domain: spi chim

text:
Yao-Yin Lou

Wenyan He

Enrico Verlato

Marco Musiani

Didier Floner

Florence Fourcade

Abdeltif Amrane

Chaoyu Li

Zhong-Qun Tian

Odile Merdrignac-Conanec

Nathalie Coulon

Florence Geneste
email: florence.geneste@univ-rennes1.fr

Ni-coated graphite felt modified with Ag nanoparticles: a new electrode material for electro-reductive dechlorination

Keywords: Electrocatalytic reduction, organochlorine compound, Ag nanoparticle, graphite felt, alachlor

Reductive removal of the organochlorine herbicide alachlor on Ni-coated graphite felt modified with Ag nanoparticles (Ag/Ni@GF) was studied. Ag/Ni@GF was prepared by spontaneous deposition of silver onto Ni-coated graphite felt (Ni@GF), which was previously obtained by electrodeposition of Ni onto graphite felt (GF). Interestingly, Ag/Ni@GF exhibited a high specific surface area of 26 m 2 g -1 determined by the Brunauer-Emmett-Teller

A c c e p t e d M a n u s c r i p t

Introduction

Organochlorine compounds represent one of the most undesirable classes of contaminants, since they are biorecalcitrant and inevitably leave residues in the environment surrounding human activity. Many physico-chemical methods have been investigated to degrade them, including photocatalytic oxidation [1], Fenton oxidation [2], sonoelectrochemical oxidation [3] and electrochemical oxidation-reduction combined processes [4]. The main drawback of these methods is the generation of non-selective reactive radicals (e.g. •OH and •O 2 in electro-Fenton or oxidation on BDD) that lead to uncontrolled by-products. Reductive dehalogenation processes, especially electroreduction have been proposed as very promising approaches for the removal of hazardous organic halides, since no extra toxic chemical is needed, a high selectivity is obtained and the whole process is performed under mild conditions [5]. Metallic electrodes such as Ag [6,7], Pd [8], Cu [9,10] and Au [11] have been reported as good catalysts for the cleavage of the C-X bond (X=Cl, Br, or I). For example, 2,4-A c c e p t e d M a n u s c r i p t dichlorophenoxyacetic acid has been reduced at Pd modified Ni foam with a removal efficiency of 98% and a maximal current efficiency of 14% [12] and trichloroacetic acid was selectively reduced at Cu, due to its strong adsorption on the electrode surface via Cu-Cl and Cu-CO bonds, shown by SERS [13]. Silver is well known for its high catalytic activity toward the reduction of carbon-halogen bonds, due to its strong tendency to form Ag...X...R interactions with organic halides. For example, Rondinini et al. [14] found that CHCl 3 is converted to CH 4 at Ag nanoparticles modified-carbon powder with 100% current efficiency, and Ardizzone et al. [15] observed a remarkable 0.4-1.2 V shift in the positive direction at Ag with respect to glassy carbon, known as a non-catalytic electrode.

Nanoscale electrode materials can decrease the manufactural cost of the cathode, when dealing with a large volume of water, and can enhance the electrocatalytic performance by a high surface/volume ratio, compared to polycrystalline bulk counterparts [15]. Generally, there are two common ways to deposit Ag nanoparticles on the electrode support: electrodeposition and spontaneous deposition via galvanic displacement reactions. Although electrodeposition is a good method to control nanoparticle size and loading, it might lead to heterogeneous deposits, due to uneven current distribution in different areas of complex shape electrodes [16]. Spontaneous deposition, interesting for its simplicity and to produce large complex-shape electrodes [17], has been extensively used to prepare nanoparticle-modified Ni foam electrodes [16,18,19]. We have previously used a spontaneous deposition approach to achieve the preparation of Ag nanoparticle-modified Ni foam electrodes (Ag/Ni foam) and have shown their good catalytic properties for the electroreduction of a widely used chloroacetanilide herbicide, alachlor, known to contaminate ground and surface waters due to its biorecalcitrance [18].

In this work, we report the preparation of a new catalytic electrode material (Ag/Ni@GF) by spontaneous deposition of Ag onto Ni-coated graphite felt (Ni@GF). The use of Ni-coated A c c e p t e d M a n u s c r i p t graphite felt as support is particularly attractive owing to its high specific surface area compared with Ni foam, high electrical conductivity, good mechanical strength and low specific weight [20]. Moreover, it can be prepared in large scale at moderate cost (around 3.5 cents cm -3 ), which is suitable for environmental applications. Surface area and Ag loading of Ag/Ni@GF electrodes have been evaluated and their catalytic ability to reduce alachlor has been studied and compared to results previously obtained with a Ag/Ni foam electrode.

Experimental Section

Chemical and materials

Ni sulfate hexahydrate (NiSO 4 •6H 2 O), anhydrous sodium sulfate (Na 2 SO 4 ) 99%, sodium thiosulfate pentahydrate (Na 2 S 2 O 3 •5H 2 O), silver nitrate (AgNO 3 ), PVP (polyvinylpyrrolidone, molecular weight 10,000) were purchased from Alfa Aesar and boric acid 99% was supplied by Acros Organics. Nitric acid was obtained from Sigma-Aldrich. Graphite felt (GF, Recycled vein graphite RVG 4000) was supplied by Mersen (France).

Preparation of Ni coated-graphite felt modified with Ag nanoparticles (Ag/Ni@GF)

Ag/Ni@GF was prepared by a two-step procedure presented in Fig. 1, including: (1) pulseelectrodeposition preparation of the Ni-coated GF (Ni@GF); (2) spontaneous deposition of Ag nanoparticles onto the Ni-coated GF by a galvanic replacement reaction in the presence of PVP, a capping agent used to control the shape and size of the Ag nanoparticles [17,21].

Each step is described below.

A c c e p t e d M a n u s c r i p t Electrodeposition of Ni on graphite felt. Ni@GF was obtained at room temperature, according to a previously reported protocol [20] modified as follows: 2 L of the electrolyte solution containing 0.5 M Na 2 SO 4 , 0.25 M boric acid and 0.01 M NiSO 4 •6H 2 O was flowed through the GF at a rate of 8 mL min -1 in a home-made flow cell, shown in Fig. 1. The GF (10.7 × 5.8 × 0.3 cm), used as the working electrode, was located between two platinum grid counterelectrodes to obtain a homogeneous potential distribution in the three-dimensional GF. Before electrodeposition, the GF was successively rinsed with pure methanol and water. During the electrodeposition process, the pH of the electrolyte solution was adjusted to 4 by adding 0.5 A c c e p t e d M a n u s c r i p t M H 2 SO 4 . A periodically changing current, with an on-pulse (80 mA cm -2 , referred to the geometric area of the felt) for 30 s and then an off-pulse (0 A) for 30 s, was applied by a Power Flex generator. The electrolyte solution (2 L) was flowed through the porous GF from one side to the other side (cycle 1) and then the direction was reversed (cycle 2). The flow rate was 8 mL min -1 , so each cycle lasted 250min. After electrodeposition, the Ni@GF was carefully washed with distilled water to eliminate residual salts.

Spontaneous deposition of Ag nanoparticles onto Ni@GF. Deposition of Ag nanoparticles onto Ni@GF was carried out by a galvanic displacement reaction, Equation ( 1)

Ni + 2Ag + → Ni 2+ + 2Ag (1)
A previously reported method [17] was modified as follows. Ni@GF was etched in 0.1 M HNO 3 for 3min and then rinsed with water three times for 5min; sonication was applied during both etching and rinsing. Ag deposition was carried out in a solution containing 1-10 The solution was maintained under argon at 25 ± 0.5 o C. To estimate the Ag loading, an electrochemical method described in [18] was used. Ag and Ni present in the samples were exhaustively dissolved in a 5 M solution of HNO 3 at 80 °C; then the limiting Ag + reduction current at a gold rotating disc electrode (2000 rpm) was measured and compared with a calibration curve. Determination of the concentration of Ag + by this method agreed within 5% with ICP-MS results [17].

Flow electroreduction of alachlor

The dechlorination reaction was performed in a home-made flow cell. To ensure a good homogeneity of the potential distribution in the three-dimensional working electrodes, Ni@GF and Ag/Ni@GF electrodes were located between two interconnected DSA counterelectrodes (dimensionally stable anodes, AC-2004, supplied by ECS International Electro Chemical Services, France) [22]. The compartments were separated by cationic exchange membranes (Nafion TM 417 membrane, France). The reference electrode (mercury-mercurous sulfate -MSE) was positioned in the middle of the working electrode (10mm diameter and 1.7mm thickness) and the potential (-0.9 to -1.6 V/ MSE ) was applied by use of a VersaSTAT 3 potentiostat from Ametek/Princeton Applied (Elancourt, France). The solution (50 mg L -1 of alachlor in 0.05 M NaOH) percolated the porous electrode thanks to a Gilson minipuls 3 peristaltic pump (Middleton, WI, USA). The typical electrolysis duration was 100min. At predetermined time intervals, 200 μL samples of solution were withdrawn from the cathode compartment for HPLC analysis. The conversion of alachlor was calculated as the molar ratio between the consumed alachlor and its initial amount. The yield of deschloroalachlor was determined as the molar ratio between formed deschloroalachlor and the initial amount of alachlor. The current efficiency (CE in %) for the dechlorination reaction was calculated by use of Equation ( 2):

CE=𝑛𝑛𝑛𝑛 𝑛𝑛 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎ℎ𝑎𝑎𝑙𝑙𝑙𝑙 𝑄𝑄 × 100 ( 2
)
where n is the number of electrons (n = 2), F is the Faraday constant (96 500 C mol -1 ), n alachlor is the amount of consumed alachlor (mol), and Q is the amount of charge consumed (C).

Apparatus and analysis

The surface morphology of electrode materials was analyzed by means of scanning electron microscopy (SEM), with a Jeol JSM 7100F (10kV) instrument, equipped with a field emission gun, operating in high-vacuum conditions, at an accelerating voltage variable from 2 to 20 keV, depending on the observation needs. The crystalline structure was analyzed with a Bruker D8 Advance X-ray diffractometer equipped with Cu Kα 1 radiation source (λ = 0.15406 nm) in the θ-2θ configuration. Nitrogen adsorption measurements were carried out with a Micromeritics ASAP 2010 instrument at 77K. Prior to adsorption, the electrode

Results and Discussion

Preparation of Ag/Ni@GF

Preparation of Ni@GF

We previously reported a suitable electrodeposition method to homogeneously coat fibers of graphite felts with a Ni layer in a batch cell [20]. In this work, we report an optimized method that uses a flow electrochemical cell to enhance mass transport inside the felt (Fig. 1). A low Ni 2+ concentration (0.1 mM) and high current densities were also used to allow the electrodeposition to be limited by diffusion of ions from the bulk solution to the electrode surface. As shown in Figures S1(a) and S1(b), after four deposition cycles there were still some areas of the graphite felt not covered by Ni, but after six cycles all fibers were covered by a continuous Ni layer. To check the homogeneity of the Ni coating, the Ni@GF was cut transversally and analyzed by SEM. A similar nickel film was found for the internal and external parts of the graphite felt. However, the thickness of the Ni coating varied from ca. 0.8

A c c e p t e d M a n u s c r i p t μm in edge areas to ca. 0.19 μm in center areas of Ni@GF (respectively named Ni@GF-edge and Ni@GF-center in Fig. S1). The thickness on the edge part of the felt was higher, probably due to the higher current density on the part of felt that was closer to the contact. The density of Ni@GF-center (196 ± 4 mg cm -3 ) compared with the density of the graphite felt (92.4 ± 0.9 mg cm -3 ) allows us to estimate a Ni loading of 104 mg cm -3 , which is significantly lower than for Ni foam (220 mg cm -3 ).

