Hugues Mondésert

Frédéric Bossard

Denis Favier

Anisotropic electrospun honeycomb Polycaprolactone scaffolds: elaboration, morphological and mechanical properties

Keywords: Mechanical Anisotropy, Scaffolds, Electrospinning, Honeycomb patterns

Tissue engineering technology requires porous scaffolds, based on biomaterials, which have to mimic as closely as possible the morphological and anisotropic mechanical properties of the native tissue to substitute. Anisotropic fibrous scaffolds fabricated by template-assisted electrospinning are investigated in this study. Fibers of electrospun Polycaprolactone (PCL) were successfully arranged spatially into honeycomb structures by using well-shaped 3D micro-architected metal collectors. Fibrous scaffolds present 2x4 mm² wide elementary patterns with low and high fiber density areas. Distinct regions of the honeycomb patterns were analyzed through SEM images revealing different fiber diameters with specific fiber orientation depending on the regions of interest. Tensile test experiments were carried out with an optical observation of the local deformation at the pattern scale, allowing the determination and analysis, at small and large deformation, of the axial and transverse local strains. The honeycomb patterned mats showed significantly different mechanical properties along the two orthogonal directions probing an anisotropic ratio of 4.2. Stress relaxation test was performed on scaffolds at 15% of strain. This measurement pointed out the low contribution of the viscosity of about 20% in the mechanical response of the scaffold. An orthotropic linear elastic model was consequently proposed to characterize the anisotropic behavior of the produced patterned membranes. This new versatile method to produce architected porous materials, adjustable to several polymers and structures, will provide appealing benefits for soft regenerative medicine application and the development of custommade scaffolds.

Introduction

Natural tissues in the human body exhibit various morphologies and complex architectural structures depending on the function they fulfill within the living organism [START_REF] Engelmayr | Accordionlike honeycombs for tissue engineering of cardiac anisotropy[END_REF][START_REF] Fung | Stress, Strain, growth, and remodeling of living organisms[END_REF]. The Extra-Cellular Matrix (ECM) is formed of an intricate meshwork of pores, fibers and bridges based on collagen fibrils and elastin bundles [START_REF] Flemming | Effects of synthetic micro-and nano-structured surfaces on cell behavior[END_REF][START_REF] Ní Annaidh | Characterization of the anisotropic mechanical properties of excised human skin[END_REF]. Such natural frameworks are anisotropic and spatially organized to provide a mechanical support and to favor cell adhesion, colonization and proliferation. Biomimetic scaffolds consequently require similar anisotropic architectures to mimic in the best way the structural function of the injured tissue [START_REF] Ma | Biomimetic materials for tissue engineering[END_REF]. Previous studies have shown the relevance of membrane topography for cell activity. Cell alignment, proliferation and functional performance are triggered by structural features of scaffolds [START_REF] Tallawi | Novel PGS/PCL electrospun fiber mats with patterned topographical features for cardiac patch applications[END_REF][START_REF] Criscenti | 3D screening device for the evaluation of cell response to different electrospun microtopographies[END_REF][START_REF] Kuppan | In vitro co-culture of epithelial cells and smooth muscle cells on aligned nanofibrous scaffolds[END_REF][START_REF] Kang | Multilevel structural stereocomplex polylactic acid/collagen membranes by pattern electrospinning for tissue engineering[END_REF], suggesting that anisotropy is a key component for a healthy tissue development.

Electrospinning has been used over the last decades as an efficient elaboration technique to produce tridimensional scaffolds for regenerative medicine. Indeed, this simple and versatile process allows the fabrication of polymer networks based on fibers with micro or nano-scales diameters, forming a mat with a thickness of few hundred of microns to centimeters. Electrospun fibrous mats exhibit attractive structural properties such as high porosity, large surface to volume ratio and good pore interconnectivity that make them suitable candidates for soft tissue engineering scaffolds [START_REF] O'brien | Biomaterials & scaffolds for tissue engineering[END_REF]. Due to the instability of the polymer jet during the whipping phase, conventional electrospinning produces nonwoven fibrous mats with a random fiber deposition and consequently in plane isotropic properties. Main hypotheses on fiber structuration state that final scaffold morphology is driven by a few factors such as electrostatic forces, remaining charges within the fibers (S. Nedjari et al., 2015), speed and chaotic trajectory of fiber paths induced by the whipping instability, or surface area energy effect [START_REF] Yao | Self-assembly of electrospun nanofibers into gradient honeycomb structures[END_REF].

Anisotropy was introduced into electrospun membranes by triggering the spatial fiber organization using specific collectors such as rotating mandrels or non-flat and homogenous templates such as grids or wirenets. Early studies succeed to align fibers toward preferential directions using collectors rotating at high velocity [START_REF] Li | Electrospinning of Polymeric and Ceramic Nanofibers as Uniaxially Aligned Arrays[END_REF][START_REF] Liu | Analysis of the Effects of the Residual Charge and Gap Size on Electrospun Nanofiber Alignment in a Gap Method 8[END_REF][START_REF] Beachley | Effect of electrospinning parameters on the nanofiber diameter and length 17[END_REF][START_REF] Zhou | Fabrication of nanofiber mats with microstructure gradient by cone electrospinning[END_REF][START_REF] Li | Elastic antibacterial membranes comprising particulate laden fibers for wound healing applications[END_REF]. This first simple method, widely used to produce aligned fibers, is also known to reduce their diameter due to the rotational force applied by the drum [START_REF] Alfaro De Prá | Effect of collector design on the morphological properties of polycaprolactone electrospun fibers[END_REF]. Moreover, high rotational speed of the drum disturbs the stability of the jet and often breaks the fiber that could weaken the mechanical properties of the entire scaffold.

Collectors made with two conductive parts separated by an isolative gap can as well be used to form fiber bridges with high alignment [START_REF] Liu | Analysis of the Effects of the Residual Charge and Gap Size on Electrospun Nanofiber Alignment in a Gap Method 8[END_REF][START_REF] Beachley | Effect of electrospinning parameters on the nanofiber diameter and length[END_REF][START_REF] Beachley | Effect of electrospinning parameters on the nanofiber diameter and length 17[END_REF][START_REF] Vaquette | Increasing electrospun scaffold pore size with tailored collectors for improved cell penetration[END_REF][START_REF] Bayati | Adipose-derived stem cells could sense the nanoscale cues as myogenic-differentiating factors[END_REF][START_REF] Dippold | Novel approach towards aligned PCL-Collagen nanofibrous constructs from a benign solvent system[END_REF][START_REF] Shah Hosseini | Quantitative approaches of nanofibers organization for biomedical patterned nanofibrous scaffold by image analysis: Quantitative approaches of nanofibers organization for biomedical patterned nanofibrous scaffold by image analysis[END_REF]. Shah Hosseini et al. [START_REF] Shah Hosseini | Quantitative approaches of nanofibers organization for biomedical patterned nanofibrous scaffold by image analysis: Quantitative approaches of nanofibers organization for biomedical patterned nanofibrous scaffold by image analysis[END_REF] produced aligned PLLA fibers onto 3D static collectors based on a 2-bands gap technique. They obtained with a small tip-to-collector distance and high voltage a controlled fiber orientation.

Even if interesting methods have been studied to sustain the alignment of the fibers [START_REF] Bayati | Adipose-derived stem cells could sense the nanoscale cues as myogenic-differentiating factors[END_REF], insulating gap produces unidirectional fibers in a small quantity and over a small distance, making the use as scaffolds difficult. The two previous methods provide a way to align fibers only in one selected direction, giving a restricted flexibility in the design of more complex degree of anisotropy representative of natural tissues.

Other recent researches have shown interesting improvements in patterning fibers using 3D templates. Conductive or non-conductive supports allow the local modification of the electric field at the vicinity of the collector. Fibers follow a specific path driven by the electrostatic forces induced by the electric field and deposit onto the more attractive parts of the collector. Zhang et al. (Daming and Jiang, 2008;[START_REF] Zhang | Patterning of Electrospun Fibers Using Electroconductive Templates[END_REF] are the first to introduce patterns in their tubular electrospun structures. In their work, collected fibers formed square patterns with an order degree depending on feeding rate, voltage and solvent composition. Metal wire nets or grids were investigated as conductive collectors in a few publications [START_REF] Neves | Patterning of polymer nanofiber meshes by electrospinning for biomedical applications[END_REF][START_REF] Munir | Patterned indium tin oxide nanofiber films and their electrical and optical performance[END_REF][START_REF] Vaquette | Increasing electrospun scaffold pore size with tailored collectors for improved cell penetration[END_REF][START_REF] Titov | Facile patterning of electrospun polymer fibers enabled by electrostatic lensing interactions[END_REF][START_REF] Kang | Multilevel structural stereocomplex polylactic acid/collagen membranes by pattern electrospinning for tissue engineering[END_REF]. In these cases, fibers are deposited preferentially on the metal mesh areas and adopt the same macroscopic shape of the collectors. Fibrous mats with repetitive structure can be produced using protrusion collectors [START_REF] Zhang | Bionic electrospun ultrafine fibrous poly(L-lactic acid) scaffolds with a multi-scale structure[END_REF][START_REF] Wang | Electrospinning of Polymer Nanofibers with Ordered Patterns and Architectures[END_REF]Qiao et al., 2018). Yujie Qiao et al. (Qiao et al., 2018) fabricated polycaprolactonecollagen-silk fibroin electrospun scaffolds by collecting on probe array collectors.

Membranes produced onto such 3D templates are thin, hard to remove from their supports and show often a low level of structuration decreasing with collection time.

