Lila Chaal

Catherine Debiemme-Chouvy

Claude Deslouis

Georges Maurin

Alain Pailleret
email: alain.pailleret@upmc.fr

Boualem Saidani

Comparative AFM nanoscratching tests in air of bulk copper and electrogenerated cuprous oxide films

Keywords: nanoscratching test, atomic force microscopy (AFM), x-ray photoelectron spectroscopy (XPS), cuprous oxide, copper, erosion-corrosion

The normal and lateral spring constants of rectangular silicon AFM cantilevers bearing pyramidal silicon tips were accurately calibrated using a procedure that takes into account their tilt compared to horizontal orientation and their trapezoidal cross-section. Such systems were used to carry out nanoscratching tests in air on technical substrates presenting a moderate roughness (RMS 40 nm) and made either from bulk copper or from cuprous oxide thin films electrogenerated on copper. The various events occurring during these nanoscratching procedures were characterized in details. In particular, the features of the scars appearing on the scratched zones and SEM observations of the AFM tips used during the nanoscratching procedures are described and exploited to establish a better understanding of the effects of the nanoscratching procedures on the targeted samples. In the case of

Introduction

Erosion-corrosion phenomena are particular cases of corrosion. They result mainly from a fluid circulating at a fast flowing rate along a metallic surface. They are potentially influenced by geometrical characteristics of metallic installations. Erosion-corrosion phenomena are increasingly investigated nowadays as they potentially have a severe impact on the integrity of fluid networks in industry and pollution in environment. As an illustration, one can cite among the various installations or metallic parts exposed to such corrosion process, distribution networks of either cold or hot water, metallic structures in ports, pipelines, thermal exchangers, pumps, turbines or propellers for example. In fact, soft metals (copper and lead for example) as well as metals whose resistance to corrosion strongly depends on the stability of a superficial film (aluminum, stainless steels, other steels) are all expected, and were actually shown, to be indeed victims of erosion-corrosion phenomena. In the latter cases, turbulent flows may lead to a systematic and progressive destruction of the protective films (such as oxide films for example), which obviously results in very high

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ACCEPTED MANUSCRIPT 3 corrosion rates for the bare metals. Such situation was indeed found to occur for stainless steels in artificial chlorinated media [START_REF] Colombier | Aciers inoxydables -Aciers réfractaires[END_REF], carbon steels in seawater [START_REF] Tousek | Theoritical aspects of the localized corrosion of metals[END_REF] as well as copper and copper alloys [3][4][5]. In a previous experimental investigation related to erosion-corrosion phenomena, cuprous oxide (Cu 2 O) was chosen as an experimental model of moderately adherent protective coating of copper. It was exposed to erosion-corrosion risks produced by corrosive aqueous solutions under measurable and controlled flow conditions using a socalled rotating cage setup [6]. This choice was made on purpose so as to characterize the beneficial effect of hydrodynamic drag reducing agents, such as cationic surfactants, in erosion-corrosion situations.

Cuprous oxide is the most frequently encountered corrosion product of copper [7]. Its formation is known to depend on solution pH, potential of copper and composition of the electrolytic solution. For example, chloride traces deeply complicate and accelerate copper corrosion at the copper/aqueous solution interface. Cuprous oxide is also a well-known p-type semiconductor that presents moreover interesting optical and magnetic properties [8]. Lately, cuprous oxide received considerable attention due to its potential applications in solar energy conversion, nanoelectronics, magnetic storage devices, catalysis, biosensing and lithium ion batteries [8].

Our long term objective is to correlate characteristic mechanical parameters of electrogenerated cuprous oxide films and bulk copper with the mechanical resistance they present during erosion-corrosion experiments carried out in our research group. During these latter experiments, critical shear stresses exerted by the moving fluids led to the partial removal of the cuprous oxide films electrodeposited on bulk copper.

In the frame of this contribution, our purpose was i) to show the feasibility of AFM nanoscratching tests on rather rough electrogenerated cuprous oxide films and bulk copper samples (RMS 40 nm), ii) to identify the various events occurring during these tests, iii) to

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establish from these tests strategies leading to characteristic mechanical parameters of the targeted materials as well as to a better understanding of the observed wear mechanisms.

Experimental Methods

Bulk copper samples were prepared from copper rods (diameter: 5 or 10 mm) purchased from Goodfellow (Purity 99.99 %), which were coated with an insulating cataphoretic lacquer and subsequently embedded in an epoxy resin. The cross section of these copper rods was first polished on emery SiC paper up to 1200 grade, then on a microabrasive paper made from alumina deposited on polyester film (grain size 9 μm (MOA 9), ESCIL, Chassieu, France). The resulting copper electrodes were rinsed with demineralized water, then placed in an ultrasonic bath for 10 minutes, rinsed again, dried, and finally stored in a dessicator until testing. They were then used as samples for nano-scratching experiments of bulk copper or as working electrodes for the electrogeneration of cuprous oxide thin films. In this latter case, the retained electrochemical procedure implied a usual three electrode electrochemical cell including a platinum counter electrode and a mercury/mercurous sulfate saturated reference electrode (SSE). The three electrodes were connected to a SOTELEM potentiostat/galvanostat and used for the electrodeposition as well as for the electrochemical characterization of the resulting Cu 2 O films.

Cu 2 O films were electrogenerated by oxidizing anodically rotating copper disks in the galvanostatic mode in a corrosive aqueous solution containing Na 2 SO 4 (0.1 M) and NaCl (1 mM) at pH 8. In this purpose, the copper electrode was polarized for one hour at a current density of 1 mA/cm 2 while its rotation speed was kept constant at 100 rpm. These experimental conditions were close to those used in an earlier work dealing with the behaviour of copper covered with a copper oxide layer in neutral media in erosion-corrosion experiments [9]. Moreover, pH was raised to slightly alkaline values so as to promote

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efficiently the formation of cuprous oxide layers according to Pourbaix' diagram of copper.

Little NaCl amounts were also added to the electrolytic solution in order to prevent cuprous oxide films from self-healing once they were partly pulled out during the erosion-corrosion experiments [6]. The presence of sodium chloride was thought to provoke a few morphological, and possibly mechanical, heterogeneities that were necessary to allow the resulting cuprous oxide films to offer a moderate mechanical resistance to the erosioncorrosion tests.

In a second step, these electrogenerated Cu 2 O films were thoroughly characterized using ex-situ techniques such as X-Ray Diffraction (XRD), and X-Ray Photoelectron Spectroscopy (XPS) as well as voltammetry.

XRD analyses were carried out using a horizontal goniometer (Philips) and a filtered MoK K source. The resulting diffractograms were interpreted by comparing them with JCPDS files.

XPS analyses were carried out at ITODYS laboratory (Paris, France) using a VG ESCALAB 250-iXL spectrometer. The X-ray source was monochromatic Al K radiation (1486,6 eV). Before analysis, the surface of the samples was etched with Ar + cations in order to remove the native oxide and/or the contamination layer ( 1 nm). The high-resolution spectra were recorded with a pass energy of 20 eV in the constant analyzer energy mode. The detection angle was 90° with respect to the sample plane. The photoelectron peaks were analysed after subtraction of the background calculated using Shirley's method.

AFM scratching and imaging experiments were carried out on copper working electrodes either freshly polished or coated with electrogenerated cuprous oxide thin films.

For this purpose, these substrates were immobilized below the AFM tip of the Molecular Imaging AFM setup using a home made sample holder. This setup was composed of a Pico-LE base equipped with a micro-positioning device allowing the precise positioning of the The scanning electron microscopy (SEM) images were obtained with the help of an Ultra55 Zeiss field-emission gun-SEM (FEG-SEM) microscope.

Results

Theoretical considerations and experimental calibration of the tip/cantilever system

An accurate calibration of the cantilever/tip systems is crucial if one wants to extract quantitative and comparable information from force measurements or nanoscratching experiments carried out using an AFM equipment. The calibration procedure reported hereafter is easily applicable to most rectangular cantilevers as it does not necessitate any substrates or cantilever/tip systems elaborated especially for calibration purposes. As only rectangular cantilevers were used throughout this work, the force calibration was relatively simple. On its own, a cantilever immobilized at one end and bearing a pyramidal tip at the opposite end displays a mechanical behaviour that requires eight spring constants to be described accurately [10]. Although an analytical expression has been proposed for each of them in literature, only two of them, namely the normal spring constant, k N , and the lateral spring constant, k L , are systematically exploited for the quantitative determination of

4 Lh I G k Si L
4 mechanical properties of materials using AFM [START_REF] Gnecco | Chap 11: Friction and Wear on the Atomic Scale[END_REF]. They express the ability of the cantilever for simple normal bending and lateral friction (or torsion) respectively. These two major constants can be calculated for a cantilever beam with a rectangular cross section from the following expressions, respectively [10,[START_REF] Gnecco | Chap 11: Friction and Wear on the Atomic Scale[END_REF]:

3 3 L I E k Si N 3 (1)

And

(2)

I is the moment of inertia of the cantilever beam. In the case of a rectangular cross section, I can be calculated using the following expression:

(3)

In these expressions, E Si is Young's modulus of the cantilever (1.69 x 10 11 N/m 2 or 169 GPa for silicon [START_REF] Gnecco | Chap 11: Friction and Wear on the Atomic Scale[END_REF]), G Si , its shear modulus (0.5 x 10 11 N/m 2 or 50 GPa for silicon [START_REF] Gnecco | Chap 11: Friction and Wear on the Atomic Scale[END_REF]), w, its width, L, its length, t, its thickness while h is the height of the tip. These four latter geometrical parameters can be accurately measured using FEG-SEM images of the cantilever/tip systems. Moreover, it has been argued in literature that a non-rectangular cross section makes expression (3) invalid for the calculation of the moment of inertia [START_REF] Poggi | [END_REF]. As it was observed from FEG-SEM observations that the cantilevers used in this work actually have a trapezoidal cross section, a more adequate expression of I, the moment of inertia, was used instead of (3), according to literature [START_REF] Poggi | [END_REF]:

Here, a and b are respectively the width of top and bottom surfaces of the cantilever.