Modification of Ni@GF with Ag nanoparticles

Modification of Ni@GF with Ag nanoparticles was performed by use of thiosulfate solutions, as previously reported for the preparation of Ag/Ni foam [18]. Preliminary tests carried out in the same concentrations (1 mM AgNO 3 , 80 mM Na 2 S 2 O 3 , and 0.2 mM PVP) showed that the conditions previously used to modify the Ni foam with Ag nanoparticles were not directly adaptable to Ni@GF. Indeed, a low density of nanoparticles was obtained probably due to the high surface area of Ni@GF compared with Ni foam. Furthermore, the distribution of Ag nanoparticles was more homogeneous on Ni@GF-center than on Ni@GF-edge. Hence, Ni@GF-center with Ni thickness around 0.19 μm was preferred for further deposition of Ag nanoparticles.

According to the literature [23][24][25], sonication can enhance the chemical activity of reacting sites and the mass transport of compounds to the electrode surface, resulting in an increased number of nucleation sites. Therefore, the influence of sonication on the deposition of Ag nanoparticles on Ni@GF was checked. As detailed in Table S1 The resulting samples were submitted to SEM (Fig. 2 and Fig. S2). Although, under some conditions, an improvement in the homogeneity of the Ag nanoparticles distribution was observed, prolonged sonication time was found to cause also a significant decrease in the Ag To improve the Ag nanoparticle loading on Ni@GF, the effects of Ag + concentration and deposition time were investigated. No significant difference in Ag nanoparticles distribution was observed between samples prepared in 10 mM AgNO 3 , 80 mM Na 2 S 2 O 3 , and 0.2 mM PVP (Fig. S2 (c-d)) or in 5 mM AgNO 3 , 40 mM Na 2 S 2 O 3, and 0.1 mM PVP (Fig. 2). Hence, lower reactant concentrations (1 mM AgNO 3 , 8 mM Na 2 S 2 O 3 and 0.1 mM PVP) were tested, but with longer deposition times (30, 45 or 60min) with magnetic stirring (500 rpm). SEM images of the samples, named Ag(30)/Ni@GF, Ag(45)/Ni@GF, and Ag(60)/Ni@GF for 30, 45 and 60min deposition times, respectively, are shown in Fig. 3. For all deposition times, uniform distributions of Ag nanoparticles were observed, with minor differences between the internal and external parts of the Ni@GF. Ag(30)/Ni@GF had a very low density of smallsized Ag nanoparticles. The Ag nanoparticles on Ag(45)/Ni@GFs were island-like, with a high density, whereas aggregation of nanoparticles occurred with Ag(60)/Ni@GF.

A c c e p t e d M a n u s c r i p t

Since the Ni thickness was only 0.19 µm, EDS analysis was able to quantify Ag and Ni on the surface of the carbon fibers and a reliable Ag/Ni ratio could be estimated (Table 1). A low Ag content was obtained after 30min deposition, which significantly increased after 45 and 60min of reaction, suggesting that there is a significant induction period during the initial phase of the deposition step. The average particle diameter was also estimated by analysis of the SEM images in Fig. 3 (Table 1). For the Ag(45)/Ni@GF sample, about half of Ag nanoparticles had diameters between 30 and 50 nm (Fig. S3). Ag(45)/Ni@GF electrodes were preferentially used in studies on the electrocatalytic activity (see below). External: (a,c,e,g) and internal: (b,d,f,h).

Characterization of Ag/Ni@GF

The crystal structure and preferred orientation (texture) of Ag nanoparticles and Ni layers were studied by means of X-ray diffraction (XRD). Fig. 4 shows the X-ray diffractograms of Ag/Ni@GF, Ni@GF and GF. The diffraction peaks of Ag appear at 2θ = 38.6 o , 64.5 o , and 77.4 o , which correspond to the (111), (220), and (311) planes, respectively. Peaks at 2θ = 44.6 o , 51.9 o and 76.4 o , testify to the presence of metallic Ni on the GF [26][27][28]. Fig. 4 shows that the Ag nanoparticles have a predominant crystalline (111) facet [15], known for its strong catalytic activity [START_REF] Ardizzone | [END_REF]. Indeed, PVP is well known to control the shape and size of Ag nanoparticles during their formation. It has been reported [30] that PVP forms a strong bond with the (100) facets of Ag nanoparticles, lowering their free energies and growth rates, and resulting in favoring the formation of Ag (111) facets. The mean Ag crystallite size could be estimated to be around 17 nm, calculated from the broadening (𝛽𝛽) of XRD peaks, according to the Scherrer relation, Equation (3):

𝑑𝑑 = 𝑘𝑘𝑘𝑘 𝛽𝛽 1/2 𝑐𝑐𝑐𝑐𝑐𝑐(𝜃𝜃) ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• (3)
where 𝑑𝑑 is the average crystallite size (nm), 𝑘𝑘 the wavelength of X-ray radiation (0.15406 nm), 𝜃𝜃 is the angle at the position of the peak maximum, 𝛽𝛽 1/2 is the width of the diffraction peak at half height, and 𝑘𝑘 is 0.94. The 17nm crystallite dimension is significantly lower than the particle size estimated by SEM (Table 1). This suggests that most nanoparticles consisted of assemblies of tinier crystallites.

It is also possible that the average crystallite size obtained by XRD was underestimated owing to the porosity and light weight of the graphite matrix responsible for a large penetration depth for X-rays, leading to a non-negligible peak broadening.

We have previously reported that, after the modified electrode is dissolved in a 5 M solution of HNO 3 at 80 °C, Ag loading could be estimated by linear sweep voltammetry at a gold rotating disc electrode (2000 rpm). The concentration of Ag + ions determined with this method agreed within 5% with results given by ICP-MS [17]. Using the same electrochemical method, we found a Ag loading in Ag(45)/Ni@GF of 13.7±1.3 mg cm -3 (Table 2). RDE voltammograms and calibration curve for the determination of Ag loading are shown in Fig.

A c c e p t e d

M a n u s c r i p t S4. A comparison with the results previously obtained for Ag nanoparticle-modified Ni foams [17] highlighted that Ag loading was almost 7 times higher for Ag(45)/Ni@GF and that the average size of Ag nanoparticles (40 nm) was less than half. To check whether PVP, necessary to achieve the desired morphology of Ag deposits, remained adsorbed on the nanoparticles surface, possibly influencing their catalytic activity, Ag(45)/Ni@GF was investigated by means of Surface-enhanced Raman spectroscopy (SERS).

The black curve in Fig. 5 clearly shows the presence of PVP adsorbed on Ag nanoparticles, highlighted by characteristic peaks of the N-C=O ring deformation stretching at 690 cm -1 and of the C=O stretching at 1764 cm -1 [31]. Broad peaks appearing around 1330 and 1580 cm -1

were due to amorphous carbon from the graphite felt support. The red curve, recorded for a Ag(45)/Ni@GF electrode after its use in an electrolysis, is discussed below. A c c e p t e d M a n u s c r i p t Owing to its importance in electrocatalysis, the electrode surface area was determined by BET measurements and by electroanalytical methods (Table 3). The BET surface area of Ni@GF was 19 m 2 g -1 and increased to 26 m 2 g -1 after modification with Ag nanoparticles. The area increase was probably caused by the additional Ag area and by the roughening of Ni due to the galvanic displacement reaction [32].

The specific surface area given by BET takes into account the surface of all the pores that nitrogen gas can penetrate. However, since organic molecules cannot penetrate into tiny pores, the electrochemically active surface area (EASA) was determined, to compare Ni@GF and Ni foam. Two electrochemical methods were used to measure the EASA: (i) cyclic voltammetry in 1 M KOH, with measurement of the charge corresponding to the Ni(OH) 2 /NiOOH couple (Fig. S5) [17]; (ii) cyclic voltammetry in 1 mM K 3 [Fe(CN) 6 ] as a redox probe and application of the Randles-Sevcik equation for a one-electron process (Equation ( 4)) [START_REF] Bard | Electrochemical Methods: Fundamentals and Applications[END_REF]:

A c c e p t e d M a n u s c r i p t

𝐼𝐼 𝑝𝑝 = 0.446𝑛𝑛𝑆𝑆𝑆𝑆 0 �𝐷𝐷 0 � 𝑛𝑛 𝑅𝑅𝑅𝑅 √ѵ (4)
with I p peak current, F Faraday constant, S electrode surface, D 0 diffusion constant, C 0 concentration of Fe(CN) 6

, R universal gas constant, T absolute temperature and ѵ scan rate.

In the latter method, the linear dependence of the oxidation peak current vs. scan rate allowed the estimation of the EASA of Ni foam and Ni@GF (Fig. S6) [START_REF] Bard | Electrochemical Methods: Fundamentals and Applications[END_REF][START_REF] Ensafi | [END_REF]. Measurements of the EASA of Ni@GF with the electrochemical methods gave results of the same order of magnitude, though significantly different, whereas the BET area was approximately 20 times higher than the EASA. BET area for Ni foam was not determined, so a comparison between Ni foam and Ni@GF was possible only on the basis of EASA. The ratio between the surface areas of Ni@GF and Ni foam depended on the method: it was ca. 2.8 (Randles-Sevcik equation) or ca. 12 (Ni(OH) 2 /NiOOH redox couple). Since the typical Ag loading per unit volume was about 7 times higher on Ni@GF than on Ni foam (Table 2), the Ag loadings per unit EASA were comparable for both systems.

On the basis of data reported in Table 2 (Ag loading and average particle dimension), it is possible to estimate an approximate Ag surface area, by assuming the Ag nanoparticles to be spherical, all with the same diameter, identical to the average value. Under these assumptions, the Ag surface area per unit volume of the catalyst (S Ag ) is given by:

S Ag = 6L ρd ( 5
)
where L is the Ag loading, ρ is its density and d is the particle diameter. Calculations according to Equation (5) yield ca. 1960 cm 2 cm -3 for Ag-modified Ni@GF and ca. 110 cm 2 cm -3 for Ag-modified Ni foam. Thus, it may be concluded that, thanks to the larger surface area of the Ni@GF support, the smaller size of the Ag nanoparticles and the higher Ag loading per volume, Ag-modified Ni@GF had ca. 20 times larger Ag surface area than Ag-A c c e p t e d M a n u s c r i p t modified Ni foam, which was a favorable feature for its application as electrocatalyst.

Another interesting feature of Ni@GF was its low density compared with Ni foam, owing to the presence of graphite inside the Ni fibers.

Table 3. Specific surface area and density of Ni@GF and Ni foam.

Electrode

Electrode surface Density (mg cm -3 ) BET area (cm 2 cm -3 ) EASA (cm 2 cm -3 )

K 3 [Fe(CN) 6 ] Ni(OH 2 )/NiOOH Ni@GF 37240 a 1060 2780 196±4 Ni foam - 380 225 b 328±4 a
This BET area value expressed in cm 2 cm -3 is calculated from the measured 19 m 2 g -1 value. b In previous work [35], EASA of 235 cm 2 cm -3 (before etching) and 285 cm 2 cm -3 (after etching) was measured for a similar Ni foam.

Dechlorination of alachlor TM

Voltammograms obtained at a Ag disk in 0.05 M NaOH, with and without 100 mg L -1 alachlor are compared in Fig. 6. A significant reduction peak at -1. Bulk electrolyses for the reductive dechlorination of 50 mg L -1 alachlor in 0.05 M NaOH were performed in a flow electrochemical cell. Fig. 7 shows the alachlor conversion (a) and deschloroalachlor production (b) in six successive electrolyses carried out at -1.6 V/ MSE with the same Ag(45)/Ni@GF and Ag(60)/Ni@GF working electrodes, used to compare the influence of the Ag loading. In the first electrolysis, alachlor conversions was slightly lower (95.0% vs. 98.3%) and the selectivity for deschloroalachlor production was slightly higher (70.2% vs. 66.1%) on Ag(45)/Ni@GF than on Ag(60)/Ni@GF. Thus, the Ag loading, twice higher for Ag(60)/Ni@GF than for Ag(45)/Ni@GF, had a minor influence on the catalytic activity. Results obtained with both Ag-modified Ni@GF electrodes were significantly better than those previously found, under the same conditions, with Ag-Ni foam electrode (51.0% alachlor conversion with 36.4% deschloroalachlor production, after 60min electrolysis [17]).

