

Figure 4 .

 4 Figure 4. Side (a) and top (b) view of the NiWO4 structure along the [100] direction. The NiWO4(100) unit cell and the main crystallographic directions are indicated (W-blue, Ni-green, O-red); (c) lattice model visualizing the epitaxial alignment of the NiWO4(100) and Ni(110) lattices, with the corresponding unit cells indicated. The large grey spheres correspond to the substrate Ni atoms, the small blue spheres to the NiWO4 film Ni (or W) atoms.

  The most often occurring facets are the top one (indicated A) and the side facet B, which are both atomically flat and exhibit characteristic stripe patterns, with the stripes running along the Ni[110] substrate direction, as discussed in more detail below. The top facet is separated from the side facets by straight edges, which include angles compatible with the angles between highsymmetry directions parallel to NiWO4(100) planes, as sketched in Fig. 5c. In particular, the NiWO4[011] direction is parallel to the Ni[110] substrate direction, which is in line with the results of the XRD analysis. The edge parallel to the NiWO4[011] direction is also the one, where the top facet A and the side facet B meet each other.

Figure 5 .

 5 Figure 5. Large-scale STM images (1000 x 1000 Å 2 , U = +1.0 V, I = 10 pA) of 2 MLE (a) and 5 MLE (b) NiWO4 films on Ni(110). The dominant facets are indicated A (top) and B (side); (c) and (d) are schematic drawings, highlighting the edges (black line) between the top (gray shaded) and the side facets in the STM images in (a) and (b), respectively. The black arrows indicate the rotation sense of the edge directions, while the white arrows indicate which NiWO4 <011> direction is parallel to the Ni substrate [110] direction.

  ) revealed that the stripes of the A-facet (which are parallel to the Ni[110] direction) are now aligned along the other diagonal of the NiWO4(100) unit cell, which is in the NiWO4[011] direction, in good agreement with the XRD analysis. At intermediate oxide coverages (between 2 and 5 MLE) the NiWO4 film displays an intriguing growth pattern reminiscent of the "hill-and-valley" reconstruction of vicinal surfaces, which is driven by the lowering of the surface energy. Figure 6a shows an STM image of a 3.3 MLE NiWO4 film surface (Fig. 6b is a 3D view of the image in Fig. 6a), containing a staircase of alternating A-and B-facets, separated by atomically sharp and straight edges running in the Ni[110] direction. The A-facets are distinguished in STM by their single stripe pattern, whereas the B-facets exhibit a double stripe structure (see Fig. 7 for more detail). A line profile taken along the A-B-A-B staircase is plotted in Fig. 6c and allows us to determine the angle between the A-and B-facets, which measures 6° ± 1°.

Figure 6 .

 6 Figure 6. (a) STM image of a 3.3 MLE NiWO4 film on Ni(110) (450 x 700 Å 2 , U = +0.5 V, I = 50 pA). The A and B facets are indicated; (b) 3D view of the STM image in (a) to emphasize the staircase film morphology; (c) line profile across the Aand Bfacets, including an angle of 6°.

Figure 7 .

 7 Figure 7. High-resolution STM images of the A (a) and B (b) facets: (a) 100 x 100 Å 2 , U = +1.0 V, I = 100 pA, (b) 100 x 100 Å 2 , U = -1.0 V, I = 150 pA. The corresponding unit cells are indicated with black lines; (c) and (d) display line profiles taken along the directions indicated in (a) and (b) respectively.

  O broken N W-O broken

  with low Miller indexes: their polar character (NP = non-polar, P = compensated polar), their surface energy (in J/m 2 ), the area of their 2D unit cell (in Å 2 ), the number of broken Ni-O and W-O bonds per unit cell and per surface area (in parenthesis, in Å -2 ).As a general trend, the orientations of lowest energy are non-polar and involve the smallest number of broken bonds per surface area. This is particularly clear in case of W-O bonds, and highlights their larger strength compared to the Ni-O bonds. As a consequence, the most stable surfaces are Ni-terminated. If W atoms are present in the outermost layers, they are

Figure 8 .

 8 Figure 8. Top: side views of the Niterminated (left) and W=Oterminated (right) NiWO4(100) (1x1) surface. W, Ni and O atoms are represented in blue, green and red spheres, respectively. Only five top layers are shown in the upper panels and three in the lower panels. Bottom: their LDOS projected on surface atoms (solid lines) and central atoms (dashed lines).