Another parameter influencing substantially the value of the normal spring constant is the tilt angle of the cantilever by comparison with horizontal once it is placed in its work position, as recently noticed in literature [13]. Heim et al. indeed showed that this spring constant is actually divided by a factor cos 2 (1 -(2h tan )/L) -1 . In this correction factor, is the tilt angle of the cantilever by comparison to the horizontal direction. In our experimental conditions, cantilevers were found to be tilted by an angle of 7 degrees once they are placed on the nose of the AFM scanner.

By taking into account each of the adjustments reported above in the expression of k N

(1), one obtains the following expression.

Once k N is experimentally determined, k L can easily be deduced using a well established relation linking them [14]. Alternatively, one can use equation ( 2) in which the moment of inertia will be replaced by the expression given in ( 4). (6) If one wants to determine the normal and lateral forces applied or undergone respectively by the cantilever/tip system, other important parameters that need to be precisely determined are the angular sensitivities, S N and S L expressed in nm/V, of the four quadrants photodetector, with respect to change in the angles for vertical and torsional deformation of the cantilever. The determination of S N can be achieved by exploiting force vs. distance curves obtained on incompressible surfaces such as glass or alumina for example, where elastic deformations of the substrate are negligible [10,[START_REF] Gnecco | Chap 11: Friction and Wear on the Atomic Scale[END_REF]. In such cases, the vertical movement of the scanner equals the deflection of the cantilever. The relation between the difference of the vertical signals on the four-quadrant detector, V N , and the distance from the surface, z, is therefore deduced from the slope of the elastic part of the resulting curve. In our experimental conditions, S N was found to be 10 nm/V from force distance curves obtained on glass (Fig. not shown). As the four quadrants of the photodetector are located symmetrically

A C C E P T E D M A N U S C R I P T N N N N V S k F k L L L L V S L h k F 2 3 2 3
in the same plane perpendicularly to the laser beam, one can consider in a first approximation that S N and S L take identical values.

Once k N , k L , and S N are determined experimentally, it is then possible to calculate accurately the normal and lateral forces, namely F N and F L respectively. The former is calculated as a function of V N , the voltage measuring the deflection of the cantilever while the latter involves V L , the voltage expressing the friction of the cantilever that is read during the nanoscratching experiments, using the following expressions [START_REF] Gnecco | Chap 11: Friction and Wear on the Atomic Scale[END_REF]:

(7) (8)
The procedure detailed above was applied for each of the AFM tips used in this contribution in order to determine accurately the normal force used for each of the nanoscratching tests reported hereafter (see Table 1). Zones A and B were both scratched with the help of a unique silicon tip by carrying out five consecutive scans with an applied normal load of 0.729 μN and 1.21 μN respectively as calculated from Eq. ( 5) (see Table 1). In both cases, the fast scan direction was orthogonal to

Nanoscratching tests of bulk copper

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the axis of the cantilever and the scan rate of the tip was 4 μm.s -1 . Both zones are clearly delimited by scratching residues, possibly copper and/or silicon dusts produced presumably from the wear of the substrate and/or the tip (this issue will be discussed later on). By comparison with the portions of the image that are outside the scratched zones, it appears clearly that the scratched zones underwent damages whose extent is a consequence of the load applied with the AFM tip during the nanoscratching tests, as no other difference exists between the two corresponding nanoscratching procedures. In zone A, one can notice that copper ridges have been slightly worn (see Fig. 1b) but no square based well was created in the copper substrate in spite of the five consecutive nanoscratching scans. In zone B, copper ridges were almost completely erased, as confirmed from the vertical cross section shown on Fig. 1c as an illustration. Moreover, a weakly deep square based well was dug in the copper substrate (see Figure 1d).

In order to elucidate more deeply the various events occurring during these nanoscratching tests, an SEM image of the AFM silicon tip used is shown on Fig. 2a. This side view of the AFM tip clearly shows that a significant amount of matter was accumulated at the apex of the AFM tip during the nanoscratching tests and conserved there during the subsequent imaging experiments. An EDS analysis of the matter accumulated on the tip due again to electrostatic interactions allowed the identification of copper as the main constituent (see Fig. 2b). As expected, silicon was also identified as it is the initial constitutive material of the AFM tip. Besides these two elements, gold was detected. Indeed, a thin gold film was initially deposited on the whole tip in order to make it sufficiently conductive for SEM imaging. More surprisingly, aluminum was detected, possibly as a consequence of the presence of nanometric polishing residues brought on the copper substrate by alumina based polishing paper. In parallel, one must admit that a thorough comparison of the SEM images obtained before (image not shown) and after use (see Fig. 2a) did not allow observing a thorough wear of the silicon tip. This is an important point as it implies that silicon based tips appear to be approximately non-deformable and resistant to wear by comparison to copper during these nanoscratching tests.

A further insight in the scratching tests reported above can be achieved by using the contact radius to line step ratio (CRLS), a parameter introduced recently in literature [15]. Its use requires a theoretical description of the geometry of the tip/sample contact. By considering the substrate as a flat elastic surface in a first approximation and a spherical AFM tip apex, one can use the Hertz model to calculate R c , the radius of the disk shaped contact zone between the tip and the substrate, which can be approached by [16]: (9) In this expression, r, F N and E * are respectively the radius of curvature of the AFM tip, the applied normal force and the combined elastic modulus which can be calculated from the following equation: (10) In this equation, Cu and Si are the Poisson's ratios for copper and silicon respectively whereas E Cu is Young's modulus for copper. The numerical values used in our calculations for these three parameters are 0.351 [17], 0.278 [18], and 1.31 10 11 N/m 2 [17] respectively. By using the R c value calculated from Eqs. ( 9) and ( 10), one can then determine the ideal length of the slow scan axis that is necessary to carry out exclusively load-dependent, and therefore easily exploitable nanoscratching tests, from the following equation [15].

The coulombic charge q measured below this reduction peak can be used to calculate the thickness e of the corresponding cuprous oxide film using Faraday's law:

zFS qM e ( 13
)
In this equation, M is the molar mass of Cu 2 O (143.09 g.mol -1 ), z, the number of electrons exchanged in the electrochemical reduction reaction (z = 2 from reaction (12) given above), F, the Faraday constant (F = 96490 C.mol -1 ), , the density of cuprous oxide ( = 6 g.cm -3 ), S, the electrode surface (S = 0.2 cm 2 ). Using these different values, the thickness calculated from Eq. ( 13) and Fig. 3 is 366 nm. Let us emphasize that this value may provide an underestimation of the real thickness as it does not take into account a likely porosity of these electrogenerated oxide films.

XRD technique allowed the determination of the chemical identity and the crystallographic orientation of the electrogenerated film. A typical diffractogram as well as the interpretation of each of its various peaks are shown in Fig. 4. Let us notice first that the penetration depth of X-rays in our experimental conditions was shown to be largely sufficient to cross the electrogenerated film, indicating thus that the resulting diffractogram mainly contains a contribution of this latter as well as another one from the underlying copper substrate. This latter was cristallographically identified as a bulk copper sample made of (111)-and (200)-oriented crystals in the face centered cubic lattice. XRD analysis also allowed us to conclude that cuprous oxide Cu 2 O (or cuprite) was identified as a main constituent of the whole film, as expected. One can moreover conclude that it displays a

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ACCEPTED MANUSCRIPT 14 preponderant (111) orientation in the simple cubic lattice although it must be noticed that a very minor (110) texture was also observed (see peaks corresponding to cuprite in Fig. 4).

Besides cuprite, nantokite (CuCl) was also identified undoubtedly with a predominant (111) crystallographic orientation in the face centered cubic lattice (see peaks corresponding to nantokite in Fig. 4). Its presence in the electrogenerated film is often assumed to be due to its low solubility. However, due to the large Cu 2 O intensity, it can be concluded in a first approximation that CuCl is a minor component.

In order to further confirm the identity of the bulk constituents of the electrodeposited film, XPS technique was also exploited as it expectedly allows differentiating the various oxidation degrees for a given chemical element, namely copper in our case. Literature indeed shows that the presence of copper (II) is justified by a Cu 2p 3/2 peak located at 933.6 eV and accompanied with satellite peaks that appear in the energy range 940-944 eV [22-24a]. On the other hand, Cu (I) and Cu (0) species cannot be differentiated as the 2p electrons have the same binding energy. Such purpose is actually achievable on the basis of the LMM Auger peaks as the shape of these latter differ from Cu(0) to Cu(I) [24a]. With the help of these data, XPS spectra obtained either at bare copper electrodes or at copper electrodes coated with a electrogenerated cuprous oxide film were analyzed and compared (see Fig. 5). In the region corresponding to Cu 2p, both spectra show two photopeaks appearing at 932.7 and 952. Electrochemically generated cuprite based coatings whose chemical composition was thoroughly investigated and reported above were further characterized from a morphological point of view by using AFM in the tapping mode in air (see topography image in Fig. 6 outside the central square zone). This AFM image shows that the cuprous oxide films are mainly continuous although they are not sufficiently thick to hide the valleys and ridges that were produced on copper during the polishing procedure. In other words, the topography existing immediately after polishing on the naked copper disk is maintained after the electrodeposition of a cuprous oxide based coating although this latter creates a granular aspect on the surface.