Higher Ag loadings and larger EASA, resulting in larger Ag surface areas, were probable causes of the better catalytic activity of Ag-modified Ni@GF.

As shown in Fig. 7, during successive electrolyses, alachlor conversion became higher for Ag(45)/Ni@GF than for Ag(60)/Ni@GF and the selectivity for deschloroalachlor remained higher for the former, with a progressively larger difference. A 5.1% decrease in alachlor conversion and 9.7% decrease in descholoralachlor production, after 6 electrolyses, showed the significantly better stability of Ag(45)/Ni@GF, compared to Ag(60)/Ni@GF (ca. 17% decrease in both alachlor conversion and deschloroalachlor production). The morphology of Ag(45)/Ni@GF and Ag(60)/Ni@GF electrodes before and after 6 electrolyses was compared by SEM (Fig. 8). Ag(45)/Ni@GF exhibited no obvious changes of A c c e p t e d M a n u s c r i p t both Ni layer and island-like Ag nanoparticles. Ag(60)/Ni@GF showed some areas with exfoliation of the Ni layer and, unavoidably, loss of the Ag nanoparticles present on it, which can explain the observed loss of catalytic activity. In the case of Ag(60)/Ni@GF, the more extensive deposition of Ag certainly caused a higher consumption of Ni, and possibly a stronger strain of the residual Ni layer, rendering it more fragile towards the stress caused by hydrogen evolution that cannot be avoided at the applied potential of -1.6 V/ MSE [20].

A Ag(45)/Ni@GF electrode used in a 100min electrolysis was characterized again with the aid of SERS. The red curve in Fig. 5 shows the disappearance of characteristic peaks of PVP after the electrolysis, indicating that the electroreduction led to complete desorption of PVP from the Ag nanoparticles. Since the Ag(45)/Ni@GF electrode retained the same level of electrocatalytic activity during several electrolyses, one can conclude that the initially adsorbed PVP had neither positive nor negative influence on the dechlorination reaction. To improve the current efficiency, a less negative potential was applied to favor the reduction of alachlor and to decrease hydrogen evolution. Fig. 9 shows the effect of the applied potential on the reductive dechlorination of 50 mg L -1 alachlor, performed in 0.05 M NaOH at -0.9 and -1.6 V /MSE on Ag(45)/Ni@GF, and compared with results obtained with an unmodified Ni@GF electrode. As previously observed [35], Ni@GF could reduce alachlor, but with slower kinetics and much lower selectivity for deschloroalachlor. It is worth noting that, for the three-dimensional electrode, a potential distribution occurs opposite to a planar electrode, explaining that at -0.9 V the electroreduction of alachlor already happened, whereas no current was observed on the voltammogram of Fig. 7. Table 4 shows the effect of applied potential and NaOH concentration on the current efficiency, in addition to the data on alachlor conversion and deschloroalachlor formation, after a 100min electrolysis. When the potential was decreased from -1.6 to -0.9 V /MSE , the current efficiency for Ag(45)/Ni@GF increased from 0.8% to 36.6%, whereas a similar conversion of alachlor and the yield of deschloroalachlor were maintained. The increase of the pH (0.1 M NaOH solution) also slightly increased the current efficiency, due to the less strong competition by the hydrogen evolution reaction, and both alachlor conversion and deschloroalachlor production slightly increased.

A c c e p t e d M a n u s c r i p t

Conclusions

New Ag/Ni@GF electrodes for the dechlorination of organic chlorides have been prepared by modifying Ni-coated graphite felts with Ag nanoparticles. These particles were deposited through a galvanic displacement reaction that would be easy to scale up for the preparation of large-size electrodes for environmental applications, at moderate cost. The experimental conditions previously reported to modify Ni foam with Ag nanoparticles [18] had to be adapted to Ni@GF, owing to its high surface area. Whereas the increase of mass transport by sonication did not allow a homogenous repartition of nanoparticles, an increase of Ag + concentration and of deposition time led to uniform distributions of Ag nanoparticles inside the Ni@GF. PVP was required as capping agent to control the shape and the size of Ag nanoparticles, leading to main crystal facets [111]. SERS experiments showed that PVP is adsorbed on Ag particles on Ag/Ni@GF electrodes, but is totally desorbed during electroreduction.

Interestingly, Ag/Ni@GF electrodes had high Ag loading (13.7 mg cm -3 ), high specific surface area (26 m 2 g -1 ) and low density, since graphite felt was used as support. The Ag surface area in these electrodes could be estimated, from Ag loading and nanoparticles average dimension, to be close to 2000 cm 2 cm -3 , which is 20 times larger than those of Ag nanoparticle-modified nickel foam.

Due to its interesting properties, Ag/Ni@GF electrodes exhibit good catalytic activity in the dechlorination of alachlor, with high conversion yields of 96-99% and current efficiency up to 36.6%. Good selectivity was also achieved, with the formation of 76.1% deschloroalachlor.

Furthermore, the Ag/Ni@GF electrode displayed good stability after 6 successive electrolyses.

Fig. 1 .

 1 Fig. 1. Preparation of Ag/Ni@GF electrodes.

A c c e p t e d M a n u s c r i p t

A c c e p t e d M a n u s c r i p t

  materials were out-gassed under vacuum at room temperature. Their surface area was calculated according to the Brunauer-Emmett-Teller (BET) method. Surface-enhanced Raman scattering (SERS) was performed with a XploRA Raman instrument Jobin Yvon-Horiba, France, equipped with an 50 X, NA 0.5 objective, that used a 532nm Laser line, with a 0.3 or 3.0 mW power at sample for as-prepared and used electrodes, respectively. The concentration of alachlor and deschloroalachlor were determined with a Waters 996 high performance liquid chromatography (HPLC) system equipped with a Waters 996 PDA (Photodiode Array Detector) and a Waters 600 LCD Pump. The separation was achieved on a Waters C-18 (5 μm; 4.6 × 250 mm) reversed-phase and the mobile phase consisted of a mixture of acetonitrile/ultrapure water (70/30, v/v). The flow rate was 0.4 mL min -1 and 50 μL injection was used. Detection was carried out at 195 nm.

A c c e

  p t e d M a n u s c r i p t loading, probably due to detachment and release of Ag nanoparticles. For this reason, no sonication was applied in further Ag deposition experiments.

Fig. 2 .

 2 Fig. 2. SEM images of Ag-Ni-graphite felt samples prepared in 5 mM AgNO 3 , 40 mM

Fig. 3 .

 3 Fig. 3. SEM images of Ag/Ni@GF samples prepared in 1 mM AgNO 3 , 8 mM Na 2 S 2 O 3 and

Fig. 4 .

 4 Fig. 4. XRD analysis of Ag(45)/Ni@GF, Ni@GF, and GF

Fig. 5 .

 5 Fig. 5. SERS spectra recorded with as-prepared Ag/Ni@GF (black curve) and the same electrode after 100min electrolysis in 0.05 M NaOH solution containing 50 mg L -1 alachlor

  28 V /MSE , visible only in the presence of alachlor, shows the expected electrocatalytic activity of Ag towards dechlorination processes.

Fig. 6 .

 6 Fig. 6. Voltammograms recorded at Ag disk in 0.05 M NaOH solution with and without 100 mg L -1 alachlor. Scan rate 100 mV s -1 .

Fig. 7 .

 7 Fig. 7. Effect of Ag loading on the conversion of alachlor (a) and production of

Fig. 8 .

 8 Fig.8. SEM analysis of Ag(45)/Ni@GF (a-d) and Ag(60)/Ni@GF (e-h) before (a-b,e-f) and

Fig. 9 .

 9 Fig. 9. Effect of cathode potential on the electrochemical conversion efficiency of alachlor (in

A c c e p t e d M a n u s c r i p t

Table 1 .

 1 Ag loading and average particle size of Ag nanoparticles on Ni@GF for different deposition times in 1 mM AgNO 3 , 8 mM Na 2 S 2 O 3 and 0.1 mM PVP.

	Electrode	Ag loading by SEM-EDS analysis Atomic % Mass %	Average particle diameter (nm)
	Ag(30)/Ni@GF 0.9	1.6	--
	Ag(45)/Ni@GF 9.8	16.6	~ 40
	Ag(60)/Ni@GF 22.1	33.6	~ 100

Table 2 .

 2 Characteristics of different Ag nanoparticle-modified porous electrodes

a Estimated from SEM analyses b Calculated from the Scherrer equation c Ag loading in mass % (w.%) and atomic % (at %) determined by SEM-EDS. d Mass content controlled during electrode preparation.

Table 4 .

 4 Influence of applied potential and NaOH concentration on alachlor conversion, deschloroalachlor production, and current efficiencies in 100min electrolyses.

	Electrode Potential	NaOH	Current	Removal of	Production of
		(V /MSE )	concentration	efficiency	alachlor	deschloroalachlor
			(M)	(%)	(%)	(%)
	Ni@GF	-1.6	0.05	0.4 ±0.05	82.8	39.3
	Ag/Ni@GF -0.9	0.05	36.6 ±2	96.0	76.1
		-1.6	0.05	0.8 ±0.08	93.8	71.6
		-1.6	0.1	3.7 ±0.8	99.0	74.1

Acknowledgements

The authors thank the China Scholarship Council for a Ph.D. grant. We also thank our joint laboratory, LIA CNRS NanoBioCatEchem, for supporting the collaboration with Xiamen University.

Author Contributions: † Y.Y. Lou and W.Y. He contributed equally to this work. S1,S2 6. References

Supporting material

Figures S1 to S6 and Tables


Simon Grelet

Vanessa Andries

Myriam Polette

Christine Gilles

Katrien Staes

Anne-Pascaline Martin

Claire Kileztky

Christine Terryn

Véronique Dalstein

Chun- Wen Cheng

Chen-Yang Shen

Philippe Birembaut

Frans Van Roy

Béatrice Nawrocki-Raby

The human NANOS3 germline gene contributes to lung tumour invasion by inducing epithelial-mesenchymal transition

Keywords: Nanos3, Lung cancer, Tumour invasion, Epithelial-mesenchymal transition, Posttranscriptional regulation

In this study, we explore the role of the human NANOS3 germline gene in lung tumour progression. We report that Nanos3 is overexpressed by invasive lung cancer cells and represents a new prognostic marker for non-small cell lung carcinomas (NSCLC). We also show that Nanos3 gene expression is restricted in testis and brain and is regulated by epigenetic events. Moreover, we demonstrate that Nanos3 is upregulated in cultured cells undergoing epithelialmesenchymal transition (EMT). Nanos3 overexpression in human NSCLC cell lines enhances their invasiveness by upregulating EMT, whereas its silencing induces mesenchymalepithelial transition. Nanos3 represses E-cadherin at the transcriptional level and upregulates Vimentin post-transcriptionally. Also, we show that Nanos3 binds mRNAs encoding Vimentin and regulates the length of the 3' poly(A) tail of VIM mRNA Finally, Nanos3 can also protect Vimentin mRNA from microRNA-mediated repression. We thus demonstrate a new role for Nanos3 in the acquisition of invasiveness by human lung tumour cells and propose a new mechanism of post-transcriptional regulation of EMT.