Scaffolds with honeycomb-like structures have promising properties for cell tissue engineering. They provide an anisotropic structure with a large surface area and zones with low and high porosity which can promote cell development [START_REF] George | Differentiation of mesenchymal stem cells into osteoblasts on honeycomb collagen scaffolds[END_REF][START_REF] Yao | Self-assembly of electrospun nanofibers into gradient honeycomb structures[END_REF]. [START_REF] Yao | Self-assembly of electrospun nanofibers into gradient honeycomb structures[END_REF] described in their work the elaboration of self-assembled honeycomb structures on flat collectors. Gradient honeycomb patterned meshes with a 100 µm thickness were induced by the opposite actions of surface tension and electrostatic repulsion. Similar structures can be achieved by the fluctuation of fiber diameter in a beadslike morphology or by combining electrospinning and electrospray techniques [START_REF] Ahirwal | From self-assembly of electrospun nanofibers to 3D cm thick hierarchical foams[END_REF][START_REF] Garcia | Poly(ε-caprolactone)/Hydroxyapatite 3D Honeycomb Scaffolds for a Cellular Microenvironment Adapted to Maxillofacial Bone Reconstruction[END_REF][START_REF] Nedjari | Electrostatic template-assisted deposition of microparticles on electrospun nanofibers: towards microstructured functional biochips for screening applications[END_REF]. Thicker fibers, easily discharged in contact with the collector, would define attractive areas for new incoming fibers whereas smaller overhanging fibers would repulse them inducing a self-assembly organization. In these cases, patterns adopted a hexagonal like configuration that resembles more to a Voronoi tessellation than to a regular honeycomb pattern. More symmetrical honeycomb shaped scaffolds can be produced by template assisted electrospinning. [START_REF] Rogers | A novel technique for the production of electrospun scaffolds with tailored three-dimensional micro-patterns employing additive manufacturing[END_REF] used patterned resin formers covered by thin gold layer to reproduce reentrant honeycomb structures.

Mechanical properties of isotropic random electrospun fibers are commonly assessed with axial tensile tests [START_REF] Lee | Characterization of nano-structured poly(1caprolactone) nonwoven mats via electrospinning 8[END_REF][START_REF] Katsube | Mechanical Characterization of Electrospun Polycaprolactone (PCL): A Potential Scaffold forTissue Engineering[END_REF][START_REF] Croisier | Mechanical testing of electrospun PCL fibers[END_REF][START_REF] Domaschke | Predicting the macroscopic response of electrospun membranes based on microstructure and single fibre properties[END_REF]. Silberstein et al interestingly demonstrated that mechanical properties of random electrospun mats were closely linked to the fiber/fiber interactions.

Bending of transverse fibers would influence densification and alignment of fibers and consequently determine mechanical behaviors of the mats such as stiffness and transverse contraction [START_REF] Silberstein | Elastic-plastic behavior of non-woven fibrous mats[END_REF]. Similarly, Simonet et al have shown that low temperature electrospun matrices exhibit higher porosity and void space between fibers that lower globally the mechanical properties of the scaffolds [START_REF] Simonet | Tailoring the void space and mechanical properties in electrospun scaffolds towards physiological ranges[END_REF].

Only a few of publications discuss about mechanical properties of patterned fibrous membranes made by electrospinning [START_REF] Kang | Multilevel structural stereocomplex polylactic acid/collagen membranes by pattern electrospinning for tissue engineering[END_REF][START_REF] Neves | Patterning of polymer nanofiber meshes by electrospinning for biomedical applications[END_REF][START_REF] Vaquette | Increasing electrospun scaffold pore size with tailored collectors for improved cell penetration[END_REF][START_REF] Wang | Electrospun nanofiber meshes with tailored architectures and patterns as potential tissue-engineering scaffolds[END_REF][START_REF] Yan | Plasma treatment of random and aligned electrospun PCL nanofibers[END_REF]. Basically, the introduction of a fiber organization affects the global mechanical behavior of the electrospun scaffolds. In the case of fiber bundles, mechanical anisotropy can be evaluated by comparing tensile stress in directions along and perpendicular to fiber orientation [START_REF] Chou | Relationships between mechanical properties and drug release from electrospun fibers of PCL and PLGA blends[END_REF][START_REF] Jamadi | Synthesis of polyester urethane urea and fabrication of elastomeric nanofibrous scaffolds for myocardial regeneration[END_REF][START_REF] Wu | Preparation of aligned poly(glycerol sebacate) fibrous membranes for anisotropic tissue engineering[END_REF]. [START_REF] Shamsah | Mimicking the Annulus Fibrosus Using Electrospun Polyester Blended Scaffolds[END_REF] investigated the mechanical response of aligned PCL fibers undergoing a loading in the direction of 0, 30 or 60° to fiber directions. They showed that tensile properties are directly connected to the loading direction.

The greater the angle is between the load axis and the direction of fiber alignment, the weaker is the cohesion between fibers resulting in reduced mechanical properties. Aligned electrospun scaffolds are rarely characterized mechanically along the transverse direction (90°), without taking into account the important anisotropy of the scaffolds.

Electrospinning is used with a large range of polymers. Among them, Polycaprolactone PCL is a well-known synthetic polymer widely used for biomedical applications. Its bio compatibly and bioresorbability properties, low cost and easy electrospinnability make PCL electrospun fibers attractive materials for tissue engineering [START_REF] Janmohammadi | Electrospun polycaprolactone scaffolds for tissue engineering: a review[END_REF].

In this study, a technique with new electro-conductive collectors to create anisotropic scaffolds is described and used to fabricate PCL fibrous mats with a repetitive honeycomb pattern. The aim of this work is to introduce a new anisotropic custom made scaffolds, evaluate their mechanical properties and link them to their structural aspects. Firstly, heterogeneous fibrous membranes are characterized depending on the different areas of the pattern. Then mechanical properties of these new biomaterials are investigated to highlight their anisotropic behaviors, with respect to the local deformation of the pattern. Finally, an orthotropic linear elastic model is introduced to model the mechanical behavior at small strains of the structured scaffolds.

Material and methods

Materials

Poly(ε-caprolactone) (PCL) with a mean molecular weight of 80 000 g/mol, Dichloromethane (DCM ≥ 99.9 %) and Methanol (MeOH > 99.5 %) were purchased from Sigma Aldrich. All polymer solutions for electrospinning were obtained by dissolving PCL pellets in a two solvents blend (DCM and MeOH) with a volume ratio of 4/1. Solutions were stirred with a roller mixer device for 24 hours minimum at room temperature in order to obtain a clear and homogeneous solution. Polymer solutions within a range of 8 to 16 wt% were investigated in reference to previous works of Lancuski et al. [START_REF] Lancuški | Carbohydrate-Decorated PCL Fibers for Specific Protein Adhesion[END_REF][START_REF] Lancuški | Electrospun Azido-PCL Nanofibers for Enhanced Surface Functionalization by Click Chemistry[END_REF]. A solution at a concentration of 16 wt% providing uniform and bead-free fibers was experimentally selected.

Electrospun membranes elaboration

A vertical electrospinning apparatus was used to produce PCL polymer fibers. Set up consists of a syringe pump (model: KDS Legato 200, KD Scientific, Holliston, MA, USA) containing the polymer solution and a voltage source (±30 kV , iseq GmbH, Radeberg, Germany) as shown in figure 1.a. A 5 mL syringe with a 21 Ga metal needle tip was loaded with the polymer solution and a constant flow rate of 5 mL/min was applied. Distance between the tip of the needle and the collector was adjusted at 20 cm and the applied voltage varied from 12 to 20 kV. Fibers were collected for 10 to 15min on their templates at a temperature of 21°C, with a humidity controlled between 40 to 60 %.

Morphological characterization set up

Optical microscopes (Olympus, Keyence) were used with transmission mode to observe honeycomb micro-structures and assess local arrangement of the fibers. A fieldemission-scanning electron microscope (FESEM ZEISS ULTRA55) was used to characterize the structure at the fiber scale. Fibrous samples were previously coated with a thin carbon layer by sputtering for SEM imaging. Measurements over 100 different fibers were made to estimate the average fiber diameter (AFD). Fiber orientation analysis was performed with ImageJ software using the plugin OrientationJ to extract the local 2D orientations of the fibers based on structure tensors [START_REF] Püspöki | Transforms and Operators for Directional Bioimage Analysis: A Survey[END_REF]. Profilometer scan of the patterned scaffolds was performed with a numerical microscope, Keyence, VHX-7000, (magnification x50, numerical aperture 0.9, motorized xy plate (1 µm presision). The produced membrane exhibit an irregular profile that makes the determination of an accurate thickness delicate. However, this parameter is required to determine the nominal stress = 〈 〉 of the membrane tensile test, with F the measured axial force, l0 the initial width and 〈 〉 the initial apparent thickness of the sample. It is worth noting that the true stress cannot be used as the thickness of the membrane 〈 〉 , thus the exact cross section, cannot be evaluated at every step of the tensile deformation.

Mechanical characterization set up

An alternative method is proposed in this study by measuring sample mass m. As shown in the following equation, the reverse porosity 1can be expressed as:

1 -= / 〈 〉 (1)
where denotes the bulk density of PCL (1.145 g/cm 3 ) , the porosity and , , 〈 〉 the initial dimensions of the fibrous sample. Using the first equation the nominal stress can thus be calculated as:

= " # 1 - (2)
Porosity of all samples was considered constant since electrospinning parameters are identical. It was experimentally measured with a first honeycomb sample by measuring its thickness 〈 〉 and mass m (equation 1). Initial thickness 〈 〉 was measured by a mechanical compression test with a Haake MARS III, Thermo Scientific rheometer (maximum axial force of 0.1 N with a parallel plate geometry (diameter of 20 mm). The obtained value of 94 % was calculated and considered as reference for later analysis.

In this way, we introduce a way to compare patterned samples with a mass measurement which was found experimentally more accurate than measuring apparent thickness 〈 〉 0 of each sample. This approach is also less intrusive than measuring average thickness since rectangular membranes are non-damaged by a mechanical micrometer prior to tensile tests.