At this stage, it can be reasonably concluded that the electrochemical procedure that was elaborated in this work indeed allowed the production of a perfectly covering film of cuprous oxide, in good agreement with literature [START_REF] Gal | Corrosion, passivation et protection du cuivre en solution aqueuse[END_REF][START_REF] Souto | [END_REF][25][START_REF] Millet | Etude électrochimique et photoélectrochimique de couches d'oxyde de cuivre semi-conductrices. Rôle d'un inhibiteur de corrosion du cuivre[END_REF][START_REF] Millet | [END_REF][28], while copper (II) oxide was never observed in the resulting coatings.

Nanoscratching tests of Cu 2 O oxide films

Cu 2 O thin films were scratched using a procedure close to the one used for the nanoscratching tests carried out on bulk copper. The modified zone was scratched with the help of a silicon tip (see characteristics for tip 2 in Table 1) by carrying out only one nanoscratching scan with an applied normal load of 0.62 μN (calculated using Eq. ( 5), see Table 1). The fast scan direction was orthogonal to the axis of the cantilever and the scan rate

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was 4 μm.s -1 . On the images shown in Fig. 6 (see topography image on Fig. 6a and amplitude image on Fig. 6b) that were obtained using TM-AFM, one can observe that a portion of the cuprous oxide film was indeed modified as a consequence of the scratching test (see selected framed zone on Fig. 6, 4 x 4 μm 2 ). Unlike zones A and B in Fig. 1, the scratched zone appearing on Fig. 6 is not surrounded by scratching dust. By comparison with the unmodified zone of the film, the unique consequence of the scratching test is thus a disappearance of the granular aspect, as observed mainly from Fig. 6b.

As in the cases of the nanoscratching tests carried out on copper and reported above, the AFM silicon tip used to realize and image this scratching test was observed using SEM (see Fig. 7a). This image does not reveal a significant wear of the AFM tip used to scratch cuprous oxide. As in the case of copper, a significant amount of matter was permanently immobilized at the apex of the AFM tip during the nanoscratching tests. The identification of this matter using EDS led to the conclusion that it is mainly cuprous oxide as copper and oxygen were both detected (see Fig. 7b). Chloride anions were not detected, indicating that the scratched zone was mainly constituted of cuprous oxide. The role and behaviour of this cuprous oxide based overtip were observed to be identical to those of the copper overtip observed on Fig. 2a. Gold was obviously detected again as a gold thin film was initially deposited in order to allow SEM and EDS experiments. By using experimental parameters different from those used for the nanoscratching test illustrated in Fig. 6, one can produce damages much more important than those observed on Fig. 6. The nanoscratching test illustrated on Fig. 8 was carried out on cuprous oxide films

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with the help of a silicon tip (see characteristics for tip 3 in Table 1) by carrying out three consecutive nanoscratching scans with an applied normal load of 0.81 μN (calculated using Eq. ( 5), see Table 1). The fast scan direction was orthogonal to the axis of the cantilever and the scan rate was 4 μm.s -1 . On the images shown on Fig. 8 (see topography image on Fig. 8a and amplitude image on Fig. 8b) that were obtained using the tapping mode of AFM, one can observe a hole whose depth is approximately 1,1 μm as a consequence of the scratching test (see framed zone on Fig. 8, 3 x 3 μm 2 ). This illustration shows that, in spite of i) the moderate hardness of silicon tips and ii) the moderate spring constant of the cantilevers, dramatic damages can be produced in the cuprous oxide film up to a stage allowing the AFM tip to reach the underlying copper substrate. The other conclusion of this illustration is that, by comparison to Fig. 8, the experimental conditions used for Fig. 6 are, as concluded above, very close to the set of parameters allowing the minimum damage of the cuprous films investigated in this work. As expected, it also shows the beneficial effect of the number of scratching scans as the normal forces used for the tests 3 and 4 are not so different.

Discussion

In the first step of this work dedicated to the calibration of AFM probes used for nanoscratching tests, an equation including additional terms (see Eq. ( 5)) has been established to calculate the normal spring constant of rectangular cantilevers tilted during their use and presenting a trapezoidal cross section. Advantageously, this calibration procedure based on Eq. ( 5) relies exclusively on a thorough geometrical characterisation of the AFM tip/cantilever systems using the high resolution of FEG-SEM images. The determination of the lateral spring constant of each of the AFM probes was carried out using Eq. ( 6). The values are in good agreement with those generally encountered in literature [START_REF] Giessibl | Chap 4: Noncontact Atomic Force Microscopy and Related Topics[END_REF].

AFM tips accurately calibrated with the help of the procedure discussed above were then used to carry out nanoscratching tests on rough (or technical) surfaces of bulk copper

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(RMS ≈ 38 nm, full z-scale of about 85 nm) with a known applied normal force. Various events occurring during these tests such as local wear of the copper substrate, creation of copper dust immobilized on the substrate and on the AFM tip, as well as a negligible wear of the silicon AFM tip were clearly identified, confirming thus the feasibility of these nanoscratching tests in our experimental conditions. The influence of scan overlap during the nanoscratching, known to lead to additive pressure histories distributed over various locations inside the scratched area of the substrate surface, was already discussed in the results part. A deeper exploration of this important scratching parameter would have required a more specific and larger set of experiments. Nevertheless, it can be concluded from these tests that the nanoscratching test corresponding to zone A in Fig. 1 (five consecutive scans, applied normal load: 0.729 μN) hardly damages bulk copper as it only erases copper ridges initially produced by polishing. Moreover, from the apparent correlation observed between topography and friction data corresponding to the first line of the nanoscratching test carried out in zone A (Figure not shown), it can be deduced that topography influences the friction forces exerted on the AFM tip during nanoscratching tests. This seems to be detrimental to the usual procedure allowing the determination of friction coefficients from nanoscratching tests [START_REF] Chiba | [END_REF][31][32]. Although it appears from this latter that the determination of the friction force or friction coefficient may be a way to deduce the hardness of materials on condition that some of their mechanical parameters are known, the feasibility of the determination of the hardness of materials (copper and cuprous oxide in our case) using AFM nanoscratching experiments is somewhat questionable. Let us emphasize that the hardness of materials seems to be an important criteria in erosion-corrosion tests [6]. In another approach offering a quantitative exploitation of nanoscratching tests [33], Colaço et al. suggested a correlation between the volume of matter and the normal force following:

H F k L V Q N ab k L V ( 14
)

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sliding length (L). Q can also be expressed as a function of k ab , a constant measuring a probability that the AFM tip behaving as an asperity of a surface produces a mass transfer, i.e produces an abrasive wear, during a nanoscratching procedure. In this case, the two other parameters are F N , the normal load applied during the nanoscratching test and H, the hardness of the scratched material. By using Eq. 14 in the case of the second nanoscratching test carried out on copper (see zone B on Figure 1) and H Cu = 880 MPa [34], one can deduce k ab = 4. 26 10 -3 which is a value close to the 0.005-0.05 range usually mentioned in literature for k ab [START_REF] Hutchings | Tribology: Friction and wear of engineering materials[END_REF]. Let us note that k ab is related to a two-body wear situation like those observed in macroscale scratching tests and is therefore a function of the material of the abrasive asperity (or AFM tip in our case). The calculation of V was carried out from the size of the nanoscratched zone (see Figure 1, zone B) and the estimation of the depth of the wearprint using the difference between the average level of the copper surface and that of the bottom of the wearprint. L was calculated with the help of the formula L = 2Nnl in which N is the number of consecutive scans carried out during the nanoscratching test, n is the number of lines per scan in the slow speed direction (512 in our case) and l is the length of one scanline.

After a thorough characterisation using voltammetry as well as XRD and XPS spectroscopies, cuprous oxide films electrodeposited on bulk copper electrodes were also submitted to AFM nanoscratching tests. Again, the feasibility was easily shown but the narrow correlations between topography on one side and deflection and friction on the other side are much weaker even though the roughness and z-scale take values close to those measured on the bulk copper samples. By comparison with the granular aspect of the electrodeposited cuprous oxide film [START_REF] Caballero-Briones | [END_REF], the scratched zone resulting from the softest nanoscratching test (one scan, applied normal load: 0.623 μN, see Fig. 6) appears to be smoothened, indicating a wear threshold situation. The corresponding set of nanoscratching parameters is therefore sufficient to produce only a smoothing of the grainy Cu 2 O surface above its average level whereas the more aggressive nanoscratching test carried out on bulk copper (zone A, Fig. 1) was not so efficient. Although the second test (3 consecutive scans, applied normal load: 0.813 μN, see Fig. 8) carried out on Cu 2 O was only moderately more aggressive than the first one (one scan, applied normal load: 0.623 μN, see Fig. 6) and, more interestingly, much less aggressive than the second one carried out on bulk copper (5 consecutive scans, applied normal load: 1,21 μN, see Fig. 1, zone B), it produced a much more spectacular wear scar (see Fig. 8). This set of comparison strongly suggests that bulk copper is mechanically more resistant to these nanoscratching tests than electrogenerated Cu 2 O thin films. The granular structure of our Cu 2 O thin films is likely to play an important role in the wear mechanism observed during these nanoscratching tests.