INTRODUCTION

The NANOS genes comprise a small family of evolutionarily conserved RNA-binding proteins required in diverse organisms for regulation of various developmental processes, including embryonic patterning and germ cell development. The first Nanos family member described was the unique nos gene in Drosophila melanogaster, identified as a maternal effect gene required for abdomen formation. Nos has been extensively studied and is now well known to control the differentiation of the anteriorposterior body axis, primordial germ cell migration, maintenance of germline stem cell self-renewal, and suppression of somatic cell fate during germline development [START_REF] Lehmann | The maternal gene nanos has a central role in posterior pattern formation of the Drosophila embryo[END_REF][START_REF] Gavis | Translational regulation of nanos by RNA localization[END_REF][START_REF] Asaoka-Taguchi | Maternal Pumilio acts together with Nanos in germline development in Drosophila embryos[END_REF][START_REF] Hayashi | Nanos suppresses somatic cell fate in Drosophila germ line[END_REF][START_REF] Wang | Nanos maintains germline stem cell self-renewal by preventing differentiation[END_REF]. In all species analysed, Nanos homologs are characterized by a highly conserved zinc-finger domain. This domain, which mediates protein-nucleic acid interactions, has an essential role in translational regulation of Nanos targets and is also predicted to be involved in proteinprotein interactions [START_REF] Curtis | A CCHC metal-binding domain in Nanos is essential for translational regulation[END_REF]. In humans, three homologs of Nanos (Nanos1, -2 and -3) have been characterized and shown to have conserved functions in germline development [START_REF] Jaruzelska | Conservation of a Pumilio-Nanos complex from Drosophila germ plasm to human germ cells[END_REF][START_REF] Kusz | The highly conserved NANOS2 protein: testisspecific expression and significance for the human male reproduction[END_REF][START_REF] Julaton | NANOS3 function in human germ cell development[END_REF]. In a tumour context, it was recently shown that Nanos2 and Nanos3 are meiosis regulators and are dysregulated in testicular carcinoma in situ [START_REF] Jorgensen | Dysregulation of the mitosis-meiosis switch in testicular carcinoma in situ[END_REF]. Moreover, Janic et al. previously showed that ectopic expression of germline genes such as nos drives malignant brain tumour growth in Drosophila [START_REF] Janic | Ectopic expression of germline genes drives malignant brain tumor growth in Drosophila[END_REF]. In addition, upregulation of Nanos1 and Nanos3 enables the oncogenic growth of pRb-deficient cells suggesting that Nanos has a stimulatory role in cancer cell proliferation [START_REF] Miles | Post-transcriptional gene expression control by NANOS is up-regulated and functionally important in pRb-deficient cells[END_REF]. Otherwise, we previously reported that human Nanos1 (hNanos1) is repressed by E-cadherin and is involved in the acquisition of invasive properties by lung tumour cells via regulation of matrix metalloproteinase (MMP)-14 expression [START_REF] Strumane | E-cadherin regulates human Nanos1, which interacts with p120ctn and induces tumor cell migration and invasion[END_REF][START_REF] Bonnomet | The E-cadherin-repressed hNanos1 gene induces tumor cell invasion by upregulating MT1-MMP expression[END_REF].

During tumour progression, phenotypic changes occur in epithelial tumour cells, enabling only a few of them to overcome successive barriers and develop overt metastases. This phenotypic conversion, so-called EMT (Epithelial-Mesenchymal Transition), is characterized by diminished epithelial characteristics and increased mesenchymal attributes, and contributes to enhancement of invasive properties of epithelial tumour cells [START_REF] Kalluri | The basics of epithelial-mesenchymal transition[END_REF]. During the EMT process, "mesenchymal" markers (such as Vimentin, -smooth muscle actin, fibroblast specific protein 1 and MMPs) are frequently overexpressed whereas epithelial features (such as E-cadherin and Occludin) are dramatically decreased [START_REF] Gilles | Matrix metalloproteases and Epithelial-tomesenchymal transition: implications for carcinoma metastasis[END_REF][START_REF] Polette | Beta-catenin and ZO-1: shuttle molecules involved in tumor invasion-associated epithelial-mesenchymal transition processes[END_REF]. These EMT targets genes are regulated by several specific transcriptional factors such as Snail, Slug, , ZEB2, Twist and others. These proteins can transcriptionally regulate additional targets both by repressing other "epithelial" genes (such as other components of adherens and tight junctions) but also by inducing "mesenchymal" genes such as Vimentin or MMPs [START_REF] Peinado | Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype?[END_REF][START_REF] Bindels | Regulation of vimentin by SIP1 in human epithelial breast tumor cells[END_REF].

To determine whether Nanos3 is involved in tumour progression, we analysed the expression pattern of Nanos3 in samples of human non-small cell lung carcinoma (NSCLC) and evaluated the relationship between Nanos3 and EMT process. We provide new data on the role of Nanos3 in human lung tumour progression and report evidence for a novel posttranscriptional regulation of Vimentin by Nanos3.

MATERIALS AND METHODS

Tumour tissue samples

The frozen tumour pieces used for total protein extraction and subsequent western blot analyses were obtained from samples of the Tumour Bank of the Reims University Hospital Biological Resource Collection No. DC-2008-374. Surgically resected tumours were collected after obtaining informed consent from 24 patients with primary lung adenocarcinoma and 26 patients with squamous-cell carcinoma (SCC). In parallel, nontumoural parenchyma and bronchi samples matching the tumour samples were also collected. This study was approved by the Institutional Review Board of the Reims University Hospital. For immunohistochemistry analysis, tissue array slides containing 94 NSCC and 9 matched metastases were purchased from SuperBioChips (SuperBioChips Laboratories, Seoul, Korea).

Cell culture

Human lung cell lines HBE4-E6/E7, A549, BEAS-2B, NCI-H1299, Calu-1 and SK-LU-1 were obtained from the American Type Culture Collection (Rockville, MD, USA). A description of culture reagents is provided in Supplementary materials and methods. For mRNA stability experiments, Calu-1 and SK-LU-1 were treated with actinomycin D, at 2.5 µg/ml and 10 µg/ml, respectively (Sigma-Aldrich, St. Louis, MS, USA).

Stable transfection of Nanos3 cDNA vector

The eukaryotic expression plasmid pDEST-NANOS3 was constructed by transferring the human NANOS3 cDNA to the pDEST12.2 vector by Gateway technology (Life Technologies, Carlsbad, CA, USA). Calu-1 and SK-LU-1 cells were stably transfected with pDEST or pDEST-NANOS3 vectors by electroporation and selected with G418 (50 µg/ml).

Transient transfection of Nanos3 cDNA

Transient transfection of pDEST or pDEST-NANOS3 in A549 cells was carried out with X-tremeGENE transfection reagent (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions.

Transfection of siRNAs

siRNA specific sequences described in Supplementary Table S3 were selected in the coding sequence of NANOS3 (GeneBank accession number: NM_001098622.2) to generate 21nucleotide sense and 21-nucleotide antisense strands of the type (19N)TT (N, gene-specific nucleotide) (Eurogentec, Seraing, Belgium). Corresponding scrambled duplexes that do not recognize any sequence in the human genome were used as controls. Cells were transfected with siRNA duplexes (20 nM) by the calcium phosphate precipitation method as previously described [START_REF] Polette | Membrane-type 1 matrix metalloproteinase expression is regulated by zonula occludens-1 in human breast cancer cells[END_REF].

Western blotting

Total proteins from cells or tumour samples were extracted in RIPA buffer containing complete protease inhibitor cocktail (Roche Diagnostics). The subsequent steps were performed as previously described [START_REF] Polette | Membrane-type 1 matrix metalloproteinase expression is regulated by zonula occludens-1 in human breast cancer cells[END_REF].

Immunohistochemistry

Immunohistochemistry for Nanos3 was done on paraffin tissue array sections. After antigen retrieval in citrate buffer (Dako, Glostrup, Denmark), subsequent steps were performed with the biotin free EnVision + Dual Link System-HRP kit (Dako), and HRP activity was revealed with AEC+ substrate-chromogen (Dako). A detailed description of semi-quantitative scorings is provided in Supplementary materials and methods. Immunohistochemistry for Vimentin (Dako) and E-cadherin (Dako) was performed using a Ventana Benchmark XT autostainer (Ventana Medical Systems, Inc., Tucson, AZ). Subsequent steps were performed with the UltraView universal DAB detection kit (Ventana).

Real-time qRT-PCR for in vitro analysis

Total RNA was isolated using the High Pure RNA isolation kit (Roche Diagnostics). Total RNA (1 µg) was reverse-transcribed into cDNA with the Transcriptor First Strand cDNA Synthesis kit (Roche Diagnostics). qPCR reactions were performed using the "Fast Start Universal Probe Master" kit and the UPL-probe system as recommended by the manufacturer, in a LightCycler 480 Instrument (Roche Diagnostics). Forward and reverse primers (Eurogentec) for NANOS3, CDH1, VIM, GAPDH and 18S are described in Supplementary Table S3. Each sample was quantified using a standard curve based on serial dilutions of a batch of human placenta cDNA.

Epigenetic regulation analysis

Cell cultures were treated with 1 μM 5-aza-2′-deoxycytidine (DAC) (Sigma-Aldrich), 0.1 μM Trichostatin A (TSA) (Sigma-Aldrich), 100 μM CTK7A (Millipore) or 80 μM of Sadenosylmethionine (SAM) (New England Biolabs, Ipswich, MA, USA). DAC, TSA and CTKA7 stock solutions were diluted in DMSO. The SAM solution was prepared in a filtered solution of 5 mM H2SO4 and 10% ethanol. Cells were treated for four days before RNA extraction and RT-PCR analysis.

Cell circularity analysis

Cellular circularity was measured with ImageJ software (http://imagej.nih.gov/ij/index.html) according to the following formula: circularity = 4π(area/perimeter 2 ).

Invasion assay

The in vitro invasive properties of cells were assessed using a Matrigel modified Boyden chamber assay as previously described [START_REF] Joannes | Fhit regulates invasion of lung tumor cells[END_REF].

2D migration assay

The 2D migration assay has been described [START_REF] Joannes | Fhit regulates invasion of lung tumor cells[END_REF].

EMT-inducible model

For the 2D migration model of EMT, HBE4-E6/E7 cells were seeded inside a restriction glass ring on coverslip as described above and then treated for 3 days with 20 ng/ml EGF after removal of the ring. This was followed by methanol fixation and immunofluorescence analysis.

Bioinformatic prediction of Nanos3 localization

We used the following programs to predict the subcellular localization of Nanos3: ESLPred (http://www.imtech.res.in/raghava/eslpred) [START_REF] Bhasin | ESLpred: SVM-based method for subcellular localization of eukaryotic proteins using dipeptide composition and PSI-BLAST[END_REF]; PSORT II (http://psort.nibb.ac.jp) [START_REF] Nakai | PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization[END_REF]; SubLoc v1.0 (http://www.bioinfo.tsinghua.edu.cn/SubLoc) [START_REF] Chen | SubLoc: a server/client suite for protein subcellular location based on SOAP[END_REF]; CELLO (http://cello.life.nctu.edu.tw/) [START_REF] Yu | Prediction of protein subcellular localization[END_REF]; Euk-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/eukmulti-2) [START_REF] Chou | A new method for predicting the subcellular localization of eukaryotic proteins with both single and multiple sites: Euk-mPLoc 2.0[END_REF]; YLoc+ (http://abi.inf.uni-tuebingen.de/Services/YLoc/webloc.cgi) [START_REF] Briesemeister | YLoc--an interpretable web server for predicting subcellular localization[END_REF].

Subcellular fractionation

Purified proteins from four subcellular fractions (organelle, cytosolic, cytoskeletal and nuclear fractions) of BEAS-2B cells were obtained by using the ProteoExtract subcellular proteome extraction kit (Merck, Darmstadt, Germany) according to the manufacturer's instructions.

Immunofluorescence and colocalization analysis

Subconfluent monolayers of cells cultured on glass coverslips were fixed with methanol or 4% paraformaldehyde for simple labeling or colocalization analysis, respectively, as previously described [START_REF] Joannes | Fhit regulates invasion of lung tumor cells[END_REF]. For colocalization studies, the staining was analysed using a Zeiss LSM 710 confocal laser scanning microscope with a 63x oil-immersion objective and the Zeiss operating system associated with the ZEN software program (Carl Zeiss MicroImaging GmbH, Germany). Fifty planes (0.2 μm between each plane) were combined in a reconstruction by using the Imaris software for surface reconstruction (Bitplane AG, Zurich, Switzerland).