A camera (Basler acA2000-50 gm) was installed on the tensile stress measurement these non-flat collectors, the electric field is slightly disturbed, especially at the vicinity of the collector [START_REF] Titov | Facile patterning of electrospun polymer fibers enabled by electrostatic lensing interactions[END_REF][START_REF] Zhang | Bionic electrospun ultrafine fibrous poly(L-lactic acid) scaffolds with a multi-scale structure[END_REF]. Pyramidal protrusions of the collectors enhance the electrical point effect. Once fibers are close to the surface of the collector, they are more attracted to protrusions and preferentially accumulated on their tops. Continuous fiber jet will follow a preferential path to connect to close neighbor protrusions. This path depends on the fiber flexibility and the intensity of the local electric field. During the deposition, the probability for a fiber in contact with a protrusion to connect to its neighbor is higher along the fiber direction. The fact that bridges ③ are wider than ② reinforces the deposition mechanism based on the electrostatic attraction. Protrusions represent a more attractive pole, thus the fiber will be steered with higher probability toward its closest neighbor protrusions. Only a few fibers are crossing the gap in the center of the honeycomb mesh which represents a longer distance for the fiber to overcome. A second hypothesis considers the remaining charges accumulated by fibers and the electrostatic repulsion they create. As explained by Wittmer et al. [START_REF] Wittmer | Well-organized 3D nanofibrous composite constructs using cooperative effects between electrospinning and electrospraying[END_REF], fibers in the center of the mesh have more difficulty to release the positive charges compared to the fibers situated on the protrusions. Additional electrostatic repulsion forces will appear and guide new approaching fibers, resulting in a structuration. As shown in figures 3.4 and 3.5, fibers are disposed along 0 ° and ±90 ° ④ or 0 ° and ±65 ° ⑤ directions. Alignment of fibers can be correlated to a similar process observed with a gap collection in electrospinning [START_REF] Li | Collecting Electrospun Nanofibers with Patterned Electrodes[END_REF]Qiao et al., 2018b;[START_REF] Shah Hosseini | Quantitative approaches of nanofibers organization for biomedical patterned nanofibrous scaffold by image analysis: Quantitative approaches of nanofibers organization for biomedical patterned nanofibrous scaffold by image analysis[END_REF].

Fibers are aligned along one direction to form a bridge between two conductive protrusions.

Average Fiber Diameters (AFD) were analyzed through SEM images (column 3 of figure 3). AFD depends on the location of fibers, decreasing from 3.1 µm to 2.2 µm in the zones ① and ⑤ respectively. This fiber diameter gradient is clearly related to the length of insulating gaps (here considering as the voids between protrusions). Vaquette et al. [START_REF] Vaquette | Increasing electrospun scaffold pore size with tailored collectors for improved cell penetration[END_REF]) described than AFD is affected by the void gap length in their wire collectors. The first approach is that fibers elongate more as they have to make a longer path, resulting in a higher stretching and then in a lower fiber diameter. Another theory would rely on the non-regular morphology of the fiber during the deposition. Schlater et al. [START_REF] Nedjari | Thick electrospun honeycomb scaffolds with controlled pore size[END_REF] explained that structuration in electrospun scaffolds exhibiting beads and fibers is driven by the bimodal character of the fiber. Following this approach, thicker segments of the fiber, with higher accumulated charge density, will be selectively attracted to very close protrusions and compose the main bridges observed in our honeycomb meshes. Low diameter fiber segments with lower charge density will deposit more randomly, filling the gaps of the honeycomb core.

Scaffold topology

One of the main challenges mentioned in the literature in patterning electrospun scaffolds is to keep a well-defined fiber organization in depth. Indeed, fibers are collected onto the support and start to create an insulating or repulsive layer growing with time.

Patterned structures usually vanish after a few minutes of collection, corresponding to few 100 µm layer of deposited fibers [START_REF] Liu | Fabrication and Formation Mechanism of Electrospun Spatially Defined Fibrous Patterning Structures on Conductive and Insulating Substrates[END_REF][START_REF] Zhao | Nanofibrous patterns by direct electrospinning of nanofibers onto topographically structured non-conductive substrates[END_REF]. As a consequence, fibrous samples with well-defined architectures are relatively thin with a thickness that usually does not exceed 100 to 200 µm. Manipulation and subsequent tensile measurements of such membranes are not easy to perform, which might explain the poor mechanical characterization of patterned scaffolds in the literature. Curves show a classic "stress-strain" like shape, beginning by a quasi linear part at low strain (until approximately 15 % for D1 and 30 % for D2) and followed by a plateau. This two steps behavior is specific of fibrous polymer materials [START_REF] Ridruejo | Micromechanisms of deformation and fracture of polypropylene nonwoven fabrics[END_REF][START_REF] Sinha-Ray | Meltblown fiber mats and their tensile strength[END_REF]. Failure was observed in the case of D1 direction around 55 % of global true strain. With D2 patterns, a maximum strain of 130 % was reached before complete rupture of fibers (maximum apparatus gap of the test bench). Rupture of the membranes occurred always at the vicinity of the grips.

Slope in the first linear part, indicated here as modulus E, is significantly higher for the D1 orientation, ( ) = 1.7 ± 0.1 MPa versus ( = 0.4 ± 0.1 MPa for D2. Such values of modulus are slightly lower that the ones found in the literature of PCL patterned scaffolds collected on metals grids (2 to 4 MPa for Vaquette et al [START_REF] Vaquette | Increasing electrospun scaffold pore size with tailored collectors for improved cell penetration[END_REF] or 5 to 10 MPa for [START_REF] Wang | Electrospun nanofiber meshes with tailored architectures and patterns as potential tissue-engineering scaffolds[END_REF]). This difference with literature in scaffold stiffness might be explained by several factors such as a higher average fiber diameter, a higher global porosity or a more important heterogeneity in the fibrous network with more distinct areas of high and low fiber density. Yield Strains delimit the first linear regime and the second plateau regime, i.e. where the first part of the curve becomes markedly nonlinear. They were evaluated on the basis of 2 % strain difference between the straight line and the actual curve. Tensile tests performed in the D2 direction display a high yield strain of 38 % whereas in D1 direction, yield point is reached at a lower value of 19 % (figure 6).

Anisotropic ratio, ( ) /( of moduli of the first linear part, has a value of 4.2 proving the strong anisotropic behavior of produced mats. Such difference in axial stresses between D1 and D2 directions is due to the structural fibers orientation of the honeycomb configuration. Similar results on mechanical properties were obtained with fibers collecting on a metallic screw, producing a significant anisotropy ratio of 3.4 between axial and transverse directions [START_REF] Neves | Patterning of polymer nanofiber meshes by electrospinning for biomedical applications[END_REF]. Such value of anisotropic ratio is in good accordance with most natural soft tissues (human skin, tendons, ligaments, muscular tissues)

found in the human body (anisotropy between 3 to 5) [START_REF] Manschot | The measurment and modelling of the mechanical properties of human skin in vivo-II. The model[END_REF],

showing that our strategy to reproduce natural anisotropy is appropriated and can be used for soft tissue engineering applications. The difference between local and global strains is low until a critical value of 15 % for D1 direction and 30 % for D2 direction. In these domains, local analysis is matching the global strain behavior. However, after critical values, corresponding to higher strains, the local strain gives a more accurate understanding of the scaffold mechanical behavior. direction. As D1 tensile test samples are more contracted along the transverse direction, higher stresses will be applied at the vicinity of the grips. This phenomenon would explain the failure observed at a shorter elongation in D1 direction (figure 6), due to a higher restriction nearby the grips. Moreover, the higher transverse contraction of the scaffolds in direction D2 will induce a stronger fiber densification, which would explain a higher stress of D2 scaffolds

Tensile behavior -Monotonic small deformation loading

Mechanical behaviors present two regimes in terms of stress and local strain answers at low and high strains. Curves in figures 7 and 8 expose a quasi linear regime in D1 and D2

directions until 15 % of axial strains. Considering that scaffolds for biomedical applications (skin [START_REF] Maiti | In vivo measurement of skin surface strain and sub-surface layer deformation induced by natural tissue stretching[END_REF], ligament [START_REF] Ruspi | Digital Image Correlation (DIC) Assessment of the Non-Linear Response of the Anterior Longitudinal Ligament of the Spine during Flexion and Extension[END_REF] or bone [START_REF] Ahmed | Experimental Validation of an ITAP Numerical Model and the Effect of Implant Stem Stiffness on Bone Strain Energy[END_REF] implants)

would endure relatively small local strains, this last section will detail the mechanical behavior of honeycomb scaffolds in the first 15 % of true strain corresponding to the first linear regime. However, it has to be noticed that scaffolds can stand a true strain of about 40% without breaking.

In a first part, viscosity of the membranes is investigated through stress relaxation tests to determine the elasticity properties of these first linear regimes. Then, an orthotropic elastic model is introduced to describe the mechanical behavior at low strain of the honeycomb mats.

Relaxation

Relaxation of the honeycomb scaffolds was firstly investigated in both directions D1 and D2. Figure 9.a shows the measurement of nominal stress with a two steps strain control experiment. Scaffolds were extended at a fixed strain rate of 3.3 10 -3 s -1 in order to reach a maximum strain of 15 %. Then strain was kept constant to examine the relaxation of the The relaxation parameters are exposed in table 1. The time-dependent response of these electrospun materials are well described by this 5 parameters generalized Maxwell model, employed to model electrospun scaffolds [START_REF] Sethuraman | Influence of scaffold forming techniques on stress relaxation behavior of polycaprolactone scaffolds[END_REF][START_REF] Waheed | The viscoelastic response of electrospun poly(vinyl alcohol) mats[END_REF] or natural tissue relaxation [START_REF] Machiraju | Viscoelastic studies of human subscapularis tendon: Relaxation test and a Wiechert model[END_REF]. [START_REF] Baker | The mechanical properties of dry, electrospun fibrinogen fibers[END_REF][START_REF] Waheed | The viscoelastic response of electrospun poly(vinyl alcohol) mats[END_REF]. Relaxation parameters are quasi identical for the two directions of elongation D1 and D2 which means that the viscoelastic relaxation dynamics are independent of the tensile direction. Infinite stress or equilibrium stress, is estimated around 80 %, proving that materials still have a consistent structural answer under an imposed constant strain. Therefore, viscous properties of the produced honeycomb scaffolds were neglected for a further detailed description of the mechanical behaviors.