By using the quantitative exploitation on the nanoscratching test illustrated on Figure 8 and related to Cu 2 O (see Eq. 14), one can deduce that its hardness is 36 MPa if one suggests that k ab is 0,05 according to the highest boundary value of k ab corresponding to a soft material [START_REF] Hutchings | Tribology: Friction and wear of engineering materials[END_REF]. This low hardness value seems to be too low to represent the real hardness of cuprous oxide. A higher and therefore more acceptable value of hardness would have resulted from a lower removed volume. As this latter can be easily evaluated with a satisfying accuracy from Figure 8, the value extracted for the hardness of Cu 2 O possibly suggests that the nanoscratching test may not have sollicitated the hardness of the cuprous oxide film but rather another fundamental characteristic of this material such as the adhesion between the constituting grains of the material. Indeed, in the particular case of cuprous oxide, the material structure resulting from the electrodeposition process is presumably heterogeneous due to a grainy structure [START_REF] Caballero-Briones | [END_REF]. In other words, one can suggest that nanoscratching tests actually allow appealing to the adhesion forces existing between the grains, whether they are made of nanometric Cu 2 O or CuCl (see Figure 4 and5). It is remarkable that such wear mechanism strongly resembles the one observed during erosion in erosion-corrosion tests whose consequences on the integrity of similar cuprous oxide films were reported elsewhere [6]. If such hypothesis was confirmed, adhesion characteristics of the grainy Cu 2 O materials rather than bulk mechanical properties would be necessary to investigate and predict the mechanism and consequences of erosion observed in erosion-corrosion tests from the material view-point.

It would then be common to erosion and nanoscratching for cuprous oxide whereas such situation appears strongly unlikely on bulk copper where corrosion may play a more important role than erosion. Further experiments are currently underway to better characterize key structural characteristics of our cuprous oxide films such as porosity or grain structure. This set of information led to suggest two different (surface) wear mechanisms resulting rather from the differences in chemical heterogeneity and structure of bulk copper and electrogenerated cuprous oxide thin films rather than from a given bulk mechanical property (such as hardness). This conclusion is crucial for the development of a better understanding of the degradation of these materials in the course of erosion-corrosion tests.

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

LEGENDS Table 1: set of experimental parameters related to scratching procedure or cantilever/tip system for each of the four nanoscratching tests commented in this contribution Fig. 1: a) Topography AFM image obtained using the tapping mode in air of a portion of a copper electrode that includes two zones (A and B) scratched using respectively the sets of parameters N°1 and 2 (see Table 1). b,c,d) vertical profile taken along the corresponding red line drawn on the neighbouring image. that include a portion scratched using the set of parameters N°3 (see Table 1). of a portion of a copper electrode covered with an electrogenerated cuprous oxide thin film that include a portion scratched using the set of parameters N°4 (see Table 1). 98.95 (1) 3100,12 10 0.813 3

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

Each of the AFM tips characterised in this table were imaged using FEG-SEM in order to provide plan view and side view images necessary to measure the cantilever and tip dimensions. Spring constants were calculated with (1) Eq. 5, (2) Eq. 6, (3) Eq. 7.

A

  C C E P T E D M A N U S C R I P Tbearing a photo-detector and the AFM nose adequate for tapping mode AFM (TM-AFM) experiments. A PicoScan 2100 controller connected to a computer was used to drive the scanner and to collect the data generated by the laser impact on the photo-detector. In this purpose, rectangular silicon cantilevers bearing pyramidal silicon tips were used. The resonance frequency and spring constant announced by the AFM tip provider were in the ranges 260-365 kHz and 25-50 N.m -1 respectively. Nanoscratching and subsequent imaging experiments were carried out ex-situ in air using the contact and TM-AFM modes respectively. All AFM images shown hereafter underwent a tilt correction process.

Fig. 1a shows

  Fig.1ashows an AFM topography image obtained in tapping mode of a portion of a

( 11 )

 11 In equation[START_REF] Gnecco | Chap 11: Friction and Wear on the Atomic Scale[END_REF], res is the resolution (or in other words the number of pixels per line or the number of lines per scan) used during the AFM scan corresponding to the nanoscratching test (res = 512 in our experimental conditions). By including our experimental conditions in thisA C C E P T E D M A N U S C R I P T equation, CRLS sscan Lvalues were found to be 4742 nm and 5611 nm respectively for the nanoscratching tests corresponding to zones A and B on Fig.1a(on condition that F N values were calculated from Equation (5)). From the comparison with sscan L , the length of the slow scan axis actually used for these tests (2000 nm for both tests, see Fig.1), one can conclude that calculated CRLS sscan L values were both substantially higher than the sscan L value used for these two nanoscratching tests. This observation implies that there was a systematic overlap between two consecutive line scans during these nanoscratching tests. This situation generated a large set of additive pressure histories distributed over various locations inside the scratched area of the substrate surface under investigation. The influence of scan overlap in defect generation and wear has already been investigated and identified elsewhere[15].Interestingly, the nanoscratching tests represented in Fig.1and implying different scan overlaps could have been compared more accurately by calculating the map of the real pressure history as a function of location on the nanoscratched areas A and B shown in Fig. 1. Such comparison is beyond the purpose of this work as more nanoscratching tests involving other values for yet more important key-parameters, such as normal force or number of consecutive scans for example, would have been necessary first. At this stage of our experiments, this contribution is rather focused on the feasibility of such nanoscratching tests and the comparison of the behaviours of bulk copper and thin films of electrogenerated cuprous oxide during these tests and not on the effect of scan overlap on these behaviours. Electrodeposition and characterization of Cu 2 O oxide films on copper In a first step, typical Cu 2 O films generated using the experimental electrochemical conditions reported in the experimental part were characterized using linear voltammetry in the cathodic potential range. A typical voltammogram obtained on a copper electrode modified with a cuprous oxide film is shown on Fig. 3 (see continuous line). Unlike the one A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 13 obtained in similar experimental conditions at a freshly polished copper electrode (see dashed line on Fig. 3), it clearly shows a well defined cathodic peak situated at -1.3 V vs MSE. In good agreement with literature [19-21], this peak is attributed undoubtedly to the electrochemical reduction of Cu 2 O following the reaction: Cu 2 O + H 2 O + 2 e -→ 2 Cu + 2 OH -

  5 eV and corresponding to Cu 2p 3/2 and Cu 2p 1/2 respectively, indicating thus the absence of Cu(II) (see Fig. 5a). Presence of Cu(I) was nevertheless confirmed on the basis of the Cu LMM spectra corresponding either to metallic copper or to the electrogenerated films (see Fig. 5b). The Auger peak observed for the metallic copper is indeed significantly shifted towards lower kinetic energy (917 eV for Cu instead of 919 eV for Cu 2 O). Simultaneously it undergoes a real change in its shape by comparison to the one observed on cuprous oxide coatings. The presence of Cu 2 O was also confirmed with the help of a conclusive interpretation of XPS A C C E P T E D M A N U S C R I P T spectrum in the O1s region, as this latter clearly showed a peak at 530.9 eV, which is a characteristic of cuprite (spectrum not shown) [24b]. Peaks, such as the Cl2p photopeak, clearly indicate the presence of chloride anions, these latter being obviously bound to copper (I) since no sodium was detected. A quantification attempt provides the following composition: 85% at. Cu 2 O and 15% at. CuCl.

2

 2 Calculations leading to the contact radius and CRLS sscan L using Eq. (9-11) respectively were not realised as they both necessitate the knowledge of Cu , (Young's modulus and Poisson's ratio for cuprous oxide respectively), two characteristics that are unknown for our electrogenerated films of Cu 2 O.

  The accurate determination of the normal forces used during the nanoscratching tests reported in this work also provides a conclusive qualitative comparison. Concerning the nanoscratching tests carried out on copper, it appears from the comparison of zones A and B shown on Fig.1that the set of experimental conditions beyond which copper can be efficiently scratched in our experimental conditions is actually the one corresponding to zone A (2 x 2 μm 2 , 5 consecutives scans, . 5 and 7)). In parallel, the nanoscratching test carried out on cuprous oxide and illustrated in Fig.6allows the determination of the wear threshold on cuprous oxide films (4 x 4 μm 2 , 1 scan, . 5 and 7). As a consequence, both experiments can be advantageously compared. At this stage, one can simply deduce that copper is mechanically more resistant towards nanoscratching tests than cuprous oxide, which may again reveal two different wear mechanisms resulting from two different structures, rather than various bulk properties (hardness for example). This result is already quite significant but only qualitative as it is not based on the comparison of the Young's moduli of the two materials involved.ConclusionIn this contribution, nanoscratching tests carried out in air using AFM on technical bulk copper samples (or hand-polished samples, to be differentiated from theoretical (or model) samples such as monocrystals, atomically flat surfaces, …), either bare or covered with electrogenerated cuprous oxide films are reported. The feasibility of such tests is confirmed with the help of i) the TM-AFM imaging of wear prints and ii) FEG-SEM imaging and EDS analysis of AFM tips. Their quantitative exploitation is delicate as a consequence of the significant roughness of the targeted samples (z-scale 100-200 nm) and its well known effects on friction coefficient measurements. Nevertheless, a qualitative comparison of these nanoscratching tests was made possible thanks to an accurate determination of the applied normal forces based on an accurate calibration procedure of the cantilever/tip systems employed. It allows concluding that dense bulk copper samples are mechanically more resistant than granular cuprous oxide films electrodeposited on copper towards AFM nanoscratching tests. In a more quantitative exploitation of these tests, a model extracted literature allowed us to deduce an abnormally low value of the hardness for cuprous oxide.

Fig. 2 :

 2 Fig. 2: a) SEM image of the AFM silicon tip used to scratch zones A and B in the contact

Fig. 3 :Fig. 4 :Fig. 5 :Fig. 6 :

 3456 Fig.3: Linear voltammograms obtained at a copper electrode (dashed line) and at a copper

Fig. 7 :

 7 Fig. 7: a) SEM image of the AFM silicon tip used to scratch a portion of a Cu 2 O film in the

  Figure Tip k N (N.m -1 )

FIGURES

  FIGURES

- 2 ,

 2 Fig. 4

  Fig.5

  Fig.6

ACKNOWLEDGMENTS S. Borensztajn and F. Pillier are warmly acknowledged for SEM imaging and EDS experiments. LC acknowledges the financial support of the "TASSILI" programs with Algeria (N° 03 MDU 572 and 06 MDU 686).