RNA immunoprecipitation

Cells were lysed and immunoprecipitations were performed using either control IgG or anti-Nanos3 antibody according to the instructions for the EZ-Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Darmstadt, Germany). The purification of protein complexes was checked by western blot. After phenol/chloroform treatment, RNA extracted from the complexes was subjected to RT-PCR by using the First Strand cDNA Synthesis Kit for RT-PCR (Roche Diagnostics). PCR reactions were performed with the AmpliTaq Gold PCR Master Mix kit (Applied Biosystem). Forward and reverse primers (Eurogentec) for CDH1 and VIM are described in Supplementary Table S3.

mRNA poly(A) tail length analysis

The lengths of mRNA poly(A) tails were measured using the poly(A) tail length assay kit (Affymetrix Santa Clara, CA, USA). Briefly, 1 µg of total RNA was G-I tailed and reverse transcribed using a tail-specific primer. Samples were amplified by PCR using the VIM specific forward primer described in Supplementary Table S3 and the universal reverse primer provided with the kit. An ACTIN specific forward primer provided with the kit was used as control. PCR products were analysed on a 10% acrylamide gel and visualized on LAS-1000 fluorescence imager (Fujifilm, Tokyo, Japan) after SYBR Gold Nucleic Acid Gel Staining (Invitrogen).

Dual luciferase reporter assay

WT and mutated VIM3'-UTR firefly luciferase reporter vectors have been reported [START_REF] Cheng | MicroRNA-30a inhibits cell migration and invasion by downregulating vimentin expression and is a potential prognostic marker in breast cancer[END_REF]. Both

Calu-1 and SK-LU-1 transfectants were transfected with 100 ng reporter construct containing WT or mutated 3'-UTR of VIM. The firefly luciferase/Renilla luciferase activity ratio of each sample was measured in the dual-luciferase reporter assay system (Promega, WI, USA).

Statistical analysis

For in vivo experiments, Mann-Whitney and Wilcoxon tests were used to compare Nanos3 levels by western blot and immunohistochemistry, respectively. Associations between Nanos3 score and biological data were studied using Chi-square test. Univariate analysis was performed to identify factors associated with overall survival using the KaplanMeier method and compared with the Log Rank test. Factors with a p-value < 0.05 by univariate analysis were included in a multivariate analysis using the Cox proportional hazard model. For in vitro experiments, data are expressed as a mean ± standard deviation, and the one-sample t-test or Student's t-test was used for statistical analysis. P < 0.05 was considered significant.

Analyses were performed using GraphPad Prism version 5.0 (GraphPad Software, La Jolla, CA) and Stata/IC 10 (StataCorp, College Station, TX).

RESULTS

Nanos3 is overexpressed by invasive NSCLC cells.

We analysed Nanos3 expression in a series of human NSCLC samples using frozen material and tissue array slides. Western blot analysis of protein extracts showed that Nanos3 is overexpressed in both human lung adenocarcinoma (median of Nanos3/GAPDH ratio: 0.09, range: 0.00-0.64) and squamous cell carcinoma (SCC) (median: 0.11, range: 0.00-0.92), whereas in the non-tumoural counterparts it was almost undetectable: parenchyma (median: 0.01, range: 0.00-0.06; p<0.001) and bronchi (median: 0.01, range: 0.00-0.10; p<0.01) (Fig. 1A). This overexpression in tumours was also observed at the transcript level by RT-PCR (data not shown). Immunohistochemical analysis revealed that expression of Nanos3 is a frequent event in NSCLC because only 5 cases out of 94 (5.3%) displayed no staining (Supplementary Table S1). Nanos3 was expressed in the nucleus and/or cytoplasm of tumour cells independently of the histological subtype (Fig. 1B). A stronger staining was frequently observed at the invading front of tumour clusters, especially in SCC (Fig. 1B, f, arrowheads).

Moreover, establishing a scale of Nanos3 immunostaining intensity showed that Nanos3 is expressed less in primary lung tumours than in the metastases associated with them (Fig. 1C).

The median scores were, respectively, 8 (range: 4-12) and 14 (range: 5-24; p<0.01). The Nanos3 score was not related to the histological type, differentiation status, tumour size, N status, or tumour node metastasis (TNM) stage of the primary tumours, but, interestingly, it correlated with patient outcome (Fig. 1D and Table 1). A high Nanos3 score was associated with poor survival of patients with NSCLC. Median survival time was 85.5 months (range:

2149) for patients with a score < 10, and 38.5 months (range: 4132) for those with a score ≥ 10 (p=0.0394). Moreover, multivariate analysis showed that Nanos3 is an independent prognostic marker for NSCLC (p =0.041, Table 1).

Further, comparison of NANOS3 gene expression with in vitro invasive capacity of various lung carcinoma cell lines revealed that cells producing the highest NANOS3 mRNA levels were also the most invasive (Fig. 1E).

Overall, these findings indicate an association between Nanos3 overexpression and lung cancer progression, and particularly tumour invasion and dissemination.

Human Nanos3 expression is restricted to brain and testis in normal adult tissues and is

regulated by epigenetic events.

We also wondered about the Nanos3 expression profile in normal human tissues. With the exception of testis and brain, NANOS3 transcripts were almost undetectable in healthy adult somatic tissues (Fig. 2A).

Since Nanos3 was found to be ectopically expressed in human NSCLC, we were interested in the mechanisms controlling the level of neo-expression of the NANOS3 gene in lung cancer. For this purpose, several human tumour cell lines were treated with the DNA methyl transferase inhibitor Decitabine (DAC) or the histone deacetylase inhibitor

Trichostatin A (TSA), or with both. We found that the mRNA level of NANOS3 was upregulated by DAC in HBE4-E6/E7 cells and by the combination of DAC and TSA in SK-LU-1, Calu-1 and A549 cells (Fig. 2B). In contrast, the high level of NANOS3 mRNA expressed by NCI-H1299 cells was unaffected by these inhibitors. Conversely, treatment of NCI-H1299 cells with the methyl donor S-adenosyl methionine (SAM) or the histone acetyl transferase inhibitor VII (CTK7A), or with both, induced a decrease in NANOS3 gene expression (Fig. 2B).

Taken together, these in vitro and in vivo data indicate that NANOS3 gene expression is enriched in testis and brain, and that it is overexpressed in lung cancer by epigenetic mechanisms involving both DNA methylation and histone acetylation.

Nanos3 is upregulated during experimental EMT in vitro and regulates EMT-associated invasion.

It has been proposed that EMT is very important in tumour invasion [START_REF] Nieto | Epithelial plasticity: a common theme in embryonic and cancer cells[END_REF], so we verified the relationship between Nanos3 and the EMT process. To this end, we used two in vitro models of EMT in HBE4-E6/E4 bronchial cells: induction of 2D migration (Fig. 3A) and epidermal growth factor (EGF) treatment (Supplementary Fig. S1A). In both models, we saw cell morphology changes characteristic of EMT. The loss of membrane-associated expression of the epithelial marker E-cadherin and the de novo expression of the mesenchymal marker Vimentin were used to confirm the occurrence of EMT in both models.

Interestingly, we found that Nanos3 was upregulated in cells that underwent EMT (Fig. 3A and Supplementary Fig. S1A).

To study whether Nanos3 is causally involved in tumour cell invasion, we first generated stable transfectants overexpressing Nanos3 in the squamous cell carcinoma cell line Calu-1 and the adenocarcinoma cell line SK-LU-1, which were originally moderately invasive. Nanos3 overexpression induced a drastic morphological change from a cohesive epithelial cell shape to a more scattered fibroblast-like cell shape coupled with decreased cell roundness (Fig. 3B). Western blot analysis revealed downregulation of E-cadherin and overexpression of Vimentin in Nanos3-overexpressing cells (Fig. 3C). Consistent with these findings, cells overexpressing Nanos3 were more invasive in a Matrigel modified Boyden chamber assay (Fig. 3D). Similar results were obtained with A549 cells transiently transfected with NANOS3 cDNA (data not shown).

Conversely, Nanos3 silencing in the highly invasive NCI-H1299 lung tumour cell line by transient transfection of NANOS3 small interfering RNA (siRNA) induced a more epithelioid phenotype characterized by a larger cell circularity index (Fig. 3E). Cells with transiently silenced Nanos3 showed increased expression of E-cadherin as well as decreased expression of Vimentin (Fig. 3F). This was accompanied by a strong decrease in invasive and 2D/3D migratory capacities of Nanos3-silenced cells (Fig. 3G; Supplementary Fig. S1B and S1C; Supplementary Video S1). Similar results were also obtained with BEAS-2B (Supplementary Fig. S2) and A549 (data not shown) cell lines transiently transfected with Nanos3 siRNA.

In addition, our immunohistochemical analysis of serial sections of human NSCLC showed that all tumor clusters expressing Vimentin and showing loss of E-cadherin also express Nanos3 (Fig. 3H).

Differential regulation of EMT targets by Nanos3.

To We also assessed the precise subcellular localization of Nanos3 by using a combination of bioinformatic prediction methods, subcellular protein fractionation and fluorescence microscopy. In silico, the NNCN, CELLO, Euk-mPLoc, SubLoc, ESLPred, PSORT II, and Yloc+ algorithms [START_REF] Bhasin | ESLpred: SVM-based method for subcellular localization of eukaryotic proteins using dipeptide composition and PSI-BLAST[END_REF][START_REF] Nakai | PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization[END_REF][START_REF] Chen | SubLoc: a server/client suite for protein subcellular location based on SOAP[END_REF][START_REF] Yu | Prediction of protein subcellular localization[END_REF][START_REF] Chou | A new method for predicting the subcellular localization of eukaryotic proteins with both single and multiple sites: Euk-mPLoc 2.0[END_REF][START_REF] Briesemeister | YLoc--an interpretable web server for predicting subcellular localization[END_REF] all predicted that Nanos3 is preferentially localized in the nucleus (Supplementary Table S2). Finally, we scrutinized the mechanism by which tumour-associated Nanos3 induction modulates VIM mRNA post-transcriptionally. Nanos family members are known to regulate translation by controlling poly(A) tail length [START_REF] Wreden | Nanos and pumilio establish embryonic polarity in Drosophila by promoting posterior deadenylation of hunchback mRNA[END_REF]. So, we first examined the effect of modulating Nanos3 expression on the length of the VIM mRNA poly(A) tail. We found that the length of the VIM mRNA poly(A) tail was increased by Nanos3 overexpression (Calu-1:

64 ± 28%, p < 0.05; SK-LU-1: 109 ± 35%, p < 0.05) and decreased by its silencing (A549: 31 ± 14%, p < 0.05; Calu-1: 27 ± 4%, p < 0.01; SK-LU-1: 21 ± 4%, p < 0.05) (Fig. 5A). We observed that VIM mRNAs were less sensitive to the actinomycin D treatment in Nanos3 transfectants than in their respective control, suggesting that Nanos3 stabilized mRNAs encoding Vimentin (a increase of 123 ± 23% and 97 ± 51% for Calu-1 and SK-LU-1, respectively; p < 0.05) (Fig. 5B). We also showed that the induction of Vimentin protein upon Nanos3 overexpression was abolished by cordycepin, a polyadenylation inhibitor (Fig. 5C).

Finally, because miR-30a has been shown to downregulate Vimentin expression [START_REF] Cheng | MicroRNA-30a inhibits cell migration and invasion by downregulating vimentin expression and is a potential prognostic marker in breast cancer[END_REF], we explored the link between Nanos3 and miR-30a. Using a luciferase assay, we found that binding of Nanos3 to the 3'-UTR of the VIM transcript resulted in a strong induction of luciferase activity in Calu-1 and SK-LU-1 cells. Moreover, we observed that the Nanos3 effect is not additive to the protective effect of both 3'-UTR mutations (VIM/mut2 and VIM/mut12), reported to affect miR-30a binding sites (Fig. 5D).

All together, these data suggest that Nanos3 regulates Vimentin by a polyadenylationassociated mechanism and protects VIM mRNA from microRNA repression.

DISCUSSION

This study demonstrates the involvement of human Nanos3 in the progression of NSCLC. We showed that Nanos3 expression is correlated with tumour invasion in vitro and in vivo and is associated with decreased overall survival of patients. Moreover, Nanos3 is upregulated in vitro during experimentally induced EMT and regulates cell invasion and migration of lung tumour cells via both transcriptional and post-transcriptional regulation of EMT-associated genes.