Orthotropic elastic model of one pattern

A single honeycomb pattern is modeled here mechanically as it can be assimilated as a representative Elementary volume of a bigger structural scaffolds. Developing constitutive equations for one single pattern will provide the initial elements to develop a finite element model of a more complex scaffold made of several patterns.

Considering the low viscosity properties analyzed in the paragraph above, a linear elastic model is proposed in this section to describe the anisotropic mechanical behavior of the honeycomb scaffolds considered as membranes. This model is valid only at small deformation where the linear regimes were observed experimentally.

A strict linear elasticity model is employed in which the following Hooke's law might be used:

F )) ) G = H F I )) I 2I ) G JK:ℎ H = F H )) H ) H ) H ) H H H ) H H G (4)
The 3x3 elasticity symmetric tensor H relates the stress and the strain I via the equation ( 4), where and I have 3 values referring to 11, 22 normal components and 12 shear components. In the case of orthotropic materials, the normal and shear components are decoupled meaning that normal stresses only cause normal strains and shear stresses only cause shear strains. Elasticity tensor can be simplified with only 5 free parameters:

∁ NO>PN = F H )) H ) 0 H ) H 0 0 0 H G (5)
It is simpler to define the inverse elasticity tensor ∁ QR:ℎQ -1 , which can be expressed as following:

F I )) I 2I ) G = S T T T U 1 ( ) ' - V ) ( ) ' 0 - V ) ( ' 1 ( ' 0 0 0 1 μ ) ' X Y Y Y Z F )) ) G (6)
with E Young moduli, V Poisson's coefficients and µ shear modulus Experimental shear test was not feasible considering the small thickness of the samples.

Consequently, last shear modulus value μ12 was estimated via the Huber's formula (7) [START_REF] Huber | The theory of crosswise reinforced ferroconcrete slabs and its application to various important constructional problems involving rectangular slabs[END_REF].

( ) ( V ) = - I I )) V ) = - I )) I μ ) =
[\ @ \ B )] √ _ @B _ B@ (7) 1.7 ± 0.1 MPa 0.4 ± 0.1 MPa 3.5 ± 0.4 0.6 ± 0.1 0.16 ± 0.05 MPa

Table 2 -Mechanical parameters of electrospun PCL honeycomb scaffolds

For an orthotropic linear model, the inverse elasticity tensor ∁ NO>PN =) is defined symmetric.

Thus, a relation ( 7) between Young's moduli and Poisson's coefficients can be deduced from the expression of ∁ NO>PN =) in equation ( 6).

The following equation (8) needs to be verified to insure the symmetry of the tensor ∁ QR:ℎQ -1 :

_ @B \ @ = _ B@ \ B (8)
These ratios were experimentally calculated from the parameters of table (2):

_ @B \ @ = 2.1 ± 0.4 and V 21 ( 2 = 1.6 ± 0.7 (9)

The disparity between the 2 ratios is relatively small and equation ( 8) is verified taking into account the relative errors made experimentally, induced either by the replicability of the tests or the local strain measurements. These experimental calculations reinforces the use of an orthotropic linear elastic model to represent the honeycomb mechanical behavior.

This first model of the honeycomb mats is a strong first approach in the description and future development of applied regeneration scaffolds with more complex loading constraints.

Conclusion

A new way to shape by electrospinning scaffolds with anisotropic properties was investigated in this study. Honeycomb patterned fibrous mats were successfully produced by electrospinning using new template-assisted collectors. The metal 3D collectors allowed the fabrication of well-shaped anisotropic hexagonal honeycombs. Fibers observe different average diameters, densities and orientations depending on the regions in the elementary honeycomb pattern. Mechanical tensile tests confirmed the anisotropic nature of the produced scaffolds at small and large strains. Anisotropic ratio of elastic moduli of 4.2 was found between transverse and axial directions, matching to the range of soft tissue structural properties. At small strains (up to 10%) where the viscosity aspect can be neglected, an orthotropic linear elastic model was introduced to depict the anisotropic mechanical behavior of the micro-structured scaffolds. For future biomedical applications, the produced honeycomb meshwork will give the mechanical stability needed while the center of the mesh, niche with high porosity and good pore interconnection, will create a friendly environment to host cells. The illustrated electrospinning process will provide promising tools to regenerative medicine to create artificial scaffolds to replace anisotropic targeted tissues.

Fibers

  were projected to form wide fibrous membranes onto a patterned collector made of a brass metal cross of 70 mm wide (figure 1.b). The so formed micro-structured collectors are composed of small metal peak arrays placed on the top as shown in figure 1.c. Protrusions, obtained by electro-erosion, are disposed in a specific honeycomb pattern. Distances °, °, ° and ° (figure 1.d) are equal to 4 mm, 1.4 mm, 1 mm and 2 mm respectively. Protrusions are 1mm high and have a square base (300 µm large). They were designed with a pyramidal shape at the top in order to accentuate the local modification of the local electric field.

Figure 1 -

 1 Figure 1 -Electrospinning set up: a) fiber patterning on microstructured collectors, b) fibrous membrane deposited on microstructured collectors, c) 3D view of microstructured collectors, d) elementary honeycomb mesh (top view of the collector protrusions: distances are ° = 4 mm, ° = 1.4 mm, ° = 1 mm and ° = 2 mm)

  Uniaxial tensile tests were carried out with an ARES-G2 rheometer (TA Instruments, New Castle, DE, USA). Samples were cut in rectangular shapes (l0 = 10 mm, L0 = 70 mm) (see figure 2.a) using a sharp blade. Sample extremities were placed into two axial clamps. In order to avoid local damage of the scaffolds induced by the grips, small strips of paper tape were added between the sample and the grip. Samples were stretched along one direction at a constant crosshead speed of 0.1 mm/s. Gauge length H0 was set at 30 mm. ARES-G2 rheometer is equipped with a normal force transducer of 20 N (0.001 N precision) and a positioning accuracy of 0.1 µm. Uniaxial tensile tests were performed at least on three samples in the two perpendicular directions D1 and D2 defined in figure 2.a.

  bench. A motorized device enables to follow the center of the gauge length of the sample during elongation and the deformation of a few honeycomb patterns. Patterns situated at the center of the sample are considerably less altered by the presence of the grips, which restrain locally the transverse deformation of the fibrous material. As shown in figure 6, membranes adopt from 15% of strain a dog bone shape characteristic of a non homogenous deformation. Stacks of images were processed with imageJ software in order to calculate local axial and transverse strains at the core of the mechanical sample (more details in § 4.2).

Figure 2

 2 Figure 2 -a) Electrospun cross membrane from where tensile samples D1 and D2 were extracted (l0=10 mm and L0 = 70 mm), b) Honeycomb patterns of the fibrous scaffold

Figure 3 -

 3 Figure 3 -SEM images of honeycomb scaffolds with fiber orientation and Average Fiber Diameter measurements: ① high fiber density zone (collector protrusions), ② and ③ fiber bridges, ④ and ⑤ center of the honeycomb pattern

Figure 4 .

 4 a shows the AFDs trend regarding the different areas and the relative distance d between peaks of the micro-structured collectors. Distances d2 to d5 are described on the elementary honeycomb pattern in figure 4.b. The smallest diameter is obtained in the center of the mesh ⑤ where the space d between protrusions is maximum while AFD is maximum in the dense zone ①, similar to a random collection on flat surface (here considering as a conventional electrospinning i.e. d =0). AFDs in zones ② and ③ are higher than the ones in zone ④ and ⑤, as the distance d between protrusions in ② and ③ is smaller. Figure 4 exposes then the global tendency of AFD to decrease when the distance d increases.

Figure 4

 4 Figure 4 -a) Average fiber diameter (AFD) of zones ① to ⑤ of the elementary honeycomb in function of the distance d between protrusions, b) definition of distances d1 to d5, d1 for zone ① is considered as zero.

Figure 5 .

 5 Figure 5.a reveals the heterogeneous topography of the 3D structured fibrous mats via a 3D profilometer scan. Figure 5.b shows a SEM cross section view of the membrane, section being defined by the dashed line b in Figure 5.a. Honeycomb scaffolds are produced here over 70 mm with high and low fibers zones defining a 3D characteristic structure.

Figure 5 .

 5 Figure 5.c shows the surface profile along the dashed line c in figure 5.a. Produced scaffolds displayed an apparent thickness between 350 and 500 µm, value of 381µm estimated in figure 5.

Figure 5

 5 Figure 5 -a) Optical profilometer scan of 3D honeycomb scaffolds, b) SEM image and c) profile graph of the membrane cross sections

Figure 6

 6 Figure 6 displays tensile curves of the honeycomb fibrous mats. The nominal stress, calculated from equation (2), is plotted in function of the global true strain defined as ln & & ' . Standard deviations are depicted with blurred zones in figure 6. Pictures of deformed membranes are illustrated in the two directions D1 and D2 at 0, 15, 30 and 50% of global true strain.

Figure 6 -

 6 Figure 6 -Average Nominal Stress with standard deviations vs global True Strain of honeycomb PCL scaffolds. Pictures of tensile test in D1 and D2 directions at 0, 15, 30 and 50 % of global true strain

Figure 7 -

 7 Figure 7 -Nominal stress of honeycomb scaffolds in function of global (dashed line) or local true strains (solid line). Optical images of D1 and D2 scaffolds at different global true strains. Surface S = d0 * d5 is plotted in color orange

measured in figure 6 .

 6 Indeed, as shown by Silberstein et al, transverse contraction while stretching is directly linked to a change in porosity. Fibers organized and aligned in the direction of loading increasing the mechanical stiffness of the mats (figure6).