A C C E P T E D

M A N U S C R I P T


Thi Kim

Claude Gabrielli

Alain Pailleret

Hubert Perrot

L T T Kim

Correlation between ion-exchange properties and swelling/shrinking processes in hexasulfonated calix[6]arene doped polypyrrole films : ac-electrogravimetry and electrochemical atomic force microscopy investigations

Correlation between ion-exchange properties and

swelling/shrinking processes in hexasulfonated calix[6]arene doped polypyrrole films: ac-electrogravimetry and electrochemical atomic force microscopy investigations

Introduction

Calixarenes are metacyclophanes produced from the condensation of phenol with formaldehyde [START_REF] Asfari | calixarenes[END_REF][START_REF] Vicens | Calixarenes in the Nanoworld[END_REF]. As they are poorly soluble in water, the necessity of appending ionic functional groups, such as carboxylic acid (-COOH) or sulfonic acid (-SO 3 H) groups at the *Revised Manuscript (including Abstract) Click here to view linked References para-position of the phenolic units has been reported in literature (see [START_REF] Shinkai | [END_REF]4] and Chapter 24 in [START_REF] Asfari | calixarenes[END_REF]). Sulfonated calix [n]arenes are consequently versatile water-soluble binding agents able to bind with a significant selectivity a large set of interesting species, whether these latter are molecular or mono-atomic, ionic or neutral, and organic, inorganic or biological [5]. In the case of hexasulfonated calix [6]arenes (C6S) employed in this contribution, let us cite as possible targeted analytes in a non-exhaustive list, UO 2 2+ and some transition metal (Ni 2+ , Zn 2+ , Cu 2+ , Fe 3+ ) [6,7] and lanthanide [8] cations, ferrocene and cobaltocenium derivatives [9][10][11][12], quaternary ammonium cations [13], cationic surfactants [14,15], neutral molecules [16],

as well as proteins (such as Bovine Serum Albumine (BSA)), amino-acids, oligo-peptides, drugs, and other small bioactive molecules [17][18] and even iron oxide nanoparticules [19].

Among the various applications of these versatile complexation behaviours, the elaboration of calixarene based electrochemical sensors has been thoroughly investigated although it required the exploration of strategies allowing the immobilisation of sulfonated calixarenes on classical working electrode surfaces. One way to achieve this goal consists in using these ionic calixarenes as doping anions in electronically conducting polymer matrices (films or colloids) such as polypyrrole [20][21][22], poly-(ethylene-dioxy-thiophene) (PEDOT) [23] or polyaniline [24]. The elaboration of electrochemical sensors based on this strategy will succeed only on condition that the calixarene based dopants conserve the binding character and the selectivity they display in solution once they are incorporated in the polymer matrix, which was shown to be achieved for C6S doped polypyrrole films with a targeted analyte such as uranyle cations [22]. Interestingly, the success of this strategy will also rely on the ion exchange properties of the resulting C6S doped conducting polymer films, which in turn depend, for a part, on the structural characteristics of these films possibly at the nanometric or molecular scale. Let us notice here that information on the structural characterisation is poor in literature in the case of electrodeposited electronically conducting polymers (ECP) films.

The purpose of this contribution was first to develop a deep understanding of ion (and potentially solvent) exchanges taking place in electrodeposited C6S doped polypyrrole (PPy) films during electrochemically controlled doping/undoping using ac-electrogravimetry experiments based on a well-established procedure.

Our second intention was to identify the electromechanical (swelling/shrinking) behaviour resulting presumably from these ions and solvent exchanges in our PPy/C6S films.

A few research groups have indeed developed strategies allowing the accurate in-situ measurement of these volume (and topographical) changes under electrochemical control of the doping state of the polymer film either at locale or global scale [25][26][27][28][29][30][31][32][33][34]. In this contribution, we used electrochemical atomic force microscopy (also referred to as EC-AFM),

i.e. AFM in an electrolytic solution allowing an electrochemical conditioning of the substrate (C6S doped PPy films in this contribution).

Experimental part

Electrochemical deposition of PPy/C6S thin films

Hexasulfonated calix [6]arene doped polypyrrole (PPy/C6S) thin films were electrochemically deposited from aqueous solutions containing the pyrrole monomer (0,1 M) and hexasulfonated calix [6]arene (C6S, 1,66 10 -3 M, see its structure on scheme 1). Such electrolytic solution was always prepared immediately prior the electropolymerisation was launched so as to avoid the formation of insulating polypyrrole particles via an autopolymerisation mechanism reported in literature [35]. The six sulfonic acid groups all possess a same and very low pKa value which was suggested to be below 1, as reported in [36]. On the other hand, the acidic power of the six phenolic protons (OH groups) is measured by different pKa values revealing intramolecular hydrogen bonds resulting from the removal of one or several of these phenolic protons. As a poly-acid, C6S was shown to possess only two ionisable OH groups in the pH range 2,5-7 (pKa 1 = 3,37-3,45 and pKa 2 = 4,76-5,02) whereas the remaining four phenol groups are only weakly acidic (pKa 3 > 11) [36][37][38]. As a consequence, the pH of our electrolytic solutions used for the electrodeposition step is expected to be 2, which is largely above the pKa values of the sulfonic acid groups and below that of the phenolic protons. C6S is thus expected to be hexa-anionic in this electrolytic solution. In spite of the low ionic strength of the resulting electrodeposition solution, no other background salt was added in the electrodeposition solution so as to make sure that the produced PPy films were doped with hexasulfonated calixarenes only, and display therefore ion exchange properties resulting only from the incorporation of those large anions during the electrodeposition step. The electrochemical technique used for the electrodeposition of these films was cyclic voltammetry. The potential range chosen for this electrodeposition step extends from -1 V to 0,4 V vs an aqueous K 2 SO 4 saturated mercurous sulphate (SSE) reference electrode so as to avoid the electrochemical oxidation (and subsequent degradation) of the hexasulfonated calix [6]arene anions as reported in literature [39][40][41]. PPy films dedicated to EC-AFM investigations were electrodeposited on a working electrode made of a thin layer of platinum (thickness: 60 nm, RMS : 2,5 nm, area: 0,126 cm 2 ) deposited on mica through a mask using the sputtering technique. Those dedicated to ac-electrogravimetry (and cyclic electrogravimetry) investigations were electrogenerated on a quartz bearing a key shaped gold electrode on each of its two faces. A typical voltammogram corresponding to the electrodeposition of these films in these experimental conditions is shown on Figure 1. SEM-FEG imaging of the profile of the obtained film reveals a thickness close to 500 nm. This is only an estimation as it was measured in vacuum conditions required by SEM-FEG imaging.

As a consequence, it is definitely not the thickness of a fully water-loaded and hydrated film.

Ac-electrogravimetry investigations

The experimental procedure and setup used to carry out the ac-electrogravimetry experiments have been reported elsewhere in previous publications of our group [42][43][44][START_REF] Gabrielli | Theoretical studies of Electrogravimetry in thin Electroactive Films In Modern Aspects of Electrochemistry[END_REF]. The quartz crystals used in the present investigations have a base frequency of 9 MHz.

EC-AFM measurements

EC-AFM experiments required a home made electrochemical cell in which the working electrode bearing the PPy film lies flat in the back of the cell whereas a platinum grid and a silver wire covered with an electrogenerated silver chloride film are used respectively as counter-and Ag/AgCl reference electrodes respectively. A SP300 potentiostat (Bio-Logic, Claix, France) is connected simultaneously to i) this electrochemical cell so as to apply various potential conditionings and to read the resulting current on the working electrode and ii) the AFM controller (Picoscan 2100, Agilent, USA) so as to allow this latter to read these two signals at the acquisition frequency of the data related to proper AFM imaging. This was also necessary so as to insure a perfect synchronization of the acquisition of height, potential and current data. AFM imaging was performed in-situ in a transfer electrolytic solution (0,1 M KNO 3 aqueous solution) in the contact mode on a 1 nm 2 area using triangular AFM cantilevers (PNP-TR, NanoWorld, Paris, France) characterised by a low spring constant (k N 0,08 N m -1 ). A 1 nm 2 scanned area was chosen as it is expected to be much smaller than the contact area between the tip and the polymer sample. This choice led in a first approximation to a situation where the tip can be considered immobile in the x-y plane of the sample, and behaves therefore as a profilometer measuring the variation of the thickness of the film (and not the thickness itself) regardless of the local roughness and roughness changes also expected to occur in our experimental conditions on the investigated samples. The scan rate of the AFM tip was chosen in the range 0,5 to 2 Hz in order to provide an imaging duration longer than that required for the corresponding potential conditioning (consecutive cyclic voltammetry scans or consecutive potential steps for example) applied on the polymer film.

Results

Cyclic electrogravimetry

After transfer in a KNO 3 aqueous electrolytic solution, PPy/C6S films electrodeposited in the experimental conditions described above can be electrochemically and reversibly doped and undoped using cyclic voltammetry, as revealed respectively by the oxidation and reduction waves appearing on the resulting voltammogram (see Figure 2a).