Figure 8 (

 8 Figure 8 (bottom part) displays the local density of states (LDOS) projected on surface atoms at the Ni (left panel) and W=O (right panel) terminations together with the projection on the corresponding atoms in the slab center. On the Ni termination, the main feature is a strong Ni surface state at the bottom of the conduction band (CB), leading to a gap reduction of 0.6 eV.

Figure 9 .

 9 Figure 9. Top view of the outermost atoms of the Niterminated (a) and W=Oterminated (b) reconstructed NiWO4 (100) surfaces. The simulated STM images of the models in (a) and (b), obtained at bias -1V and 3V (with respect to the VB maximum), respectively are shown in (c) and (d).The unit cell is doubled along the 𝐴 ⃗ axis.

Figure 10 shows

 10 Figure10shows two models of the vicinal (811) surface in which one recognizes a small (211) ledge facet (FigureS1in Supplementary material). Model I (Fig.10a) has a Ni-type step edge and two additional Ni atoms on the (100) terrace in a row-wise configuration per unit cell. The corresponding surface energy is equal to 1.09 J/m 2 . More stabilization can be obtained by removing one NiWO4 formula unit from each step edge (Model II -Fig.10b), Es = 0.96 J/m 2 . In this case a W=O-type edge is obtained, with an oxygen atom in an on-top position and a short W=O bond characteristic of an oxo-ligand. It is worthwhile noting that the row-wise arrangement of Ni atoms on the terraces is slightly more stable than the chessboard one, at variance with the result on the planar (100) surface.

Figure 10 .

 10 Figure 10. Two proposed models (side views perpendicular to the 𝐴 ⃗ vector of the structure) of the NiWO4 (811) vicinal surface. Ni atoms on the terraces are arranged in a row-wise configuration and the step edge is Ni-terminated (a) or W=O terminated (b). The simulated STM images of (a) and (b) obtained at bias 3V (with respect to the VB maximum) are shown in Fig. 10 (c) and (d), respectively.

Table 1 .

 1 Most stable NiWO4 surfaces

	(010)	0.62	NP	46.66	4 (0.086)	0
	(210)	0.77	NP	73.94	6 (0.081)	0
	(211)	0.77	NP	45.54	2 (0.044)	1 (0.022)
	(011)	0.82	NP	70.75	4 (0.056)	2 (0.028)
	(100)Ni	1.01	P	28.68	3 (0.105)	0
	(201)	1.06	NP	78.23	4 (0.051)	2 (0.025)
	(100)W	1.10	P	28.68	2 (0.070)	2 (0.070)
	(101)	1.18	P	60.43	3 (0.050)	2 (0.033)
	(001)	1.26	NP	53.19	2 (0.038)	2 (0.038)

Table 2 .

 2 Structural and energetic characteristics of a series of surfaces vicinal to the (100) one with an increasing proportion of (211) ledges: surface energy Es; lengths (A and B) and angle between their 2D lattice vectorstheir misorientation angle with respect to (100) surface.

ACKNOWLEDGEMENTS

This work has been supported by the FWF Project P26633-N20 and the EU COST Action CM 1104. The technical support of the Elettra staff at the synchrotron in Trieste, Italy, is gratefully acknowledged.

of stabilizing nanoparticles with high-energy facets (thus with lower-coordinated and more reactive surface sites).

ASSOCIATED CONTENT

Supporting Information

Structure of different NiWO4 low energy surfaces (Figure S1); Structural and electronic characteristics of various NiWO4(100) (1x1) surface terminations (Table S1); Simulated STM images of the Ni-and W-terminated NiWO4(100)-(1x1) surfaces (Figure S2); Top view of the bulk-truncated (100) surface with the indication of the (1x1) unit cell and the rotated cell with 𝐴 ⃗ =𝑏 ⃗⃗ + 𝑐 ⃗⃗⃗ and 𝐵 ⃗⃗ = 2 * (𝑐 ⃗ -𝑏 ⃗⃗⃗⃗ ) axis (Figure S3).

AUTHOR INFORMATION Corresponding authors:

svetlozar.surnev@uni-graz.at jacek.goniakowski@insp.jussieu.fr

Notes

The authors declare no competing financial interest.