Figure 8 -

 8 Figure 8 -Local deformation of a single honeycomb mesh (D1 and D2 directions), a) Transverse strain versus Local True Strain, b) local relative variation of the surface

  polymer membrane at 15 % for 45 seconds, time length considered sufficiently long to observe and fit correctly the relaxation curves. As illustrated in figure 9.a, scaffolds exhibit in both directions a small relaxation proving that the viscosity of the material will not drastically influence the mechanical behaviors. At the end of a 45 s loading, ratio between the maximal stress values #23 4) / #23 4 (t=45s) is equal to 4.1 . The stress ratio is preserved after relaxation at t=90 s 678 4) / 678 4 = 4.2 showing that relaxation of the polymer is sufficiently low and preserve the anisotropic properties of the scaffolds.

Figure 9

 9 Figure 9 -a) Charge of honeycomb scaffolds in D1 and D2 directions b) Stress relaxation curves: Nominal stress normalized by its highest value σmax reached at t = 45 s, dashed black curves represented the fit equation (3)

Figure 10

 10 Figure 10 exposes experimental curves needed to retrieve the specific mechanical properties required to complete the 2D orthotropic mechanical model. Axial mechanical tests figure 10.a and local strain analysis figure 10.b of the honeycomb scaffolds give all required information to characterize the anisotropy of the architected scaffolds. Linear regimes for

Figure 10 .

 10 Figure 10.b shows the local transverse strain curves. Poisson's coefficients are calculated (υ12 = 3.5 ± 0.4 and υ21 = 0.6 ± 0.1) on the same linear regime. Poisson's ratio υij (for i ≠ j) gives the contraction in direction j when the extension is applied in direction i. The behavior of the local transverse strains is rigorously linear for both directions until 10 % of local true strain, proving that the model might be theoretically valid for small deformations. It can be added than the contraction behavior is less pronounced in random membranes (Poisson coefficient of 0.46 for PCL electrospun mats [ref de Bhullar]). Patterned honeycomb scaffolds contains areas of low fibers density which allow a greater transverse contraction of the membranes.

Figure 10

 10 Figure 10 -a) Nominal stress and b) Transverse strain at small deformations, linear fits are calculated for values of true strain comprised between 0 to 15%

Table 1 -

 1

		Short relaxation time τ1 (s)	Long relaxation time τ2 (s)	Normalized infinite Stress D C
	Direction D1	2.2	22	0.8
	Direction D2	2.1	26	0.76

Relaxation parameters of electrospun honeycomb patterned membranes Short and long relaxation times D1 and D2 differ from one order of magnitude. The fast relaxation E ) time might be attributed to the local deformation of fibers while slow relaxation time E could reflect the constrained molecular dynamics of stretched polymer chains. Similar observations were collected when fitting stress relaxation of electrospun fibers by a same 5 parameters model

Table 2

 2 lists all required mechanical properties to fulfill the elasticity tensor. Young's moduli and Poisson's ratios were measured from the previous experimental curves.

Acknowledgement

The authors gratefully acknowledge M. Karrouch for the assistance in the elaboration of the microstructured collectors and R. Martin for her expertise and assistance in SEM microscopy. The present work was partially supported by the ANR2016-BIOSCAFF (ANR-16-CE09-0024) held by the University of Grenoble-Alpes.


R Castillo
email: rcastillo4@us.esodrio@us.es

E Dominguez Garcia

J L Santos

M A Centeno

F Romero Sarria

M Daturi

J A Odriozola

Upgrading the PtCu intermetallic compounds: the role of Pt and Cu in the alloy

Keywords: Platinum, copper, alumina, CO adsorption, FTIR, CO-PROX. 1. Introduction

This work is devoted to the study of the role of both metals in the intermetallic PtxCuy/γAl2O3 catalysts commonly employed in CO-PROX reaction. Therefore, monometallic Pt and Cu based catalysts and PtCu intermetallic compound with different molar ratios (Pt3Cu1 and Pt1Cu3) supported catalysts were carefully synthesized and deeply characterized. Room temperature CO adsorptions by FTIR spectroscopy were carried out on the mono-and intermetallic catalysts being the monometallic catalyst determinant for the study. From the analysis of the nature of the platinum surface in Pt/γAl2O3, we have demonstrated that the role of Pt sites is based in the CO dissociation for the CO2 formation and also how the platinum surface is partially blocked by leftovers from the synthesis. Moreover, the study of the Cu/γAl2O3 and the bimetallic catalysts PtxCuy/γAl2O3 allowed elucidating the effect of the copper in the metallic site and support interphase as well as the role of copper in the hydrocarbon oxidation.

Recently, Pt-based intermetallic compounds (IMCs) have been studied such improved alternative for the monometallic catalysts for several catalytic processes [1].

Especially, the study of PtCu alloys is taking relevance in the energy-related catalytic processes [2][3][4][5][6][7][8][9]. In all of them, authors highlight that catalytic performance depends on surface structure and composition.

The better catalytic behavior of intermetallic PtxCuy compounds respect to monometallic catalysts has been demonstrated in the preferential oxidation of CO [10].

This fact and the unknown active site nature of one of the most useful commercial catalyst for mobile devices [START_REF] Shore | Platinum group metal promoted copper oxidation catalysts and methods for carbon monoxide remediation[END_REF] justify why several authors focus their work in the search of a suitable answer. Trying to clarify the role of both metals during the catalytic reaction, several studies have been reported about CO adsorption on PtxCuy based catalysts, which are the object of this work. For instance, Komatsu et al. [1] showed the relationship of the metallic site electron density with the catalytic activity by the evaluation of the (CO) shift after the CO adsorptions on supported intermetallic compounds with different atomic ratio (Pt3Cu1 and Pt1Cu3).

However, before the study of the PtxCuy intermetallic compounds, it is necessary to perform a complete characterization of the monometallic catalysts. CO adsorption on supported platinum catalysts has been studied by many authors due to the importance of these compounds in industry. Olsen and Masel [12] carried out the CO adsorption on Pt (111) single crystal finding two different carbonyl adsorption modes on platinum sites; a band related to linear carbonyls around 2080 cm -1 and another for two-fold bridge bound CO at 1850 cm -1 . A shift to higher wavenumber was observed depending on the CO coverage.

On the other hand, CO adsorption may provide information about metal coordination, geometry or adsorption mode on the active site. Jänsch et al. [13] studied the surface migration of adsorbed CO species on Pt (335) crystal by electronic excitation (electron stimulated migration). They distinguished two different adsorption modes: adsorbed CO on step sites and adsorbed CO on terrace sites, which their vibrational frequencies of CO stretch mode are clearly separated by around 25 cm -1 . Platinum atoms with different particle size also could be located in different environments. Stakheev et al. [14] studied the electronic state and localization of supported Pt atoms by CO adsorption. Those authors observed three bands at 2000, 2060 and 2075 cm -1 and they concluded that the frequency shift was due to the CO adsorbed on platinum sites with a different environment. They explained how CO adsorption on more accessible platinum sites located near to the exposure surface involve different degrees of metal-support interaction or different particles shapes, and they related this kind of site with the band at 2075 cm -1 . The other two bands at 2060 and 2000 cm -1 were assigned to metallic sites with lower accessibility and also to CO adsorbed on high dispersed metallic platinum atoms.

In the cases of copper based catalyst, Cu n+ -CO surface carbonyls were located in a range from 2240 to 1916 cm -1 . Carbonyls formed by Cu + ions are the most stable species of Cu n+ -CO complexes [15]. Dandekar et al. [16] identified them in a wide range from 2160 to 2080 cm -1 during the CO adsorption on supported Cu catalyst, assigning the carbonyl on Cu supported on Al2O3 at around 2130 cm -1 . Conversely, Cu 0 -CO complexes formed via a bond with the copper atoms and carbonyls on Cu +2 ions are unstable at room temperature being detectable by CO adsorption only at low temperature (-196 ºC) [17].

Additionally, most of published results for PtCu based catalysts for CO-PROX reaction are based in a previous conditioning treatment under hydrogen environment in the range of 300 -400ºC [1,[18][19][20][21]. In this way, Romero-Sarria et al. [22] showed by operando FTIR study and CO adsorption on PtCu/Al2O3 catalyst, the effect of organic deposit (from the synthesis method) on the electron density of the active site and, therefore, its effect on the catalytic activity.

Although some authors argue that the organic molecules can be successfully removed without oxidative treatments, the presence of vibrational modes associated to the organic species in Raman and FTIR spectra of the catalysts surface after their conditioning process raises serious doubts on the effectiveness of their removing process [23].

In this work, we have studied the role of Pt and Cu in the active site of PtCu alloy supported on alumina using electron microscopy and probe molecules adsorption followed by FTIR spectroscopy. Especially, CO adsorption was carried out on PtCu/γAl2O3 catalysts with different molar ratio (Pt3Cu1 and Pt1Cu3) as well as on supported Pt and Cu catalysts for the total understanding of the role of both metals in the alloy.

Experimental

Synthesis

Catalysts have been prepared in two steps. First, unsupported nanoparticles (NPs) were synthetized and then, NPs were supported on commercial γAl2O3 (10% wt) by a mechanical procedure.

PtCu intermetallic compound with different molar ratios (1:3 and 3:1) catalysts as well as Pt and Cu monometallic nanoparticles were synthesized following the synthesis method reported by Taylor et al. [24]. Briefly, 25 ml of ethylene glycol (99%, PanReac) were heated at 110ºC in a bottom flask connected to a water condenser system for 1h. Then, the capping agent, which is poly(vinylpirrolidone) (PVP) (Mw = 55000), H2PtCl6•xH2O and CuCl2•2H2O (1:3:1 and 3:1:1 Cu:Pt:PVP molar ratio) were simultaneously added. For the monometallic catalysts, 1:1 metal:PVP molar ratio was employed. This solution was kept at 110ºC for 2 h. At this point, an excess of reducing agent is added dropwise and maintained under vigorous stirring for 15 min. The product was washed with acetone and dried at 60ºC. Finally, nanoparticles were supported on alumina.