Cyclic voltamassograms obtained simultaneously from cyclic electrogravimetry experiments

show that the oxidation/reduction of these films leads to substantial loss/gain of mass (see Figure 2b). By combining current and mass measurements, the atomic weight of the charged species, m i , involved in the electrochemical process can be easily estimated by using the following equation : i dm dm 1 m F F dq dt i . Following the potential scan, the atomic weight determined for the exchanged species varies between 40 g mole -1 and 0 g mole -1 without reaching any stable values. It indicates certainly a mixed contribution of cations without excluding the participation of the solvent and/or the anions. This point underlines the limitation of the cyclic electrogravimetry technique where only one scan rate was used for a large exploration of the potential. Furthermore, the molar mass of the exchanged species was not constant over the whole potential range, as shown from Figure 2c. These mass variations were therefore not quantitatively exploited here as ac-electrogravimetry experiments reported hereafter are much more powerful to establish a comprehensive understanding of ions and solvent exchanges taking place during doping/undoping processes. Both cyclic voltammograms and cyclic voltamassograms were found to be stable for a film stored in an aqueous KNO 3 solution during three days (see Figure 2a-b).

Ac-electrogravimetry

Theory

In this approach, where the PPy/C6S film is coated on the gold electrode of the quartz crystal, charge transfer at the electrode/film interface, mass transport in the solution and in the film are considered to be fast compared to the charge transfer at the film/solution interface, which is considered as the rate limiting step. This hypothesis is justified for thin films where, in addition, the quartz crystal is working in the gravimetric mode (i.e. no viscoelastic distortion occurs). For the thin films used in this work, EIS spectra did not show any 45° slope, which demonstrates that diffusion is not a limiting step [START_REF][END_REF]. Here, only one cation (solvated or not), one anion, and one free solvent, i.e. solvent not involved in the solvation shell of an ion, are considered, for monocharged ions, to be exchanged with the solution to compensate the charge displacement occurring during oxidation or reduction of the electroactive film to reach electroneutrality in the film.

The doping mechanism of an electroactive film can be described simply by:

M , P M e P ' c c k k (1) and e A , P A P a ' a k k (2)
where <P> is the host electroactive film, <P,M + >, et <P,A -> the inserted cation and anion in the film matrix. Therefore, the net instantaneous molar flux of species i (c, a or s) is i J (mol cm -2 s - 1

) and it can be obtained by applying the classical kinetic laws for heterogenous reactions. The flux of the species C i is equal for each ions to:

sol i i i i i i i i f i C C C k C C k dt dC d d J ) ( ' ) ( ) ( max min (3)
where d f is the thickness of the film and 0 i dC dt for inserted species, i max C , is the maximum concentration of the sites available for insertion, i min C is the minimum concentration of the sites occupied by the species in the host film, i sol C is the concentration of species i in the solution and

' i i k and k
are the kinetic rate constants of transfers, according to the classical Tafel laws

0 0 i i i i k k exp b E E and 0 0 i i i i k k exp b E E .
Therefore, the fluxes of the involved species i (c, a or s), i J , are positive for outgoing species:

i J > 0 for x > 0 i = a, c, s (4)
• Concerning the solvent movement, the following solvation model is considered [

s i s s i s s dC k C C k C C dt (7)
Then, the kinetic equations for the ions have the same form than those of ions.

In dynamic regime, when a small sine wave potential change, E, is imposed across the metal/film/electrolyte interfaces, low amplitude sine wave concentrations, C i , and fluxes, J i , are observed at the film/solution interface (x = d f ), such as:

E G d C K d C d j d J i f i i f i f f i ) ( ) ( ) ( where i = c, a, s (8)
As from Eq. (

' i i i i im i n i im a x i G bk (C -C )-bk (C -C ) (9) 3) where ' i
and,

' i i i i s o l K k k C ( 10
)
where G i < 0 for inserting species and G i > 0 for expelling species.

Then, according to equ. (8) :

i f i i K d j G E C ) ( (11)
Thus, the The charge variation q is related to the Faradaic current density change, F I j q , and by using eq. ( 8) it comes: (13) where m c , m a and m s are the atomic masses of the cations, anions, and solvent respectively. Therefore, the electrochemical impedance, E ( ) I , which is measured, is equal to:

) ( ) ( ) ( 1 E C E C d E I j a c f F (12) and ) ( ) ( ) ( ) ( E C m E C m E C m d E m s s a a c C f
1

F dl E ( ) I I j C ( ) E (14)
where dl C is the double layer capacitance, the electrolyte resistance being neglected here.

By using equ. ( 12) one obtains:

a f a c f c f dl K d j G K d j G d j C j I E 1 ) ( (15)
and

s f s s a f a a c f c C f K d j G m K d j G m K d j G m d E m ) ( (16)
Of course, only the charged species, anions and cations, are implied in the electrochemical impedance, E ( ) I , whereas the electrogravimetric transfer function, m ( ) E , depends on both cations, anions, and solvent all together.

Another interesting quantity can be calculated to discriminate the influence of the various species:

The electric charge/potential transfer function, q ( ) E , according to equ. ( 12) :

1 F I q ( ) ( ) E j E ( 17
) ) ( ) ( E C E C d a c f (18) a f a c f c f K d j G K d j G d (19)

Experiments

Simultaneous measurements of the electrochemical impedance E ( ) I and of the electrogravimetric transfer function m ( ) E were performed by using a technique already described elsewhere [42][43][44][START_REF] Gabrielli | Theoretical studies of Electrogravimetry in thin Electroactive Films In Modern Aspects of Electrochemistry[END_REF]. Figure 3 shows examples of the quantities of interest at various potentials, ) ( E m

, together with the q ( ) E transfer function calculated from ) ( I E and using Eq. (17). By fitting the experimental data with Eqs. ( 16) and ( 19), both the kinetic parameters i K and i G , and the atomic masses of the ions and solvent , m i , involved in the charge compensation process were obtained. Figure 4a andb show the change of the K i and i i G / K ratios with respect to the potential. Concerning the atomic masses, values of 62 g mol - 1 and 18 g mol -1 were obtained for the anions and solvent, respectively, which shows that nitrate ions and naturally water are involved. However, for the positive charges, an apparent atomic mass which varies with the potential was obtained (Figure 5). This demonstrates that the positive charges exchanged are mainly potassium in the cathodic potential domain whereas it is the solvated proton (hydronium H 3 O + ) in the anodic range. From these data, two types of quantities can be deduced. First, as from Eq. 0

i i i C G ( ) E K
, the change of the concentrations of the species in the film with respect to the potential i C E can be obtained by integrating the values of i i G E K from Fig. 4b with respect to the potential from -0,6 V to 0,2 V vs SCE. Figure 6 shows that it is mainly the cations which are involved in the charge compensation: a large quantity of cations are expelled when the potential increases.

Concomitantly, as shown in [START_REF][END_REF], some free water is also expelled with the cations whereas nitrate ions are inserted in the film when the potential increases, but in relatively small quantity compared to the cations.

So, knowing the total concentration of cations, and the relative quantity of potassium and hydronium ions (Figure 5) the concentrations of these two cations released from the film are obtained with respect to the potential. Figure 6 shows that, starting in the cathodic potential range, potassium ions are first expelled. When the potential increases hydronium ions begin to be expelled and then for larger potentials they constitute the major part of the positive species exchanged up to the anodic potentials where they are practically the only ions expelled.

EC-AFM

The variation of thickness occurring in ECPs during doping/undoping processes can be measured with a nanometric resolution using EC-AFM. Advantageously, this methodology resulting from the coupling of in-situ AFM in the contact mode with an electrochemical technique applied on the investigated samples is yet more promising due to the wide choice of electrochemical techniques that can be selected (cyclic voltammetry or chronoamperometry for example). Figure 7 shows the dynamics of the thickness variation of a PPy/C6S film during a series of consecutive potential steps applied vs an Ag/AgCl reference electrode. This potential conditioning is made of a series of four consecutive potential steps (-0,3 V, -0,7 V, 0,4 V, -0,7 V) whose duration was 30 seconds (see thin line on Figure 7d). This series of potential steps was repeated five times. -0,7 V and 0,4 V are two potential values encompassing the doping/undoping process of the film whereas -0,3 V is a potential value corresponding to the transition of identity (K + instead of H 3 O + ) observed for the exchanged cations. Figures 7a, 7b 7d, one can first conclude that there is an anticorrelation between the thickness of the film and the potential, and thus between its thickness and its oxidation state.

More precisely, the more the film is oxidised, the thinner it gets. Interestingly, the thickening dynamics almost reaches a steady state over the 30 sec duration of each of the potential steps from -0,3 V to -0,7 V whereas during the large oxidation step from 0,4 V to -0,7 V, the steady state is not reached over the same duration. The lowest and highest thicknesses were observed for the most oxidising (+0,4 V) and reducing potentials (-0,7 V) respectively. The maximum thickness increase observed after reduction of an oxidised film over a 30 sec duration is approximately 500 nm, which would correspond to a 100 % variation of the thickness measured for the dry film using SEM. This 100 % value is likely to be an overestimation as the thickness measured for this film (in its reduced state) using SEM-FEG is likely to be underestimated. Indeed, one can easily admit that such films will substantially thicken upon water uptake when transferred from primary vacuum to an electrolytic aqueous solution, regardless of the doping/undoping processes regulated by a potential perturbation. The fact that the -0,4 V potential value corresponding to the transition of the exchanged cations (from K + to H 3 O + ) led to an intermediate thickness value indicates that the film keeps thickening as it goes from the oxidised state to the reduced state whatever the identity of the incorporated cations is (K + or H 3 O + ). The thickness variations observed on Figure 7d appear to be fairly reversible over five consecutive scans, indicating a good stability for the electromechanical behaviour of this film. One must also notice from Fig. 7d that for a potential step from -0,7 V to 0,4 V, the thickness of the PPy film decreases during the expulsion of cations before it starts increasing again possibly due to the insertion of anions [START_REF][END_REF].