First, we showed the ectopic expression of the germline gene NANOS3 in NSCLC and its association with a poor outcome. Indeed, ectopic high level expression of Nanos3 is an independent marker of overall survival and may therefore be helpful in prognosis of lung cancer. There is mounting evidence for the ectopic activation of germline genes during progression of several human cancer types [START_REF] Wang | Sustaining cancer through addictive ectopic gene activation[END_REF]. Thus, our study supports this concept and the close similarity between developmental EMT and metastasis-associated EMT. We also showed that Nanos3 reactivation in lung cancer cells is epigenetically regulated by DNA demethylation and histone acetylation. These data are consistent with the fact that tissuespecific patterns of CpG methylation, which are established during embryonic development, are well preserved in adult somatic cells, but become profoundly altered in cancer cells [START_REF] Muntean | Epigenetic dysregulation in cancer[END_REF].

For instance, DNA hypomethylation in tumours has been associated with transcriptional activation of several genes, most of which are expressed only in the germline. This group of genes was named cancer-germline (CG) genes and is best known in the lung tumour context [START_REF] Loriot | Five new human cancer-germline genes identified among 12 genes expressed in spermatogonia[END_REF][START_REF] Rousseaux | Ectopic activation of germline and placental genes identifies aggressive metastasis-prone lung cancers[END_REF]. Interestingly, Nanos3 is strongly expressed by adult testis germ cells but is not expressed in most healthy adult somatic tissues, with the exception of the brain. These findings are in agreement with the previous study of Julaton et al. [START_REF] Julaton | NANOS3 function in human germ cell development[END_REF]. Taken together, our data suggest that Nanos3 may be considered a CG gene.

This study also demonstrates and depicts in part the differential mechanisms by which Nanos3 regulates EMT-associated genes. Indeed, we observed that, in accordance with its dual localization in both nucleus and cytoplasm, Nanos3 transcriptionally downregulates CDH1, but regulates Vimentin post-transcriptionally. Post-transcriptional regulation of EMT is a topic of growing interest, and several mechanisms have emerged as critical in the regulation of EMT during tumour progression [START_REF] Evdokimova | On translational regulation and EMT[END_REF][START_REF] Aparicio | Posttranscriptional regulation by RNAbinding proteins during epithelial-to-mesenchymal transition[END_REF][START_REF] De Craene | Regulatory networks defining EMT during cancer initiation and progression[END_REF]. It is now well known that the EMT program can be strongly influenced by post-transcriptional mechanisms governed by microRNAs and RNA-binding proteins (RBPs) [START_REF] Aparicio | Posttranscriptional regulation by RNAbinding proteins during epithelial-to-mesenchymal transition[END_REF]. The NANOS gene family encodes a group of RBPs believed to function as translational repressors, at least in model organisms such as Drosophila melanogaster [START_REF] Curtis | A CCHC metal-binding domain in Nanos is essential for translational regulation[END_REF]. Indeed, the translational repression of hunchback in the fruitfly involves Nanos and Pumilio [START_REF] Tautz | Regulation of the Drosophila segmentation gene hunchback by two maternal morphogenetic centres[END_REF][START_REF] Irish | The Drosophila posterior-group gene nanos functions by repressing hunchback activity[END_REF][START_REF] Barker | Pumilio is essential for function but not for distribution of the Drosophila abdominal determinant Nanos[END_REF][START_REF] Sonoda | Recruitment of Nanos to hunchback mRNA by Pumilio[END_REF]. Here, we found that Nanos3 binds mRNAs encoding Vimentin showing that Nanos3 act as a RBP regulating the EMT process. RBPs are well known for controlling pre-mRNA splicing, capping, and polyadenylation, as well as mRNA export, turnover, localization, and translation [START_REF] Aparicio | Posttranscriptional regulation by RNAbinding proteins during epithelial-to-mesenchymal transition[END_REF]. In classical models, RNA-binding Nanos proteins form multisubunit translation-inhibitory complexes with the RNA-binding protein Pumilio, and control the stability and the translation of target mRNAs through regulation of their poly(A) tail length [START_REF] Curtis | A CCHC metal-binding domain in Nanos is essential for translational regulation[END_REF][START_REF] Wreden | Nanos and pumilio establish embryonic polarity in Drosophila by promoting posterior deadenylation of hunchback mRNA[END_REF]. In this study, we show that VIM mRNAs are more stable in the Nanos3 transfectants and that Nanos3 regulates the poly(A) tail length of mRNA encoding Vimentin. Our findings demonstrate that the mechanism of poly(A) tail regulation of mRNAs by Nanos family members, which is conserved across multiple species, might be dysregulated during human tumour progression. While Nanos proteins are rather known to promote mRNA deadenylation in normal cell development [START_REF] Wreden | Nanos and pumilio establish embryonic polarity in Drosophila by promoting posterior deadenylation of hunchback mRNA[END_REF][START_REF] Bhandari | Structural basis for the Nanos-mediated recruitment of the CCR4-NOT complex and translational repression[END_REF], we report here the opposite effect, with VIM mRNA poly(A) tail lengthening under the influence of Nanos3.

This may be due to the involvement of molecular partners of Nanos3 other than those active in the developmental stages of model organisms. In addition, it was recently reported that Nanos targets the CNOT6 deadenylase for degradation, allowing stable retention of inherited transcripts in primordial germ cells [START_REF] Swartz | Deadenylase depletion protects inherited mRNAs in primordial germ cells[END_REF]. In agreement with the previous study showing that

Vimentin is dowregulated by miR-30a [START_REF] Cheng | MicroRNA-30a inhibits cell migration and invasion by downregulating vimentin expression and is a potential prognostic marker in breast cancer[END_REF], we also showed that Nanos3, as a RBP, protects VIM mRNAs from miR-30a repression. As miRNAs have been reported to play an important role in the control of cancer progression [START_REF] De Craene | Regulatory networks defining EMT during cancer initiation and progression[END_REF], our finding that Nanos3 can counteract their action is of particular interest.

C

Immunohistochemistry scoring

The staining intensity was graded on a scale of 03 (0, no staining; 1, low; 2, medium; 3, high)

and the extent of staining on a scale of 04 (0, no staining; 1, < 25%; 2, 2550%; 3, 5075%; 4, > 75%). The intensity score was multiplied by the extent score to generate both cytoplasmic and nuclear scores (012), which were then combined to obtain the immunostaining score (024).

Real-time qRT-PCR for in vivo analysis

Total RNA samples from 20 human tissues, included in the FirstChoice Human Total RNA survey panel (AM6000), were purchased from Life Technologies Europe (Ghent, Belgium).

cDNA was prepared with the iScript cDNA synthesis kit according to the manufacturer's instructions (Bio-Rad). qPCR reactions were performed using the "SensiFast SYBR No-ROX kit (GC Biotech, Alphen aan den Rijn, The Netherlands)" kit as recommended by the manufacturer, in a LightCycler 480 Instrument (Roche Diagnostics). Gene expression levels were normalized using the geometric mean of the most stable reference genes [44]. Forward and reverse primers for NANOS3 are described in Supplementary Table S3.

Second EMT-inducible model HBE4-E6/E7 cells sparsely seeded on coverslip coated with type-I collagen were treated with 20 ng/mL EGF for 5 days, fixed in methanol, and analysed by immunofluorescence.

3D migration assay

Cell migration analysis in a type-I collagen gel was performed as previously described [START_REF] Joannes | Fhit regulates invasion of lung tumor cells[END_REF].

  explore the mechanisms of EMT target regulation by Nanos3, we first analysed the mRNA levels of CDH1 and VIM. Contrary to VIM transcripts, CDH1 mRNA levels were dramatically decreased upon Nanos3 overexpression in Calu-1 and SK-LU-1 cells, which indicates that Nanos3 may employ different mechanisms to regulate its targets (Fig. 4A). This hypothesis was supported by RNA immunoprecipitation (RIP) experiments. In BEAS-2B and A549 cells, VIM mRNA co-immunoprecipitated with the Nanos3 protein, whereas CDH1 mRNA did not (Fig. 4B, top). These RIP results were also validated in Nanos3overexpressing Calu-1 cells (Fig. 4B, middle) and in Nanos3-silenced NCI-H1299 and A549 cells (Fig. 4B, bottom).

  By subcellular fractionation followed by western blotting, we confirmed in the BEAS-2B cell line both nuclear and cytoplasmic localization of Nanos3 (Fig. 4C). Immunofluorescence labeling of Nanos3 in BEAS-2B cells demonstrated moderate cytoplasmic and strong nuclear localization of Nanos3 (Fig. 4D, top). By confocal laser scanning microscopy, the nuclear Nanos3 signal appeared mainly as speckles colocalized with the nucleolar marker Nucleolin (Fig. 4D, middle and bottom). The strong colocalization of these two proteins was further validated by determining Manders' (M1=0.998; M2=0.819) and Pearson's coefficients (r=0.998). These data demonstrate a dual subcellular localization of Nanos3, a finding correlating with the differential mechanisms of EMT target regulation described above.

Figure 1 :

 1 Figure 1: Nanos3 is overexpressed in non-small cell lung carcinoma tumour cells and is

Figure 2 :

 2 Figure 2: Human Nanos3 expression is restricted to brain and testis in normal adult

Figure 3 :

 3 Figure 3: Nanos3 is overexpressed during EMT induced in vitro and regulates EMT-

Figure 4 :VIM 3 '

 43 Figure 4: Differential regulation of EMT targets by Nanos3.

Table 1 :

 1 Thus, our results describe new mechanisms of gene regulation by Nanos3 in the human EMT-associated lung tumour invasion context: a repressive regulatory role at the transcriptional level for E-cadherin and a promoting regulatory role at the post-transcriptional level for Vimentin. Taken together, our findings indicate that the oncofetal induction of human Nanos3 could be useful as a new prognostic marker for NSCLC. Our study also provides new insights into the post-transcriptional mechanisms of cancer-associated EMT induction. Factors associated with overall survival

	Variables	75-month survival rate	p-value	Hazard ratio	p-value
		(95% CI)	(Log-rank test)	[95% CI]	(Cox model)
	Total	0.4676 (0.3643 -0.5643)
	Age
	< 60 years	0.5217 (0.3657 -0.6565)
	≥ 60 years	0.4200 (0.2829 -0.5510)	0.4735
	Sex
	Male	0.4598 (0.3452 -0.5669)
	Female	0.5000 (0.2593 -0.7005)	0.4797
	Histological Type
	Adenocarcinomas	0.5357 (0.3293 -0.7042)
	Squamous cell carcinomas	0.4600 (0.3188 -0.5901)
	Large cell carcinomas	0.3750 (0.0870 -0.6744)
	Others	0.4000 (0.1227 -0.6702)	0.4438
	Differentiation status
	Well	0.4359 (0.2790 -0.5827)
	Mild	0.5926 (0.3863 -0.7499)
	Poor	0.3929 (0.2167 -0.5651)	0.1844
	Tumour size
	T1 + T2	0.4993 (0.3809 -0.6066)
	T3 + T4	0.3333 (0.1365 -0.5454)	0.0244
	Lymph node metastases
	N0	0.5800 (0.4318 -0.7024)
	N1 + N2	0.3333 (0.1976 -0.4750)	0.0190
	Tumour stage
	I	0.5064 (0.3834 -0.6170)		1 [reference]
	II + III + IV	0.3478 (0.1663 -0.5371)	0.0070	2.31 [1.27 -4.19]	0.006
	Nanos3 score
	Score < 10	0.5517 (0.4154 -0.6686)		1 [reference]
	Score ≥ 10	0.3310 (0.1852 -0.4844)	0.0394	1.72 [1.02 -2.90]	0.041

ACKNOWLEDGEMENTS

This study was supported by Région Champagne-Ardenne, La Ligue Contre le Cancer (Comités de la Marne, Haute-Marne, Meurthe-et-Moselle et Yonne), Lions Club of Soissons, Villers-Cotterets, Château-Thierry and Crépy en Valois, Un Euro contre le Cancer, Fond National pour la Santé ACI 2004-2010 INCa (Cancéropôle Grand-Est project), Research Foundation -Flanders (FWO-Vlaanderen), Foundation against Cancer -Belgium, Fonds National de la Recherche Scientifique (FRS-FNRS, Belgium) and Partenariat Hubert Curien-Tournesol. The authors thank Eymeric Lagonotte (INSERM UMRS 903) for technical assistance and Amin Bredan (VIB IRC) for critical reading and editing of the manuscript.