Structural characterization

XRD patterns were acquired at room temperature in a X'Pert Pro PANalytical diffractometer using Cu Kα radiation source (40 mA, 45kV). Patterns were acquired in the range of 10° ≤ 2θ ≤ 90° using a position sensitive detector with a step size of 0.05° High resolution transmission electron microscopy (HRTEM) was carried out using a FEI Talos electron microscope with a field emission source at an accelerating of 200 kV. Micrographs were taken with a side mounted Ceta 16M camera. Samples were deposited directly on 300 meshes holey carbon coated copper TEM-grid. XPS experiments were carried out at TEMPO beamline of SOLEIL Synchrotron (France).

The conditioning of the catalysts were carried out at the own analysis chamber equipped with a SPECS Phoibos 150-NAP hemispherical electron analyzer under hydrogen flow atmosphere (20 mL/min). The pressure work during the conditioning treatment was 2 mbar. Spectra after the hydrogen treatment were taken in ultra-high vacuum conditions at 5 x 10 -9 mbar. Surface copper and platinum concentrations were estimated by the analysis of Pt 4f and Cu 3p photoelectrons. The incident energy was 300 eV which account for inelastic mean free paths (IMFP) of 5Å. Spectra were collected with pass energy of 20 eV. Shirley background was used and signal fitting was performed with Doniach-Sunjic for platinum photoelectrons and Gaussian/Lorentzian for copper photoelectrons.

Characterization by FTIR spectroscopy

Catalysts characterization by in situ FTIR spectroscopy was performed in a glass cell connected to a vacuum system, temperature controller and probe molecule system. O2 and mainly CO as probe molecules were used in this work. The samples in a selfsupported wafer form were introduced in the cell. Firstly, catalysts were conditioned under 6.5 mbar H2 (99.97% Air Liquide) at 350ºC for 2h (heating rate of 10 C•min -1 ).

Then, temperature was cooled down until room temperature. H2 environment was pumped at 200ºC to enhance the evacuation efficiency and for avoiding the organic rests deposition on the surface catalysts. Secondly, CO (99.99997%, Air Liquide) adsorption at room temperature was carried out when the vacuum in the cell got 10 -6 mbar. CO probe molecule was added by aliquots of ~10 -2 µmol until surface saturation.

Similarly, O2 (oxygen 5.0, Linde) addition experiment was carried out by the same method until saturation. IR spectra were recorded by a Nicolet 6700 Fourier Transform IR spectrometer equipped by DTGS and MCT detectors. Spectra were collected using 128 scans with 4 cm -1 of resolution. Pellets were constituted by 15-20 mg of sample and 2 cm 2 of area.

All spectra were normalized by weight (10 mg).

RESULTS AND DISCUSSION

Structural catalyst characterization

Alumina diffraction lines dominate over the diffraction peaks of the metallic phase making difficult the structural study of the nanoparticles. However, as described above, single-phase PtCu nanoparticles were first obtained, and afterwards supported on alumina allowing the structural analysis of the nanoparticles before supporting them.

Figure 1 shows the XRD data of synthesized PtCu nanoparticles with different molar ratio (1:3 and 3:1) as well as the monometallic nanoparticles. 1.

Table 1. Chemical composition and crystallite sizes (by XRD analysis) obtained for the synthesized unsupported nanoparticles.

In turn, XRD data of copper nanoparticles show a mixture of two phases: Cu2O (JCDPS #00-001-1142) and metallic copper species (JCDPS #00-001-1241). The crystallite sizes of nanoparticles were estimated by Sherrer's equation. Since copper concentration increases in the alloy, particle size became smaller.

After the structural analysis, nanoparticles were supported on γAl2O3. Figure 2 shows a HRTEM image of Pt1Cu3 supported alumina nanoparticles. The average particle size is similar to the one calculated using XRD (~1,5 nm). As the observed average size of the supported nanoparticles obtained by HRTEM is similar, within the experimental error, to the ones estimated by XRD it may be concluded that nanoparticles were not affected by the supporting step. Although, unsupported copper nanoparticles present an average size of 20 nm, according to the Sherrer's method, once supported on alumina these copper nanoparticles were not detected by HRTEM. Nevertheless, STEM analysis in mapping mode (Figure 3) revealed a high homogeneous distribution of copper onto the alumina support. This account for a very small average diameter of the copper nanoparticles and points to the difficulty of estimating average sizes by XRD particularly when overlapping diffraction lines are present. Probably, the size of the nanoparticles is close to the detection limit of the TEM microscope in the bright field imaging mode being the analyzed crystallite size by XRD the one corresponding to Cu2O species.

CO adsorption on Pt/γAl2O3 catalyst

Figure 4 shows the IR spectra upon successive CO adsorption experiments on previously H2-reduced Pt/γAl2O3 catalyst. Carbon monoxide was progressively introduced in the FTIR system after the conditioning treatment at 350ºC for 2 hours under hydrogen atmosphere. Spectra showed in figure 4.a present a shift of the absorbed carbonyls species on the platinum surface from the initial band at 2055 cm -1 to a predominant band at 2065 cm -1 assigned to CO adsorbed on metallic platinum species [15,25]. This band shows a shoulder at 2010 cm -1 assigned by Bazin et al. [26] to adsorbed CO on small platinum nanoparticles (average particle sizes below 1.5 nm) (Figure 4.b). Also, those authors assigned the observed band at 1943 cm -1 to bridged CO species adsorbed at the metal-support interface. However, this later assignment remains uncertain since the different nature of the support. The weak band observed at 2130 cm -1 can be related with the possible presence of absorbed CO on Pt atoms present at platinum nanoparticles/alumina interface.

Finally, bridged CO adsorption species were not found in the region between 1800 cm -1 and 1850 cm -1 (Figure 4.a), in the form of a broad and weak feature. Moreover, bands at 2173 cm -1 and 2223 cm -1 assigned to CO absorbed on alumina surface were observed in the subtracted spectra. The characterization of Lewis and Bronsted acid sites by CO adsorption on the γAl2O3 surface has been extensively reported by many authors [27][28][29][30]. Physisorbed CO results in a band at 2140 cm -1 , whereas CO interacting with surface OH groups gives rise to a band at 2155 cm -1 , while another massif from 2180 cm -1 to 2245 cm -1 accounts for CO interacting with Lewis acid sites. Therefore, the bands at 2173 cm -1 and 2223 cm -1 are assigned to CO adsorbed on Al 3+ cations in octahedral, (Al +3 -CO)oct, and tetrahedral, (Al +3 -CO)tet, coordination, respectively [31].

An additional set of bands in the 1000-1800 cm -1 regions associated to the presence of residual carbonaceous species adsorbed on the support surface are also observed (Figure 5a). Weak bands at 1494 and 1420 cm -1 are ascribed to C-H bending modes in CH3 ( (CH3)as) and CH2 ( (CH2)) [32]. The ascription of the bands at 1684, 1577, 1460, 1378 and 1290 cm -1 is controversial. Borodko et al. [33] claims that the bands at 1680, 1450 and 1370 cm -1 correspond to C-H deformation modes of amide carbonyls from residual PVP decomposition products, while Romero-Sarria et al. [22] assigned bands around 1577 and 1460 cm -1 to acetate species, and bands around 1370 cm -1 to the presence of different hydrocarbons on the alumina surface. However, Finocchio et al. [32,34] assign the band at 1684 cm -1 and the weak shoulder around 1640 cm -1 to C=C stretching ( (C=C)), and the bands at 1577, 1460, 1378 and 1290 cm - 1 to OCO stretching modes of carboxylate species and CH deformations. In this way, Busca et al. [35] observed the presence of the same set of bands during the oxidation of propane and propene on Co3O4 and MgCrO4. These authors related the aforementioned set of bands to the presence of acrylate species that are intermediates in the oxidation of hydrocarbons on the alumina surface (Figure 5b). Moreover, the presence of bands corresponding to C-H vibrational modes in the 2800-3100 cm -1 region (not shown), confirms the previous assignments. Bands at 2924 cm -1 and 2838 cm -1 were assigned to the symmetric ( (CH3)s and asymmetric (CH3)as) C-H stretching modes, respectively [36]. Busca et al. [37] explained the origin of the found bands during the formaldehyde adsorption and transformation on different oxide surfaces. They related the band at 2960 cm -1 to CH vibrational modes, which can be shifted by Fermi resonance effects. Finally, bands around 2955 and 2983 cm -1 would correspond to (CH2)s and (CH2)as stretching modes.

Modification of the vibrational features of surface OH in the 3800-3500 cm -1 region (not shown) during CO adsorption on Pt/ Al2O3 were not observed since bands overlap as a result of the presence of adsorbed water. This spectroscopic evidence points out the complex situation of the surface, presenting a large number of residual species from synthesis, as well as transformation products of those residuals upon reducing treatments.

