Marion Mathelié-Guinlet

Laure Béven

Fabien Moroté

Daniel Moynet

Christine Grauby-Heywang

Ibtissem Gammoudi

Marie-Hélène Delville
email: marie-helene.delville@icmcb.cnrs.fr

Touria Cohen-Bouhacina
email: touria.cohen-bouhacina@u-bordeaux.fr

Historical perspective Probing the threshold of membrane damage and cytotoxicity effects induced by silica nanoparticles in Escherichia coli bacteria

Keywords:

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

For the past decades, nanotechnologies field has experienced a fast and growing development in many areas (microelectronics, medical imaging, cosmetics…) because of the specific physico chemical proper ties of nanoparticles (NPs). Though, this enthusiasm began to be questioned as NPs might put environmental safety and human health at risk by interacting with biological systems and affecting their behavior at the cellular level [ 1 7]. The diversity of NPs in terms of composition, shape and size/diameter (Φ) challenges politicians for regulating their safe use, and scientists for understanding how they interact with living organisms [START_REF] Beddoes | Understanding nanoparticle cellular entry: a physicochemical perspective[END_REF][START_REF] Tolaymat | An evidence-based environmental perspective of manufactured silver nanoparticle in syntheses and applications: a systematic review and critical appraisal of peerreviewed scientific papers[END_REF]. Such regulation and understanding are, though, necessary for the safe development of various applications like diagnosis in medicine [10 12], smart textiles conception [START_REF] Dastjerdi | A review on the application of inorganic nano-structured materials in the modification of textiles: focus on anti-microbial properties[END_REF][START_REF] Yetisen | Nanotechnology in textiles[END_REF], environmental treatments and detection sensors [START_REF] Oliveira | Nanomaterials for diagnosis: challenges and applications in smart devices based on molecular recognition[END_REF][START_REF] Sanvicens | Nanoparticle-based biosensors for detection of pathogenic bacteria[END_REF].

Among widely used biological systems, Escherichia coli is the most thoroughly studied species of bacteria. Ubiquitous in the environment, E. coli are Gram negative bacteria which, along with the cytoplasmic membrane and a peptidoglycan layer, possess an outer membrane (OM) composed of a phospholipid bilayer, containing lipopolysaccharide (LPS) molecules and proteins [START_REF] Nikaido | Molecular basis of bacterial outer membrane permeability revisited[END_REF]. Potential human infections and diseases need to be considered as a consequence of exposure to several E. coli strains. The strain E. coli MRE 162 that we used in this work was first studied during the Lyme Bay trials in 1966 in order to mimic real biological attacks and to determine how United Kingdom (UK) popula tions could be at risk. Releasing massive amount of such bacteria, UK militaries reported chest and blood infections, caused by inhalation of E. coli MRE 162, especially in individuals highly susceptible to disease and exposed to contaminated clouds [START_REF] Spratt | Independent review of the possible health hazards of the large-scale release of during the dorset defence trials n[END_REF]. Studies on the antibacterial activity of NPs towards other E. coli strains showed that diverse metal oxides [START_REF] Kaweeteerawat | Toxicity of metal oxide nanoparticles in Escherichia coli correlates with conduction band and hydration energies[END_REF], iron [START_REF] Lee | Bactericidal effect of zerovalent iron nanoparticles on Escherichia coli[END_REF] copper and silver [START_REF] Yoon | Susceptibility constants of Escherichia coli and Bacillus subtilis to silver and copper nanoparticles[END_REF] based nanomaterials exhibit a bactericidal activity. Major toxicity mechanisms include the disruption of the bacterial membrane integrity leading to the leakage of intracellular components [START_REF] Sahoo | Probing the cellular damage in bacteria induced by GaN nanoparticles using confocal laser Raman spectroscopy[END_REF], the production of reactive oxygen species (ROS) damaging bacterial con stituents [START_REF] Yoon | Susceptibility constants of Escherichia coli and Bacillus subtilis to silver and copper nanoparticles[END_REF] and/or the dissolution of NPs into their constituting ions interfering with the bacterial metabolism [START_REF] Singh | Evaluation of antibacterial activity of ZnO nanoparticles coated sonochemically onto textile fabrics[END_REF]. These mechanisms depend on multiple factors (composition, chemical functionalities, shape), especially the size and charge of NPs [ 29 33]. For instance, Beddoes et al. have presented an overview of in vivo and in vitro studies on human cells, and membrane models simulations, reporting that (i) small NPs (tens of nm) translocate very efficiently through the membrane causing cellular damage while large NPs (hundreds of nm) exhibit strong cellular uptake without toxicity; (ii) positively charged NPs disrupt membrane integrity and are more toxic than negatively charged NPs [START_REF] Beddoes | Understanding nanoparticle cellular entry: a physicochemical perspective[END_REF].