Figures 8a, 8b and 8c have the same meaning as Figures 7a, 7b and 7c although they correspond to another potential conditioning of the film. As figure 7d, Figures 8d-e show the variation of the applied potential and of the thickness (these data were simply split into two graphs so as to facilitate the reading of the curves). The potential conditioning consists in five consecutive cyclic voltammetry procedures including each five consecutive scans at three different scan rates. This therefore leads to a conditioning made of 15 consecutive potential scans carried out over the adequate potential range (-0,7 V to 0,4 V vs Ag/AgCl). In the chronological order, the scan rates were 200, 100 (see Fig. 8d) and 50 mV s -1 (see Fig. 8e).

One can first notice that a reduction or an oxidation of the film following a linear potential scan produce respectively a linear increase (swelling) or a linear decrease (shrinking) of its thickness (see mainly Figure 8d-e). Moreover, these thickness variations seem to be constant and reversible in a first approximation and also independent from the scan rate. For a very slow scan rate such as 10 mV s -1 (Figure not shown), the variation of the thickness over a potential ramp is lower and seems to follow a more complex law made of two variation regimes separated by a short inversion of the direction of variation (.i.e two increase regimes separated by a short decrease for example, see Figure 8f)). The potential at which this separation occurs seems to correspond to the potential range in which the transition of the identity of the exchanged cations was observed from ac-electrogravimetry.

By plotting the thickness variation as a function of the applied potential for five consecutive scans at a given potential scan rate, one can establish new graphes showing swelling/shrinking cycles over the potential range used for these experiments. As an example, Figures 9a and9b show the resulting graphes for 100 and 20 mV s -1 scan rates respectively.

By analogy with cyclic voltamperograms or cyclic voltamassograms that show respectively the variation of current (in amperes) and mass (in grams) as a function of the potential, Figures 9a and9b show voltathicknograms as they show the variation of thickness (in nanometers) as a function of the applied potential. Both of them show again a swelling of the film upon reduction and its shrinking upon oxidation, as already pointed out from Figure 8a-f but moreover they show an hysteresis loop comparable to that observed on cyclic voltamassograms, indicating that swelling and shrinking processes do not follow the same electromechanical path.

Discussion

One can reasonably expect that electrodeposited films of electronically conducting polymers undergo a variation of their thickness (swelling/shrinking) as a consequence of the ion exchanges constituting the electrochemically driven doping/undoping processes. Let us emphasise that exchanges of either free or solvating water molecules may also be observed at the film/electrolytic solution interface, with or without a directional and/or quantitative correlation with the ion exchanges. The sum of these solvent and ion exchanges is likely to result in a volume change of the films, although this latter may well depend on other simultaneous phenomena. Indeed, volume (and thus thickness) variations may also be influenced by changes of conjugation lengths produced by changes of the oxidation state of the polymer chains, or modifications of relative orientations of monomer molecules in the polymer chains due to changes of the conformational flexibility inside these latter. A variation of the hydrophilic character of polymer chains as a function of their oxidation state may also alter significantly chemical interactions between them and their subsequent stacking.

Unfortunately, the consequence of this list of parameters and phenomena producing swelling/shrinking phases makes their extent hardly predictable. Obviously, one should also keep in mind that the identity of the solvent and background salts used during the electrodeposition step and during the electrochemical doping/dedoping processes will bring another contribution, yet often unpredictable, on these swelling/shrinking processes due to their role on the interactions between the chains and their packing. From the conclusions of our experimental results obtained with the ac-electrogravimetry technique, it appears that the insertion of the cations occurs first, followed by that, slower, of free water molecules, during the reduction step from -0,3 V to -0,7 V and from 0,4 V to -0,7 V vs Ag/AgCl. This fast redox process involving in parallel cation exchanges, slower as deduced from Figure 3b, is represented by the top reaction appearing on scheme 2 [48][49][50]. This insertion of cations is clearly correlated with a thickening of the film (see Figure 7d). This is also particularly well illustrated by the obvious similarities existing between the voltamassogramms and voltathicknogramms shown respectively on Figures 2b and9a-b. All show hysteresis loops with the same orientation and the same clockwise rotation with the applied potential.

Concerning the oxidation step from -0,7 V to 0,4 V and from -0,7 V to -0,3 V, where cations and water are expelled, the film thickness decreases. When the film shrinks, it can be assumed that the porosity of the film decreases as well. This can explain why hydronium ions, which are smaller [51], are substituted to potassium ions to be the relevant ions that compensate the electric charge when the potential increases [52].

On the other side, during the reduction step from -0,3 V to -0,7 V, the cations, mainly potassium, are rapidly exchanged, which allows a steady state to be reached with a relatively short time constant of a few tens of seconds. On the opposite, during the oxidation step from -0,7 V to 0,4 V, the cations, mainly potassium first and then mainly hydronium, are expelled in a first step, which leads to a fast decrease of the film thickness, but after some time an increase of this latter is observed. Two origins can be envisaged for this tendency, either some nitrate anions which are slower than cations and water, as shown from Figure 3b, but much heavier than hydronium ions, may be inserted in the film or this slow thickening of the film may be due to some reorganization, or relaxation, of the film.

The exchange of anions (nitrate ions) revealed by the ac-electrogravimetry experiments can hardly be understood straightforward and it is obvious that the top reaction reported on scheme 2 does not allow to explain it. The ability of hexasulfonated calix [6]arenes to bind potassium cations can provide a precious help for this purpose. The vertical reactions on the left and right hand sides of scheme 2 reflect the complexation/decomplexation of potassium cations respectively in the oxidised and reduced forms of C6S doped polypyrrole films. Indeed, C6S may well bind, only weakly, potassium cations as i) the ionic radius of these latter allows them to fit in the cavity offered by C6S, and ii) para-sulfonated phenol units of sulfonated calixarenes are known to participate in cationinteractions with alkaline metal cations [5]. In our polypyrrole films, potassium cations are exchanged in their unsolvated form (molar mass = 39 g mol -1 ), which may substantially improve their affinity for complexation by C6S although this latter depends also on the availability of C6S anions for this role once they are trapped inside polypyrrole films. In spite of that, one can take as a reasonable hypothesis that complexation of unsolvated potassium cations by C6S indeed occurs in these films, whether PPy/C6S films are in the oxidised or reduced state. It must then be noticed that the reduced form of C6S doped PPy films (see the PPy/C6S form in the bottom right corner of scheme 2) includes two different forms of potassium cations, i.e. free potassium cations available for transfer at the film/electrolyte interface and potassium cations strongly bound in the cavity of sulfonated calix [6]arenes. As a consequence, soaking of our PPy/C6S films electrodeposited in the reduced form from acidic solutions in aqueous potassium nitrate solutions would lead to the complexation of potassium cations in the C6S cavities, and thus to the incorporation of nitrate ions simultaneously for the need of electroneutrality. The electrochemical oxidation of the films would lead to an excess of cationic charges in the films by comparison with the amount of anionic charges provided by the irreversibly trapped C6S anions. The respect of the electroneutrality of the film would then require the incorporation of nitrate anions from the transfer electrolytic solution. This mechanism and the bottom reaction in scheme 2 are indeed advantageous as they provide a convincing explanation for the exchange of cations (potassium or hydronium cations) and nitrate ions in opposite directions, identified from our ac-electrogravimetry observations.

Conclusion

Electrodeposited polypyrrole films doped with hexasulfonated calix [6]arenes (C6S)

were investigated in potassium nitrate aqueous solutions using cyclic electrogravimetry, acelectrogravimetry and electrochemical atomic force microscopy (EC-AFM). In the course of electrochemically driven doping/undoping processes, the two former techniques provided an accurate identification of ion and free solvent exchanges occurring in these films whereas the latter one allowed in-situ characterisation of swelling/shrinking processes.

The investigated films were found to be mainly cation exchangers, as a result of the irreversible incorporation of bulky C6S anions in the PPy matrix during the electrodeposition step. The identity of these cations (K + or H 3 O + ) depends on the potential inside the potential range encompassing the doping/undoping reactions. Free solvent (water) molecules are also exchanged simultaneously, in much lower amounts, in the same direction than cations. Nitrate anions were found to be exchanged, but in an opposite direction, as they penetrate inside the films upon oxidation and are expulsed out of it upon reduction. This minor anion exchanger character is possibly due to the complexation of potassium cations inside the trapped C6S cavities whether the polymeric matrix is in an oxidised or reduced state.

EC-AFM investigations were carried out by using the AFM tip as a profilometer during oxidation/reduction cycles driven either by consecutive potential steps or by cyclic voltammetry scans. They showed that the expulsion of cations and free solvent molecules upon oxidation implies an important shrinking of the films whereas their insertion upon reduction leads to an equally important swelling. These thickness variations appear to be reversible, independent of the potential scan rate and of the order of magnitude of the initial thickness (probably underestimated as it was measured in the dry state using SEM-FEG).

More importantly, an obvious correlation between mass and thickness variations was established from the striking similarities observed between cyclic voltamassograms and cyclic voltathicknograms.

These observations open new perspectives, not only for the applications of PPy/C6S

films, but also and mainly for the development of new methodologies allowing a better evaluation and understanding of ion insertion in conducting polymers and more widely in insertion electroactive materials.