The interaction of CO with the catalyst surface at RT allows the evaluation of the efficiency of the employed conditioning treatment. Analysis of the gas phase upon CO adsorption allows observing the production of CO2. On increasing the number of CO doses, an oscillation of the bands intensity of CO2 gas phase (around 2348 cm -1 ) was observed. Figure 6 shows the CO2 band area evolution during the CO adsorption after the conditioning treatment. It shows a small CO2 production during the first doses of CO adsorption after H2 treatment. The CO2 production decreases when the added CO amount increases, being zero at CO saturation coverages. The number of edge and kink surface sites as well as the relative occurrence of (100) and (111) faces has been estimated assuming cubo-octahedron particle shape and the model developed by Van Hardeveld and Hartog [38]. The total amount of CO2 species evolved is estimated in orders of magnitude below the number of edge and kink sites and (100) surface sites of 1.5 nm cubo-octahedron particles, respectively. As the CO2 production is well below the number of step and kink sites that are the most reactive ones the presence of surface carbon overlayers from the synthesis step after H2 treatment is suggested. Thus, these carbon deposits block nanoparticle active sites being the Pt surface only partially available for CO adsorption. Adsorbed CO may dissociate on platinum nanoparticles forming adsorbed oxygen and additional deposits of carbon on the nanoparticles surface [39,40]. The increment of the CO2 concentration in the atmosphere might be due to the reaction between the CO and the so-formed oxygen for producing CO2 gas (Eq. ( 1)). McCrea et al. [40] showed different pathway for the CO2 formation by CO dissociation on Pt crystal surfaces, as well as for the ignition of CO (Eq. ( 4)), CO oxidation via a Langmuir-Hinshelwood kinetics mechanism (Eq. ( 5)) or surface carbon oxidation by an ignition process (Eq. ( 6)), but most of them consider the presence of molecular oxygen for the oxidation process. These authors state that CO dissociation, more favorable on the (100) surface than in the (111) one, occurs just at temperatures above ~225ºC. On the other hand, Minot [41] demonstrate the influence of CO pressure on the formation of CO2 (Eq(7)) through the formation of Pt-CO-CO multilayers leaving behind adsorbed carbon species but again high temperatures are required for the reaction. The very low CO conversion into CO2 accounting for less than 0,1% of the most reactive surface sites is in accordance with the high temperature requirements for any of these reactions, while at the same time suggests a large blocking of Pt surface sites by the presence of carbon.

(1) CO (g) + O 2 (g) → CO 2 (g)

O2 introduction on Pt/ Al2O3 catalyst

The ignition of residual carbon deposits was analyzed by introducing O2 doses in the IR cell. Successive oxygen doses were submitted to the non-conditioned (fresh catalyst) or H2-treated Pt/ Al2O3 catalyst to evaluate the presence of surface carbon deposits. The goal of this experiment was to estimate the amount of surface carbon deposits in the conditioned catalyst and the effects of adsorbed oxygen from the CO dissociation on the platinum oxidation state. First, the evolution of the CO2 bands was followed during the oxygen small doses introduction (Figure 7a). CO2 production increases on increasing the amount of added oxygen being higher the amount of CO2 for the fresh catalyst, which may indicate that the H2 treatment results in a decrease of surface carbon. In addition to gas phase CO2, adsorbed CO species appear, Figure 7.b. The spectra in the CO stretching region show bands at 2064, 2130 and 2174 cm -1 assigned to linearly adsorbed CO on metallic platinum nanoparticles, CO adsorbed on platinum atoms with low electron density (Pt atoms) and absorbed CO on (Al +3 )Oct, respectively. The spectrum of the adsorbed CO phase is irrespective of the treatment but the intensity for the H2-treated sample is lower indicating that carbon deposits are partially removed with this treatment and that the Pt oxidation state is not modified by adding O2 doses at room temperature. The presence of adsorbed CO has been observed upon O2 adsorption on carbon covered Pt surfaces while adsorbed oxygen is not detected when the adsorption is carried out on clean surfaces [40]. Therefore, the presence of carbon deposits that may be partially removed upon H2 reduction treatment is stated.

In both case, for the non-conditioned catalyst and after H2 treatment, there is a remarkable surface carbon amount that can be removed by O2 adsorption at RT. The total amount of gas phase CO2 observed upon introduction of 1 µmol O2 in the IR cell is ~7,0•10 14 and ~2,5•10 14 molecules for the fresh and the H2-treated catalysts, respectively. However, the reaction conditions during the oxygen addition (exposure time, temperature, oxygen amount, etc.) are not enough for the total removal of surface carbon through a gasification process, so CO2 bands are present for all the experiment.

CO adsorption on Cu/γAl2O3 catalyst

FTIR difference spectra recorded during CO adsorption on Cu/ Al2O3 after H2 treatment are plotted in Figure 8. Together with the presence of adsorbed CO species, Figure 8.b, there is a clear modification of the OH stretching region. In Figure 8.a spectra of the OH stretching region during CO adsorption after H2 treatment are plotted showing bands at 3622, 3650, 3684 and 3755 cm -1 ascribed to OH stretching modes of newly formed bi-and mono-dentate hydrogen carbonates [42], OH groups linked to tetrahedral and octahedral Al +3 cations [43] or alternatively bridged on pentacoordinated aluminum centers [29] and terminal OH groups on surface tetrahedral aluminum sites [44] or alternatively on octahedral aluminum centers [29], respectively. It is clearly seen that CO adsorption induces a perturbation on the hydroxyls coordinated with the tetrahedral aluminum sites, demonstrating that one of the carbonyl features interacts more with the support than with the copper species. Quantitatively, lower intensity of the bands assigned to the OH vibrational modes was observed after H2 treatment, being the intensity of the bands witnessing for the coordination of CO on different sites (from groups of bands is related with the strength of the support-metal interaction, i.e. copperalumina interaction is stronger after H2 treatment.

According to Bechoux et al. [43] conclusions that studied CO adsorptions on Ag/Al2O3 after reducing and oxidizing pretreatments, copper atoms interact with the closer OH groups (neighboring basic oxygen) decreasing the available copper sites for the CO adsorption. Also, neighbor basic oxygen atoms transfer electron density to the copper atoms allowing the formation of Cu + -CO complexes and their characteristic band at 2122 cm -1 . Additionally to the expected metallic site of copper nanoparticles after reduction, the presence of Cu + species could be related to the electrophilic behavior of copper atoms in the presence of CO molecules proposed by Komatsu et al.

[1] that concluded that Cu atoms adsorb available oxygen atoms from CO dissociation originating Cu species.

Interestingly, a main band can be observed at 2123 cm -1 with a shoulder to lower wavenumber at 2098 cm -1 (Figure 8b). In addition, a weak band at 2165 cm -1 can be observed. Different possibilities could be proposed for the nature of such Cu sites. K.I.

Hadjiivanov and G.N. Vayssilvov [15] described frequencies of the surface carbonyls absorbed on the copper atoms as a function of the copper oxidation state, concluding that the characteristic band of linear carbonyls adsorbed on Cu 2+ (Cu 2+ -CO) is located between 2220 and 2150 cm -1 , band of carbonyls adsorbed on Cu + (Cu + -CO) is located between 2160 and 2080 cm -1 , while carbonyls adsorbed on metallic copper (Cu 0 -CO) show a weak band lower than 2130 cm -1 . On the other hand, Zecchina et al. [45] carried out a CO adsorption on silica supported copper catalyst after progressive reduction steps under H2 environment at different temperatures. When Cu/SiO2 was reduced at 400ºC, they observed two bands; a weak band at 2163 cm -1 and a sharper band at 2136 cm -1 . They concluded that both bands are due to CO adsorbed on small Cu + atoms on extended faces and edges and steps respectively. Also, Hadjiivanov et al. [15] proposed the band at 2165 cm -1 due to carbonyls absorbed on Cu 2+ species. Moreover, when the reduction temperature was increased during the CO adsorption on Cu/SiO2, a new shoulder around 2100 cm -1 was observed and they assigned this band to CO adsorbed on metallic copper or CO adsorbed on a Cu-rich non stoichiometric Cu2O phase. Thus, the CO adsorption on Cu 2+ species, carbonyls adsorbed on small particles of Cu + (probably located in the metalsupport interface) and CO absorption on metallic copper could be assigned to the found band at 2165, 2122 and 2098 cm -1 , respectively, in agreement with Zecchina et al. [45] and Hadjiivanov's [15] conclusions. However, the presence of Cu 2+ species is unlikely after the reducing H2 treatment. It means that band at 2165 cm -1 could be due to carbonyls adsorbed on Cu + species or CO absorbed on the Al 3+ cations in octahedral coordination ((Al +3 -CO)oct) on the alumina support. Note that tetrahedral sites on alumina are blocked by copper particles avoiding the CO adsorption. Thus, the assignment of bands at 2122 cm -1 to Cu + species after a reducing treatment could be explained with support-metal interactions upon -OH groups evacuation. In summary, observed bands at 2098, 2122 and 2163 cm -1 after both treatments are related with the CO adsorption on metallic copper species, CO adsorbed on Cu + species and adsorbed CO on Al 3+ cations with octahedral coordination ((Al +3 -CO)oct), more than on Cu 2+ sites, unexpected here. Moreover, an important detail is the absence of CO2 bands during the CO adsorption on Cu/ Al2O3.

As expected, the CO adsorption at room temperature after H2 treatment on Cu/ Al2O3 exhibits also several bands in the region between 1100 and 1800 cm -1 . First, during the CO adsorption after H2 treatment, bands at 1229, 1264, 1374, 1396, 1432, 1468, 1579, 1619 and 1657 cm -1 were observed (Figure 9). Strong carbonate bands were observed during the CO adsorption on Cu/γAl2O3 after H2 treatment at 1657, 1432 and 1229 cm -1 . Their assignment is not simple due to the different structures showed by carbonates. The main observed bands correspond to hydrogen-carbonates species; νas(COO) at 1657 cm -1 , νs(COO) at 1432 cm -1 and (OH) at 1229 cm -1 . However, different carbonates species on the surface of alumina should be expected and may be present as traces. Weak bands at 1579, 1468 and 1264 cm -1 correspond to υas(COO), υs(COO) and υ(C-C) vibrational modes of adsorbed acetates species, while bands at 1374, 1396 and 1579 cm -1 characteristic of formate species were found [START_REF] Davydov | Molecular Spectroscopy of Oxide Catalyst Surfaces[END_REF]. However, the intensity of the latter is extremely weak, even not showing the CH stretching vibrational mode. Thus, only hydrogen-carbonates species were considered for the surface analysis.