Because of their relatively low toxicity towards eukaryotic cells, their biocompatibility and their easy surface modifications, silica NPs (SiO 2 NPs) are good candidates for biomedical imaging, drug delivery, and biosensor applications [START_REF] Tan | Bionanotechnology based on silica nanoparticles[END_REF][START_REF] Wang | Mesoporous silica nanoparticles in drug delivery and biomedical applications[END_REF]. Interestingly, SiO 2 NPs, not per se but functionalized with either photosensitizing molecules or antibiotics, or anchored to nanohybrid materials are promising both in bacterial detection [START_REF] Pham | Synthesis, photophysical properties and application of dye doped water soluble silica-based nanoparticles to label bacteria E. coli O157:H7[END_REF] and antibacterial activity [START_REF] Liu | Antibacterial mechanisms of silica/ polydopamine/silver nanoparticles against gram positive and gram negative bacteria[END_REF]. Despite this great potential, the interactions of non functionalized SiO 2 NPs with bacteria are not well documented in the literature. Most studies on toxicity of such NPs towards microorganisms rely on colorimetric methods or on Colony Forming Units (CFU) counting which allow the quantification of the minimum bactericidal concentration and the determination of the percentage of dead cells [START_REF] Adams | Comparative eco-toxicity of nanoscale TiO2, SiO 2 , and ZnO water suspensions[END_REF][START_REF] Lewinski | Cytotoxicity of nanoparticles[END_REF]. However, even if the death of cells is a key parameter, their behavior in the presence of bactericidal com pounds is also crucial to investigate and to understand the underlying toxicity mechanisms and to distinguish between the effects of different parameters such as NPs size and charge. Some authors used transmission electron microscopy (TEM) to observe the morphological damages induced to E. coli cells by ZnO and Ag NPs [START_REF] Brayner | Toxicological impact studies based on Escherichia coli bacteria in ultrafine ZnO nanoparticles colloidal medium[END_REF][START_REF] Sondi | Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gram-negative bacteria[END_REF]. Confocal Raman spectroscopy molecular inter pretation for such damages was also performed for GaN NPs interacting with different bacterial strains [START_REF] Sahoo | Probing the cellular damage in bacteria induced by GaN nanoparticles using confocal laser Raman spectroscopy[END_REF]. In this context, atomic force microscopy (AFM) is also another technique which provides comple mentary opportunities (i) to observe biological systems at a nanometer scale, both in air and in physiological environment, and (ii) to probe their nanomechanical properties in real time [42 44]. Under these conditions, AFM is thus a pertinent technique to analyze bacteria morphological and rheological damages induced by potentially anti bacterial agents, such as NPs, with details at the nanoscale.