AUTHOR CONTRIBUTION STATEMENT

SG, PB, FVR and BNR designed the study. SG, VA, KS, APM, CK, CT, C-W C, C-Y S and BNR performed the experiments. SG, VA, MP, CG, VD, C-W C, C-Y S, PB, FVR and BNR analysed data. All authors were involved in writing the manuscript and had final approval of the submitted version. matrix (CSK) fractions, and the fractions were analysed by western blotting for Nanos3.

Efficiency of the fractionation was controlled using the cytoplasmic marker GAPDH, the membrane marker α1-Na/K-ATPase, the nuclear marker PARP, and the cytoskeletal marker

Supplementary materials and methods

Cell culture

All culture media and reagents were from Gibco (Invitrogen, Carlsbad, CA, USA). Cells used for experiments were passaged for fewer than six months after resuscitation. A549 and BEAS-2B were cultured in DMEM containing 10% fetal calf serum (FCS), NCI-H1299 in RPMI containing 10% FCS, Calu-1 and SK-LU-1 in MEM containing 10% FCS and supplemented with 1x MEM non-essential amino acids and 1 mM sodium pyruvate, and HBE4-E6/E7 in Keratinocyte-SFM supplemented with 0.2 ng/ml EGF and 25 µg/ml bovine pituitary extract.

Antibodies

Antibodies used for western blotting (WB), immunohistochemistry (IHC), Abcam, Cambridge, UK), α1-Na + -K + (WB: 1:5000; clone 464.6, Abcam) and GAPDH (WB: 1:75000; clone 6C5, Chemicon, Millipore, Billerica, MA, USA). Nanos3 antibody was characterized for its specificity and sensitivity in western blotting and immunohistochemistry (Supplementary Fig. S3).

Supplementary figure legends

Supplementary video

Supplementary video S1: Nanos3 silencing decreases 2D migration.

Comparison of migratory capacities of NCI-H1299 cells transfected with scrambled (left) or

Nanos3 siRNA 1 (right) in a 2D migration assay. A phase-contrast image, observed at a 16-fold magnification, was recorded every 5 min for a total of 20 h. Video duration: 12 s.

Supplementary reference

Supplementary Table S1

Description of the population studied by immunohistochemistry.

Variables


domain: spi chim phys

text:
Xin Zhao

Zhaogang Nie
email: zgnie@gdut.edu.cn

Yuhua Feng

Weiren Zhao

Jiahua Zhang

Wenchun Zhang

Paolo Maioli
email: paolo.maioli@univ-lyon1.fr

Zhi-Heng Loh

Ultrafast Acoustic Vibrations of Au-Ag Nanoparticles with Varying Elongated Structures

Keywords: Noble-metal nanoparticles, Nanoacoustics, Pump-probe spectroscopy, Finite element modeling

Acoustic vibrations of Au and Ag elongated nano-objects with original morphologies, from Ag-Ag homodimers to Au@Ag-Ag heterodimers and Au@Ag eccentric core-shell spheroids, have been experimentally investigated by ultrafast time-resolved optical spectroscopy. Their frequencies, obtained by the analysis of time-dependent transient absorption changes, are compared with results from Finite Element Modeling (FEM) numerical computations, which allow assignment of the detected oscillating signals to fundamental radial and extensional modes. FEM was further used to analyze the effects of morphology and composition on the vibrational dynamics. FEM computations indicate that (1) the central distance between particles forming the nanodimers have profound effects on the extensional mode frequencies and a negligible influence on the radial mode ones, in analogy with the case of monometallic nanorods, (2) coating Au with Ag also has a strong mass-loading-like effect on the dimer and core-shell stretching mode frequency, while (3) its influence on the radial breathing mode is smaller and analogous to the non-monotonical frequency dependence on Au fraction previously observed in isotropic bimetallic spheres. These findings are significant for developing a predictive understanding of nanostructure mechanical properties and for designing new mechanical nanoresonators.

Introduction

Over the past few decades, noble-metal nanoparticles have received an increasing attention due to their original optical and mechanical properties. [1][2][3][4][5] The most intriguing one is represented by their localized surface plasmon resonance (SPR), whose characteristics are sensitive to size, shape, environment and composition. [1][2][3][4][5][6][7] The optical absorption and scattering enhancement induced by the SPR allows to investigate their vibrational dynamics by detecting their time-resolved optical response with ultrafast laser techniques. 3,8,9 Information carried by the study of the vibrational dynamics is of both fundamental and technological interest. For example, it allows to address the influence of particle size, morphology, interface conditions and mutual mechanical contacts on the nanostructure vibrations, 8 to characterize the coupling with the surrounding environment, [9][10][11][12] and to design ultrasensitive mechanical nanobalances that detect frequency shift of vibrational modes when a small mass deposits on the particle surface. [13][14][15][16] Currently, there is a rapidly growing interest in Au-Ag bimetallic nano-objects since they present not only a combination of properties associated with monometallic Au or Ag, but also novel performances due to their synergistic effects. [17][18][19][20] However, their acoustic signatures have been little investigated in experiments. [21][22][23][24][25] So far, some Au-Ag bimetallic nanosystems, such as alloyed spheres, 21,23,25 nanoboxes and nanocages, 22 and segregated spheres, 25 nanorods 24 and bipyramids 16 were studied by Raman spectroscopy and time-resolved pump-probe experiments.

Other experimental reports focused on concentric spherical and rod-shaped hetero-structures with Au or Ag as the core, but with other metal or dielectric as the shell. [25][26][27][28][START_REF] Kirakosyan | Vibrational Modes of Metal Nanoshells and Bimetallic Core-Shell Nanoparticles[END_REF] All these results show excellent mechanical coupling of bimetallic components and an increasing quantitative understanding of their interactions.

Here, we report on the coherent excitation and detection of vibrational modes in Au-Ag composite nano-objects using femtosecond time-resolved pump-probe spectroscopy. The object structures (see Fig. 1) are varied from Ag-Ag homodimers to more singular bimetallic morphologies such as Au@Ag-Ag acorn-shaped heterodimers, made of an Au core-Ag shell Au@Ag sphere and a second monometallic larger Ag sphere, and Au@Ag eccentric core-shell spheroids, made of an eccentric Au core embedded in an Ag shell. Two vibrational modes are systematically detected, attributed to vibrations along radial and axial directions. Finite Element Modeling (FEM) analysis supports the vibrational mode assignments and permits us to finely analyze the influence of particle morphology and composition on the acoustic response of elongated nanoparticles.

Methods

Sample synthesis and characterization

The samples were chemically synthesized using a seed-mediated growth technique in aqueous solution, 20 described in detail in Section 1 of the Electronic Supplementary Information (ESI).

Citrate-stabilized Au or Ag nanoparticles with 2-mercaptobenzoimidazole-5-carboxylic acid (MBIA) ligands were used as seeds for growing Ag. By varying ligand conditions (MBIA concentration and incubation temperature) during the growth of Ag on Au or Ag seeds, the morphology of composite nanoparticles can be continuously tuned from concentric to eccentric core-shell nanoparticles, acorn-like shapes and dimers. 20 This allowed the synthesis of three samples containing nanoparticles with different structures (Fig. 1e): (1) Ag-Ag homodimers, (2) Au@Ag-Ag acorn-shaped heterodimers (also referred to as acorns) and (3) Au@Ag eccentric core-shell prolate spheroids (i.e. ellipsoids with two equal minor axes). Analysis of Transmission Electron Microscopy (TEM) images (Fig. 1a-c) indicate mean particle sizes in the 50-100 nm range with standard deviations around ~10 nm. Morphological parameters are listed in Table 1.

Table 1 Sizes of Ag-Ag dimers, Au@Ag-Ag acorn-shaped dimers and Au@Ag eccentric coreshell nanospheroids. The Au@Ag core-shell sphere forming Au@Ag-Ag acorns is made of a 51 nm diameter inner Au sphere surrounded by a 5 nm thick Ag shell. The 65 nm seed Ag sphere in Ag-Ag dimers contains an inner Au core with a diameter of around 10-15 nm. For the synthesis of the Ag-Ag dimers (sample 1), Au@Ag concentric core-shell nanoparticles with ~65 nm external diameter were firstly synthesized following a literature report. 30 The small Au core, with a diameter varying from 10 to 15 nm, was necessary to grow large Ag spheres with low size dispersion. The Au@Ag nanoparticles were subsequently used as seeds for growing a second Ag sphere, resulting in the formation of dimers (Fig. 1a). As discussed later, the impact of the small Au core (corresponding to a volume equivalent to less than 1% of the volume of the sphere) on the periods of lowest-frequencies vibrational modes, which are the main focus of this work, is insignificant. Thus, for simplicity, these dimers will be simply referred to as monometallic Ag-Ag dimers in the following. For synthesizing Au@Ag-Ag acorn-shaped dimers (sample 2)

and eccentric core-shell nanospheroids (sample 3), citrate-stabilized pure Au nanoparticles, 6 synthesized by a citrate-reduction method, were used as seeds. The thickness of the Ag shell embedding the Au nanospheres in Au@Ag-Ag acorns and the one of the thinnest Ag shell in Au@Ag eccentric core-shell nanoparticles is around 5 nm.

Steady-state absorption spectra of nanoparticles in water are shown in Fig. 1d. Three absorption bands are distinguishable: longitudinal SPR, centered around 600 to 650 nm, 20 Ag transverse plasmon-induced absorption around 425 nm and weak Au transverse plasmon-induced absorption around 510 nm (superposed to Au interband absorption 31 and absent in the Ag-Ag dimer sample).

Prior to deposition on carbon TEM grids, to avoid aggregation and better distinguish their boundaries, nanoparticles were encapsulated with a ~12 nm thin polymer shell of PSPAA (polystyrene-block-poly(acrylic acid), white layer around the dark metal particles, in Fig. 1a-c).

The Au seeds and the Ag islands grown on the seeds are all polycrystalline. Purity of the synthesized nanoparticles was very high (~98% out of ~500 nanoparticles surveyed). Azidefunctionalized Au nanorods with similar dimensions were also prepared and used as a reference for time-resolved optical experiments and their analysis (ESI Section 5).

Time-resolved optical spectroscopy

Vibrational dynamics of nanoparticles in water was investigated by ultrafast time-resolved optical spectroscopy. Broadband femtosecond pump pulses, with a fluence of around 0.1 mJ/cm 2 and covering the spectral range from ~500 to 900 nm, were used for impulsive excitation, the sample optical response being followed by monitoring the relative variations ∆T/T of the optical transmission of a time-delayed probe pulse with wavelengths tunable from 540 to 590 nm (on the blue side of the strongest absorption band, see Fig. 1d, refer to Section 2 of ESI for details of the optical setup). Pump and probe beams are orthogonally polarized to minimize the effects from coherent artifacts. A time resolution of ~52 fs fwhm for the apparatus was revealed by a second-order intensity cross-correlation between pump and probe pulses (See Fig. S1c). For time-resolved measurements, nanoparticles solutions (with no polymer) were contained in 1-mm thick fusedsilica cuvettes.

Results and Discussions

Time-resolved detection of mechanical vibrational modes

Time-resolved relative transmission changes ∆T/T for Ag-Ag dimers, Au@Ag-Ag acorns and Au@Ag eccentric core-shell spheroids are portrayed in Fig. 2a-c and Fig. S2a-c. After electron excitation by absorption of the pump pulse and internal thermalization, energy is transferred from electrons to the lattice by electron-phonon interactions, within a timescale of a few picoseconds.