Scheme 1: Molecular structure of hexasulfonated calix [6]arene (C6S)

  and 7c are EC-AFM images showing respectively the variation of height, potential (with its sign inverted) and current measured on the PPy/C6S film. Let us notice that the bottom half of these images is featureless because it corresponds to a time range that followed the end of the potential conditioning applied on the film. The top half of these three images all show horizontal stripes, indicating that there is a correlation (or anticorrelation) between the height (and therefore film thickness), potential and current. On the potential image, each stripe corresponds to a potential step and each color change between two consecutive stripes results from a transition between two consecutive potential steps. Similar features are observed on the height image, indicating that height follows variations similar with those undergone by the potential applied on the working electrode/sample. Even though current image also shows stripes, small color variations can be observed inside individual stripes. They represent slight current variations occurring during potential steps that are nevertheless very weak by comparison with those occurring during potential transitions. Due to the size of the scanned area (1 nm 2 ) and knowing that the potential and current are relative to the whole film, Figures 7a, 7b and 7c should rather be considered as images showing the variation of these three parameters (height, potential, current) as a function of time. A better representation of these variations is thus given by graphs plotted as a function of time, as shown on Figure 7d. The current variation i= f(t) was not plotted for the need of clarity. By comparing Figures 7a-b between them or by comparing the two curves plotted on Figure

Figure 1

 1 Scheme 1

Figure 2 :Figure 2

 22 Figure 2 : a) typical cyclic voltammograms, b) corresponding cyclic voltamassograms obtained after transfer of the PPy/C6S film resulting from Figure 1 in a KNO 3 aqueous electrolytic solution (0,1 M) for different aging durations of the film in this solution. c) relationship between mass and charge over the whole potential range after a 16 hours aging duration. WE: golden quartz covered with a PPy/C6S film. CE : Pt. Ref : SCE. V = 100 mV s - 1 .

Figure 3 :

 3 Figure 3: a) charge-potentiel transfer function and b) mass-potentiel transfer function at two different potentials, -0,35 V vs SCE and +0,1 V vs SCE, in 0,1 M KNO 3 aqueous solution.

Figure 3

 3 Figure 3

Figure 4 :

 4 Figure 4: Changes of a) K i and b) i

Figure 4

 4 Figure 4

Figure 5 :

 5 Figure 5 : Apparent atomic mass of the exchanged cations

Figure 5

 5 Figure 5

Figure 6 :

 6 Figure 6: Variations of the concentrations of the expelled potassium and hydronium cations with respect to the potential.

Figure 6

 6 Figure 6

Figure 7 :Figure 8

 78 Figure 7 : a) Height, b) potential (with its sign inverted), and c) current images obtained by EC-AFM on a PPy/C6S film. Scan size: 1 nm 2 . d) plot of height (bold line) and applied potential vs a Ag/AgCl reference electrode, extracted respectively from a) and b), as a function of time. The electrochemical conditioning is made of consecutive potential steps.

Figure 8

 8 Figure 8

Figure 9 :Figure 9 [Scheme 2 :

 992 Figure 9 : Cyclic voltathicknogramms (5 consecutive scans) plotted from data represented on Figure 8 for a PPy/C6S film. a) 100 mV s -1 , b) 20 mV s -1 .


Phosphonate PEG Copolymers to control the USPIO stealthiness

G. Ramniceanu 2 , B-T Doan 2 , C. Vezignol 1,2 , A. Graillot 4 * Corresponding author Nathalie.Mignet@parisdescartes.fr; Jean-Francois.Berret@univ-paris-diderot.fr

Abstract -For nanomedicine applications, there is a need to design advanced and cost-effective coatings, resistant to protein adsorption leading to increased biodistribution in vivo. In this study, phosphonate poly(ethylene glycol) copolymers were synthesized and used to coat 6nm and 13nm iron oxide particle. In vitro, PEGylated particles exhibit exceptional stability and low cellular uptake, of the order of 100 femtograms of iron per cell. In vivo, we developed an in vivo Dynamic Susceptibility Weighted MRI (7T) protocol to monitor the hepatic capture and clearance of the particles injected to mice as a function of the time. PEGylated coat of molecular weight 2000 gmol -1 show a 2hrs delay to liver uptake as compared to the commercially available USPIO Cliavist, indicating a direct correlation between the surface properties of the contrast agents and their stealthiness.

Index terms-Magnetic Resonance Imaging, Nanomedicine, Contrast agents USPIO, PEG, polymer coating.

I. INTRODUCTION

Cancer is the second leading cause of premature deaths in Europe, after cardiovascular diseases [START_REF] Murray | Alternative projections of mortality and disability by cause 1990-2020: Global burden of disease study[END_REF]. Magnetic Resonance Imaging (MRI) enables to have precise information about the size, the location and the spread of the tumor in order to diagnose it as fast as possible. In most applications intrinsic contrast is sufficient to obtain a well contrasted image with MRI, but in almost 40% of clinical studies, extrinsic contrast agents able to enhance the signal of the lesions are required. Iron oxide nanoparticles offer many properties that make them attractive as MRI contrast agents [START_REF] Sun | Magnetic nanoparticles in MR imaging and drug delivery[END_REF]. When disperse in biological fluids (plasma, lung fluid), engineered nanoparticles are selectively coated with proteins, resulting in the formation of a protein corona, recognition by the RES and a rapid cellular capture. For imaging applications, there is a need to design advanced and cost-effective coatings that are resistant to protein adsorption and that increase the biodistribution in vivo. In this study, engineered copolymers made with poly(ethylene glycol) and phosphonate anchors were synthesized and used to coat 6 nm iron oxide particles [START_REF] Murray | Alternative projections of mortality and disability by cause 1990-2020: Global burden of disease study[END_REF]. MRI dynamic methods have been developed to study their biodistribution in the liver, especially their liver uptake and release, giving indications on the nanoparticle stealthiness.

II. MATERIALS AND METHODS

II.1. Nanoparticles: the iron oxide nanoparticles were received from Paris 6. They were synthesized according to the Massart method (3). The nanoparticles were then size-sorted by subsequent phase separation. For this study, two batches of -Fe 2 O 3 nanoparticles with diameters of 6.8 nm and 13.2 nm were synthesized.

Polymers and coating:

The phosphonate PEG copolymers were synthesized by SPECIFIC POLYMERS (http://www.specificpolymers.fr/) to supply mixed dispersions of 6.8 and 13.2 nm iron oxide nanoparticles. The copolymer composition was optimized to provide simple and scalable protocols as well as long-term stability in culture media.

Nanoparticles characterization: Dynamic Light Scattering (DLS) was performed to measure the DH of nanoparticles. Ultra-Violet (UV) spectrometry was performed on a Varian Cary 50 scan to measure the iron concentration.

II.3. Cytotoxicity Assay

The cytotoxicity of USPIOs are analyzed in vitro by a WST assay on hepatocytes. II.4. Stability: All the samples were diluted to obtain a concentration of 1 mM to be analyzed for D H during 2 h and at 24 h, and 1 week. The stability of the nanoparticles was assessed by measuring the DH with DLS as a function of the time. II.4. In vitro studies Relaxivities r 1 and r 2 of USPIOs were measured in saline and cells culture medium at 7T.

II.5. In vivo studies

In vivo, we developed an in vivo Dynamic Susceptibility Weighted MRI (7T) protocol to monitor the hepatic capture and clearance of the particles to Balbc C mice as a function of the time.

III. RESULTS

In vitro, PEGylated particles exhibit long-term stability in biological media and low cellular uptake, of the order of 100 femtograms of iron per cell. In vivo, Dynamic Susceptibility Weighted MRI (7T) protocol to monitor the hepatic uptake and clearance of the particles injected to Balbc C mice as a function of the time demonstrated that commercial contrast agents (such as Cliavist) were captured by the liver within 5 minutes after injection. With a PEGylated coat of molecular weight 2000 g mol-1, the capture was postponed by 2 hours and the uptake level lower, showing a direct correlation between the surface properties of the contrast agents and the biodistribution.

IV. DISCUSSION -CONCLUSION

The present survey demonstrates that the surface chemistry of engineered particles is a key parameter in the interactions with cells. This work demonstrates that the functionalization by PEG and phosphonate groups to be attached to the particles is an effective way to develop highly stable nanoparticles and to increase their circulation time in blood. The RES uptake is delayed, allowing the particles to stay longer in the blood pool. Thus, these new PEG polymer coated nanoparticles open up new avenues for detection of tumors with MRI. Beyond applications to imaging and nanomedicine, the approach also opens up new avenues for the efficient functionalization of inorganic surfaces.

Figure 1 :

 1 Figure 1 : Photos of the nanoparticles in DMEM. Macro aggregates can be seen for the bare nanoparticles and the 13.2 nm citrate nanoparticle (red arrows).

Figure 2 :

 2 Figure 2: Liver sections of mice at different time points (pre-injection, 30min, 1h and 3h). On the left hand-side, Cliavist was injected, on the right hand-side, 6.8 nm PEGylated iron oxide nanoparticles was injected. An hyposignal is observed in both cases but at different time point.

  , Q. Crouzet 4 , C. Loubat 3 , N. Mignet 4 *, and J.-F. Berret 1 *

	1 Matière et Systèmes Complexes, UMR 7057 CNRS Université Denis Diderot Paris-VII, Batiment Condorcet, 10
	Rue Alice Domon et Léonie Duquet, F-75205 Paris, France
	Unité de Technologies Chimiques et Biologiques pour la Santé, UMR8258/INSERM U1022 CNRS Chimie
	ParisTech, 4 avenue de l'Observatoire, 75006 Paris, France.
	4 Specific Polymers, ZAC Via Domitia, 150 Avenue des Cocardières, 34160 Castries, France
	4 Unité de pharmacologie Chimique génétique et d'imagerie, UMR8151/INSERM U1022 CNRS, 4 avenue de
	l'Observatoire, 75006 Paris, France.

ACKNOWLEDGMENTS

We gratefully acknowledge ANR, INSERM, CNRS.