In the present work, our main objectives were to better understand the process of interaction of SiO 2 NPs with E. coli MRE 162, for which, to our knowledge, it is the first study of exposure to NPs. We aimed at determining a potential size and charge threshold, for which SiO 2 NPs would be toxic towards E. coli. NPs of 100 nm in diameter and larger have been shown to exhibit similar behaviors so that the herein considered range (4 nm to 100 nm) will focus on all the opened questions about size dependent biotoxicity of NPs [START_REF] Shang | Engineered nanoparticles interacting with cells: size matters[END_REF][START_REF] Bannunah | Mechanisms of nanoparticle internalization and transport across an intestinal epithelial cell model: effect of size and surface charge[END_REF]. These SiO 2 NPs were characterized in terms of size distribution and aggrega tion state in solution. We first quantified the bacterial viability upon incubation with such particles. Then, we focused on how the presence of SiO 2 NPs of different sizes and charges disturbs the bacteria morphology and the mechanical properties of their membrane, by taking advantage of AFM in tapping mode and finally correlated these data with the viability results.

Results

Size and charge characterization of SiO 2 NPs

Dynamic Light Scattering (DLS) measurements (Table 1) revealed that, for the largest SiO 2 NPs -(Φ ≥ 50 nm), the average hydrody namic diameter was larger than the primary particle sizes obtained by TEM. On the contrary, for the smallest SiO 2 NPs -(Φ < 50 nm), it was underestimated as compared to values provided by manufacturers and TEM. Size distributions, obtained by statistics on TEM clichés, suggest that all SiO 2 NPs are quite monodisperse (Fig. S1).

Zeta potential measurements (Table 1) indicated that, as expected, bare SiO 2 NPs -were highly negatively charged at pH 5.5 resulting in the stability of the suspensions and a minimal aggregation state for these NPs. After functionalization with APS, at pH 5.5, SiO 2 NPs exhibited, as expected, highly positive charges (SiO 2 NPs + ), also resulting in a good colloidal stability.

Thereafter, these well defined SiO 2 NPs were studied in interaction with E. coli cells (i) at the macroscopic level with viability tests and (ii) at the nanometer scale with AFM observations.

Effects of SiO 2 NPs -on E. coli cells viability

The potential antibacterial effect of SiO 2 NPs -was estimated by counting CFUs generated after treatment by a solution of NPs at a concentration of 1 g/L, on LB agar plates, and normalizing them with those of E. coli alone (OD = 0.01, control). Though most of such experiments required buffer solutions, ours were performed in ultra pure water for convenient reasons (AFM characterizations afterwards). No mortality is observed in this solvent (results not shown) in agreement with previous studies showing the strong resistance of E. coli in drastic environment, depending on pH and temperature for instance [47 49]. The development of CFUs is known to depend on two contributions: (i) the initial distribution of the bacteria in suspension and (ii) the toxicity of the environment, i.e. its capability, after potential treatment, to generate a CFU. Thus, the influence of the presence of SiO 2 NPs -is investigated under these perspectives.

Under static conditions, two sets of behavior were observed: (i) SiO 2 NPs -100 had no detrimental effect on the bacteria, with a CFU number close to those of the control, while (ii) the presence of smaller SiO 2 NPs -(4 nm to 80 nm) did induce an increase in these CFUs (Fig. 1a). In addition, the clustering state of E. coli cells upon SiO 2 NPs - treatment was studied by dark field microscopy (see Fig. 1b for representative images). In controls, E. coli were present as isolated cells as well as small aggregates. The same organization was observed in the presence of SiO 2 NPs -80 and 100. By contrast, in the presence of SiO 2 NPs -4, 10 and 50, the number of isolated cells was increased. Such splitting of cells clusters is likely at the origin of the CFUs increase (Fig. 1c), interactions between such SiO 2 NPs -and E. coli being stronger than cell cell interactions. Similar behaviors were observed for E. coli incubated with SiO 2 NPs -during 24 h, suggesting that their full activity is reached in 2 h (Fig. S2).

However, this technique appears insufficient to probe any toxicity phenomena of SiO 2 NPs -as they could combine, more or less, with splitting effects (Fig. 1c).