The fast lattice temperature increase launches nanoparticle mechanical acoustic vibrations, which translate into periodic oscillations of time-resolved optical transmission (black lines in Fig. 2a-c), induced by the spectral modulation of central SPR positions. 3,8,[31][32][33] As expected, oscillations are all in-phase (see Fig. S2d-f), as probe wavelengths all lie on the same side of longitudinal and transverse absorption bands. 34

= + + ∑ + (1)
where Ae-p and Ath describe the amplitudes of the decaying exponentials and Av, τv, Tv and φv are amplitude, damping time, period and phase, respectively, of the acoustic oscillations. The fast electron cooling dynamics via electron-phonon coupling has a characteristic time of a few picoseconds, 35 while the slow thermal dissipation to the surrounding water matrix shows a typical timescale longer than 100 ps. The values of vibrational periods deduced by fits (red lines in Fig. 2a-c, the two oscillating components of the fits being shown in the insets), which are the main focus of this work, are reported in Table 2. Fourier transform (FT) of the oscillating contributions to the ΔT/T signals (after subtraction of the decaying exponentials) is shown in Fig. 2d-f. For all the samples, the two frequencies obtained correspond to the periods (T1 and T2) deduced by the fits in the time domain. The shorter period for all the samples is around 20 ps (~50 GHz), longer than the time constant (τe-p) of the electron to lattice energy transfer, of a few ps, responsible for impulsive vibration launching. 3,8

Frequency analysis by comparison with FEM

To clarify the identity of the vibrational modes, FEM computations were performed introducing the samples sizes deduced by TEM images and simulating elastic response using polycrystalline elastic constants for gold and silver [START_REF] Ledbetter | Monocrystal Elastic Constants and Derived Properties of the Cubic and the Hexagonal Elements: in Handbook of Elastic Properties of Solids, Liquids, and Gases[END_REF] (also see ESI Section 4). FEM was used to (1) numerically compute the vibrational eigenmodes corresponding to each specific sample morphology and (2)

identify the ones which are most excited in time-resolved optical experiments, i.e. those whose atom displacements best match the uniform spatial dilation induced by the pump excitation. 8,[START_REF] Crut | [END_REF] Note that FEM computations are made for objects in vacuum, which is justified as vibrational periods are almost unchanged when in a liquid environment (period decrease by ~0.2% in the case of spherical nanoparticles) compared to vacuum. 38 The rationality of FEM is also verified by supplementary time-resolved investigations on Au nanorods with consistent experimental and theoretical results (see ESI, Section 5).

Ag-Ag dimers. Fig. 3a displays the FEM-computed displacement profiles of the most excited vibrational modes of Ag-Ag dimers, corresponding to a fundamental axial stretching-like mode (ASM, Fig. 3a left) and radial breathing-like mode (RBM, right). As already mentioned, all FEM computations on the Ag-Ag dimer morphology have been made considering an Ag monometallic dimer, i.e. disregarding the small Au core at the center of the 65 nm Ag sphere used as seed for growing the second Ag sphere. This is justified as the expected change of the dimer ASM and RBM frequencies due to inclusion of the small Au core (<1% Au atomic fraction), compared to the case of a monometallic Ag sphere, is negligible (within numerical precision), as preliminarily benchmarked and also in analogy with the case of the breathing mode of Au@Ag spheres previously reported (the influence of this internal structuration is expected to be much larger for higher frequency vibrational modes). 25 The detected mode with the longer period (T2, red square in Fig. 4a) is assigned to dimer ASM (Fig. 4a, open diamond corresponding to the same horizontal position), the one with the shorter period (T1, black dot) is attributed to dimer RBM (open triangle).

The dimer RBM displacement profile (Fig. 3a Conversely, the dimer ASM displacement profile (Fig. 3a left) is induced by the anisotropic quadrupolar-mode-like vibrations of the spherical components (whose displacements consist in periodical particle expansion and contractions along one direction with synchronous out-of-phase deformation in the perpendicular plane, see Fig. S4) and its computed period increases with the length of the dimer (Fig. 4a, open diamonds), in analogy with the case of extensional mode of monometallic nanorods [START_REF] Crut | [END_REF]39 . Note that individual quadrupolar modes of the two spheres forming the Ag-Ag dimers, predicted at ∼45 ps (Fig. S4), are not detected, in agreement with previous investigations on single Au-Au dimers. 40 Agreement of FEM-computed Ag-Ag dimer RBM and ASM periods (Fig. 4a, open triangle and diamond) with experimental values (solid dot and square) is good, and limited mainly by the relatively large size dispersion of the sample morphologies, used as input parameters for FEM computations, and by the choice of the polycrystalline Ag elastic constants (different sets of elastic constants predicting slightly different frequencies). 41 In addition, even though they are polycrystalline, nanodimers are composed by very few grains, 20 thus their elastic response (particularly the frequencies of radially anisotropic axial stretching modes, which are more dependent on crystallinity 3,[START_REF] Crut | [END_REF] ) may deviate from the one of purely polycrystalline nanoobjects.

Au@Ag-Ag acorn-shaped heterodimers. In the case of acorn-shaped bimetallic dimers, FEM computations (Fig. 3b) predict an ASM period depending sensitively on the composition (Fig. 4b, open diamonds). When the smaller 61 nm diameter core-shell Au@Ag sphere (Au core diameter of 51 nm and Ag shell thickness of 5 nm, corresponding to ~58% Au fraction) is replaced by a monometallic Ag (Au) sphere indeed, the dimer ASM period is expected to decrease (increase) significantly (Fig. 4b, open diamonds, horizontal axis indicate the varying composition). This behavior suggests a mass-loading-like effect on the ASM dynamics, the metal with a larger density (ρAu = 19284 Kg/m 3 larger than ρAg = 10501 Kg/m 3 ) inducing slower extensional vibrations, in analogy to previous computations on Au nanorods with Pd deposited on their tips. 27 Due to the larger difference (compared to Ag-Ag dimers) between the diameters of the two spheres composing the acorn-shaped heterodimers, different RBM vibrational modes are expected to be observable in time-resolved experiments, whose displacement profiles contain mixed contributions from both spheres but essentially correspond to mechanical breathing of the coreshell (periods indicated as left-pointing open triangles in Fig. 4b, corresponding to the displacement profile represented in Fig. 3b) and of the larger Ag sphere (Fig. 4b, right-pointing triangles, two dimer modes with breathing-like displacements of the larger Ag spheres being identified in the case of the Au@Ag-Ag composition), all in qualitative agreement with the detected shorter period (black dot). Note that, due to the significant particle-to-particle size and shape dispersion, coherent effects such as beating of oscillations induced by different predicted RBM are not observed. The dependence of the dimer RBM period corresponding to mechanical breathing of the core-shell sphere on its composition (left-pointing open triangles in Fig. 4b) is small and non-monotonic: starting from the pure Ag-Ag dimer and replacing the Ag sphere first by a core-shell Au@Ag sphere (corresponding to the experimental sample) and then by a monometallic Au sphere induces a reduction and a subsequent increase of predicted RBM periods.

Interestingly, this behavior is in agreement with the trend of the breathing mode period of isolated bimetallic spheres, as shown both by FEM computations (Fig. 4b, crosses) and also previously analytically predicted (through solution of Navier equation with continuous boundary conditions for displacement and stress at the core-shell interface) and experimentally demonstrated. 25 The predicted ASM period is 15% larger than the one observed. This discrepancy can again be attributed to the large error bars in the determination of the nanodimers morphology, i.e. in the deduction of the total length and, more importantly, of the Ag shell thickness, as considering an Ag shell thickness larger than 5 nm would induce a fast reduction of expected ASM period (Fig. 4b). Time-resolved experiments on single nano-objects (with parallel morphology determination by electron microscopy imaging) may lead to a better agreement with the predicted ASM period. 12,40 Au@Ag eccentric core-shell nanoparticles. Analogously, FEM-computed ASM periods of bimetallic Au and Ag nanospheroids (Fig. 4c, open diamonds) manifest a strong increase (~30%) when replacing Ag with Au, the ASM period of the Au@Ag eccentric core-shell spheroid (corresponding to the experimentally investigated sample, computed displacement profile shown in Fig. 3c) being intermediate. This dependence may also be viewed as a mass-loading-like effect, inclusion of the material with a larger density slowing down the extensional motion. Again, corresponding RBM periods (Fig. 4c, open triangles) show the non-monotonic dependence on composition theoretically and experimentally reported for bimetallic spheres, which underlines the analogy between the RBM motion of elongated nanoparticles and breathing mode of isotropic nanospheres. Experimental ASM and RBM periods are again in good agreement with the ones numerically computed. Interestingly, FT of the time-resolved signal on Au@Ag eccentric coreshell nanoparticles allows to distinguish an additional peak (Fig. 2f), with a frequency (∼24 GHz) intermediate between those of ASM and RBM modes. This peak is originated by a hybrid mode whose displacement profile (Fig. S5b) basically corresponds to the first harmonic of dimer axial stretching, characterized by dimer extension along the long axis with synchronous dilation of the equatorial plane and contraction of the two intermediate orthogonal planes (see also Fig. S5a for representation of this mode in the simplified case of an homogeneous spheroid).

Conclusions

In conclusion, fundamental RBM and ASM of Au and Ag elongated nano-objects, from monometallic Ag dimers to bimetallic acorn-like shapes and eccentric core-shell spheroids, were coherently excited and probed by femtosecond time-resolved optical spectroscopy. Analysis of transient absorption signals yields acoustic vibrational frequencies whose assignment to specific vibrational modes was accomplished via FEM numerical computations, which were further used to analyze the effects of particle morphology and composition on the vibrational dynamics. The axial extensional periods increase with the length of the elongated structures, and with inclusion of Au at the place of Ag, highlighting a mass-loading-like effect. The RBM mode shows a negligible frequency dependence on the nano-objects length, while its dependence on the constituting metals shows a non-monotonic trend, previously observed in isotropic bimetallic Au@Ag spheres. These findings may contribute to the understanding of mutual interactions in bimaterial nano-objetcs and to develop mechanical applications of Au-Ag bimetallic nanostructures such as mechanical nanotransducers.

  right) is similar to the one of the in-phase fundamental radial breathing mode of the two isolated Ag spheres (see Fig.S4), whose period (black cross in Fig.4a, corresponding to the breathing mode of an isolated Ag sphere with the diameter of the larger sphere composing the Ag-Ag dimer) is close to the one numerically computed for the dimer RBM (open triangle). Dimer RBM period is thus approximately independent of the dimer length (Fig.4a, open triangles, computed for three different dimer lengths, corresponding to different center-to-center distances of the two spheres composing the dimer).

Fig. 1

 1 Fig. 1 TEM images of (a) Ag-Ag dimers, (b) Au@Ag-Ag acorn-shaped heterodimers, and (c)

Fig. 2 Fig. 3 FEM

 23 Fig. 2 Time-resolved differential transmission changes ∆T/T (black lines) measured with a 570

Fig. 4

 4 Fig. 4 Experimentally detected RBM (black dots) and ASM (red squares) period for (a) Ag-Ag

Table 2

 2 Vibrational periods (T1 and T2) deduced by the fits of time-resolved pump-probe signals on the three metallic samples.

	Samples	T1 (ps)	T2 (ps)
	Ag-Ag dimers	19.7 ± 1.2	69 ± 3
	Au@Ag-Ag acorns	17.7 ± 1.0	84 ± 5
	Au@Ag spheroids	21.0 ± 2.0	82 ± 3

Acknowledgements

The authors gratefully acknowledge Aurélien Crut for his help on acoustic FEM. This work is financially supported by the National Natural Science Foundation of China (grant nos. 11774071, 11874125, and 11704079), the Science and Technology Program of Guangzhou (grant nos. 201804010451 and 201904010104), the State Key Laboratory of Luminescence and Applications (grant no. SKLA-2019-08), Pearl River Talent Programme in Guangdong Province (2017GC010251) and by a NTU start-up grant, the A*STAR Advanced Optics in Engineering Program (122 360 0008).

Conflicts of interest

There are no conflicts to declare.