Föttinger et al. [START_REF] Föttinger | [END_REF] studied the possible pathways mechanism of carbonate formations on Pd/Al2O3 upon CO adsorption, and they concluded that it takes place via "Water Gas Shift" reaction between CO and alumina hydroxyl groups followed by the reaction of the formed CO2 with other OH groups. Also, bands around 1620, 1579 and 1264 cm -1 are increasing and they could be related with the ν(C=O), ν(COO)as and ν(COO)s vibrational modes characteristic of bidentate or bridged carbonates species formation, due to the interaction of the formed CO2 with basic oxygen atoms, probably from the oxidized copper species [48].

CO adsorption on PtCu/γAl2O3 catalysts

After having characterized the materials with single metal particles, we can now address the bimetallic catalysts. For the total understanding of the PtxCuy alloy behavior and the role of both metals, CO adsorption on Pt3Cu1/γAl2O3 was carried out after H2 pretreatments. Figure 10a shows the acquired spectra during the CO adsorption on Pt3Cu1/γ l2O3 after the conditioning treatment. The Pt3Cu1 sample showed weak bands at 2096 and 2124 cm -1 . CO adsorption on Pt/γAl2O3 (Figure 4) showed a main band at 2065 cm -1 , with a shoulder at 2010 cm -1 characteristic of metallic platinum species with different particle sizes, while CO adsorption on Cu/γAl2O3 showed two bands at 2098 and 2122 cm -1 (Figure 8.b).

Surface analysis of unsupported Pt3Cu1 nanoparticles carried out by APXPS (Figure 11) with inelastic mean free path of 5Å reveals a surface composition of the nanoparticles of 57% Cu and 33% Pt, i.e. there is a high surface copper enrichment after the conditioning treatment. Therefore, the Cu-rich surface leads us to assign the bands at 2096 and 2124 cm -1 during the CO adsorption on Pt3Cu1/γAl2O3 after H2 treatment to Cu + and metallic copper species (Figure 10a). The low intensity of the carbonyl bands impeaches the observation of the bands related to platinum sites. During the CO adsorption on Pt1Cu3/γAl2O3 (Figure 10b), bands characteristic of adsorbed carbonyls on copper species appear at the same frequency, 2098 and 2122 cm - 1 , as observed on the monometallic samples. Considering the Cu-rich surface composition between 63 and 83% of copper species calculated by APXPS of the unsupported Pt1Cu3 nanoparticles (spectrum not shown), the surface of Pt1Cu3/γAl2O3 catalyst is extremtly similar to the Cu/γAl2O3 surface and the observed bands at 2098 and 2122 cm -1 could be related to carbonyls adsorbed on metallic copper species and Cu + species respectivily. The different intensities are due to the greater interaction of copper atoms in the Pt3Cu1/γAl2O3 catalyst with the alumina OH groups, which supposes less available sites for the CO adsorption. Finally, the band at 2172 cm -1 was assigned to carbonyls adsorbed to Al +3 cations in octahedral coordination ((Al +3 -CO)oct). Figure 12 shows a comparison of the carbonates spectral region between FTIR spectra of CO adsorption on Pt/γAl2O3 after H2 treatment and CO adsorption on the Pt3Cu1/γAl2O3 and Pt1Cu3/γAl2O3 catalysts.

After the CO adsorption on Pt/γAl2O3 pretreated under hydrogen treatments, bands at 1684, 1578, 1378 and 1290 cm -1 assigned to acrylates species were observed (Figure 4). However, the addition of small amounts of copper produces different surfaces species. After the CO adsorption on Pt1Cu3/γAl2O3 pretreated under H2 atmosphere, remarkable bands at 1656, 1432 and 1229 cm -1 assigned to hydrogencarbonates were observed. However, after the CO adsorption on Pt3Cu1/γAl2O3, the decrement of bands at 1578 and 1460 cm -1 characteristic of absorbed acetates species is showed. Thus, copper species in PtCu alloy (3:1 molar ratio) promotes the surface hydrocarbons oxidation. Initial acrylates species formed by platinum atoms are oxidized to acetate species [49,50]. With the increment of the oxidation degree, acetate species are oxidized to CO2 and H2O, which leads to an increase of the sites availability.

Finally, CO2 and H2O form hydrogen-carbonates (Figure 13). The appearance of the decreasing bands of acetate species in samples pretreated under hydrogen environment means a lower reaction rate for the hydrocarbons oxidation. Small CO2 production was observed during the CO adsorption on Pt3Cu1/γAl2O3 after H2 treatment. On the other hand, after the CO adsorption on Pt1Cu3/γAl2O3 catalyst, only the final product is appreciable. Therefore, according to the above comparison, Pt1Cu3/γAl2O3 catalysts shows a low reaction rate for the surface hydrocarbons oxydation respect to the PtCu alloy 3:1 molar ratio. The acetate species oxidation to CO2, clearly evidenced with the decrement of bands at 1470 and 1580 cm -1 , and the absence of CO2 transformation into hydrogencarbonates could explain the higher CO conversion and selectivity to CO2 for the intermetallic PtCu 1:3 molar ratio catalyst during the preferential oxidation of CO [22,51].

Conclusions

The role of both metals in the intermetallic compound PtCu was studied by CO adsorption. PtCu nanoparticles alloy with different molar ratio supported on alumina as well as the monometallic solids were synthesized and characterized. The monometal platinum catalysts characterization revealed the role of platinum atoms on the CO dissociation to form CO2 by different pathways, evidencing a remarkable effect of the organic residuals from the synthesis step. The influence of the metal-support interaction on the active sites was evaluated by the correlations between the alumina OH groups and carbonyls absorbed on the active site of supported copper nanoparticles. Moreover, the effect of copper on hydrocarbons oxidation was highlighted considering the different reaction properties.

  and acquisition time of 300 s. Structural determination was performed by comparison with the database PDF2 ICDD2000 (Powder Diffraction File 2 International Centre for Diffraction Data, 2000).

Figure 1 .

 1 Figure 1. XRD data of prepared nanoparticles; Cu NPs (wine line), Pt NPs (blue line), Pt3Cu1 NPs (green line) and Pt1Cu3 NPs (yellow line)

Figure 2 .

 2 Figure 2. HRTEM image of Pt1Cu3/γAl2O3 catalyst. The insert shows the size distribution of supported Pt1Cu3 nanoparticles.

Figure 3 .

 3 Figure 3. STEM images of Cu/γAl2O3 catalyst; bright-field (BF), High-angle annular dark-field (HAADF) and EDX elemental mapping of Cu, O, Al and C.

Figure 4 .

 4 Figure 4. a) Difference FTIR spectra during CO adsorption on Pt/γAl2O3 from (a) 0.1 μmol CO to (ay) 13 μmol CO. An isosbestic point due to the species transformation is observed. Difference spectra displayed, obtained by subtracting the corresponding spectra acquired before gas exposure. b) FTIR spectra recorded after CO adsorption on Pt/γAl2O3.

Figure 5 .

 5 Figure 5. Acquired FTIR spectra during the CO adsorption on Pt/ Al2O3 from (a) 0.1 μmol CO to (ay) 13 μmol CO. a) Evolution of the region between 1000 cm -1 and 1800 cm -1 . b) Observed vibrational modes in the acrylates species formation.

Figure 6 .

 6 Figure 6. Evolution of the CO2 gas phase area bands during the CO adsorption on Pt/ Al2O3 after H2 treatment. a) FTIR spectra region from 2290 to 2440 cm -1 . b) CO2 gas phase band intensity evolution as a function of the added CO doses. Note that the time between doses is always the same and the CO amount added is also the same in each dose (0,009 μmol CO/dose).

( 2 )

 2 CO (g) + * → CO* (3) CO* → C* + O* (4) CO (g) + O* → CO 2 (g) + * (5) CO* + O* → CO 2 (g) + * + * (6) C* + 2O* → CO 2 (g) + * + * + * (7) CO 2 (g) + C* ↔ 2CO (g) + *

Figure 7 .

 7 Figure 7. a) Evolution of the CO2 band area during the small doses oxygen addiction on non-activated Pt/ Al2O3 (yellow color) and on Pt/ Al2O3 after H2 treatment (blue color). b) Absorbed CO after oxygen addiction on non-activated Pt/ Al2O3 (yellow color) and on Pt/ Al2O3 pretreated under H2 atmosphere (blue color).

Figure 8 .

 8 Figure 8. Difference FTIR spectra during the CO adsorption on Cu/ Al2O3 after H2 treatment from (a) 0.1 μmol CO to (ay) 20 μmol CO: (a) ν(OH) vibrational modes of the OH groups (b) CO adsorbed on copper sites.

Figure 9 .

 9 Figure 9. FTIR spectra during CO adsorption on Cu/ Al2O3 after H2 treatment from (a) 0.1 μmol CO to (ay) 20 μmol CO.

Figure 10 .

 10 Figure 10. Difference FTIR spectra during CO adsorption at room temperature on: (a) Pt3Cu1/γAl2O3 and (b) Pt1Cu3/γAl2O3.

Figure 11 .

 11 Figure 11. Pt 4f and Cu 3p APXP spectrum of unsupported Pt3Cu1 nanoparticles. Blue line corresponds to platinum component and red line to the copper one.

Figure 12 .

 12 Figure 12. Difference FTIR spectra of CO adsorption at room temperature on Pt/γAl2O3 after H2 treatment (orange color), CO adsorption on Pt3Cu1/γAl2O3 (blue color) and CO adsorption on Pt1Cu3/γAl2O3 (red color). Bands pointed with solid lines correspond to species on Pt/γAl2O3 and bands pointed with dashed lines correspond to species on PtxCuy/γAl2O3 catalysts.

Figure 13 .

 13 Figure 13. Schematic mechanism for the hydrocarbon oxidation on the Pt1Cu3/γAl2O3 surface.

Acknowledgements

Financial support for this work has been obtained from the Spanish Ministerio de Economía y Competitividad (MINECO) (ENE2013-47880-C3-2-R) co-financed by FEDER funds from the European Union. Authors acknowledge to SOLEIL synchrotron for provision of beamtime for carrying out the APXPS experiments.