Consequently, AFM experiments were carried out to better under stand, visualize and characterize the type of interaction of SiO 2 NPs - with E. coli and their potential deleterious effects.

Morphological characterization of E. coli bacteria by AFM

Before investigating their interactions with SiO 2 NPs, the morphol ogy and the structure of E. coli cells alone were characterized. In air, E. coli bacteria exhibited a typical rod shape, convention name that is widely used for such bacteria [START_REF] Daniel | Control of cell morphogenesis in bacteria: two distinct ways to make a rod-shaped cell[END_REF] (Fig. 2a). E. coli length, width and height were estimated, on average, at 2.0 μm, 1.2 μm and 0.3 μm, respectively. Also, E. coli surface showed a "brain like" aspect not only from the morphology point of view (Fig. 2b) but also from the rheology point of view with nanodomains, referred as "ripples" thereafter (Fig. 2c). The nanodomains were on average 3.9 ± 1.5 nm in height and 39.9 ± 7.4 nm in width. This particular phase separation pattern is due to the presence of an outer membrane (OM) in Gram negative bacteria. Based on the AFM tip interactions with the sample, phase contrasts on E. coli stand for membrane constituents with either different mechanical properties or different molecular organizations: ripples on the one hand and inter ripples on the other hand.

Morphological and structural characteristics of E. coli membrane being characterized, its interactions with SiO 2 NPs are hereafter studied, under these perspectives.

AFM study of the damages induced by SiO 2 NPs -on E. coli cells

The potential impacts of SiO 2 NPs on the morphology and structure of E. coli cells are discussed hereafter, in terms of size dependency. In order to efficiently observe these damages we had to control the quantity of added SiO 2 NPs so as to image both SiO 2 NPs and bacteria the SiO 2 NPs concentration was then reduced from 1 to 0.1 g/L.

When treated with SiO 2 NPs -at a concentration of 0.1 g/L, E. coli cells were present on the substrate as isolated cells or aggregates, as in untreated samples. All SiO 2 NPs -accumulate not only around and on bacteria but also on the substrate, as free isolated or aggregated particles (Fig. 3a b). Because of their higher stiffness, SiO 2 NPs appear as brighter zones than bacteria in phase images, which also show their respective distribution on the substrate (Fig. 3c d).

When exposed to SiO 2 NPs -80 and 100, E. coli cells remained rod shaped and displayed the same height as control cells (Fig. 4a b). By contrast, in the presence of SiO 2 NPs -4, 10 and 50, E. coli morphology evolved from rod like to more spherical shape with up to 50% decrease in their length (Fig. 4c e).

After exposure to large SiO 2 NPs -80 and 100, E. coli cells not only exhibited an unchanged global morphology but also an unmodified phase separation pattern of its envelop, which remained similar to that of healthy unexposed cells (Fig. 5a b and Fig. S3). As soon as smaller SiO 2 NPs -(4, 10 and 50 nm) were used, strong detrimental cell membrane reorganizations were observed along with morphological changes. Indeed, the brain like structure of the OM of E. coli was completely altered as compared to the OM structure of unexposed cells (compare Fig. 5a,c and Fig. S3): the ripples nanodomains turned into island like structures and several areas in the OM even lack any regular organization. At this point, the reorganization might arise from either a (i) different molecular packing, i.e. the way molecules of the OM arrange themselves on the surface, or (ii) a different molecular conformation, i.e. the way molecules of the OM self orientate them selves at the air bacteria interface (condensed or fluid conformation).

SiO 2 NPs -not only affect the organization of the OM but also its morphology. When Φ > 50 nm, two behaviors were observed: either (i) SiO 2 NPs -were deposited on bacteria or (ii) they were trapped in between two cells (Fig. S4). In the latter case, the ripples nanodomains adapt to the NP shape, changing their orientation around the particle to partially wrap it like a "cocoon" (Fig. 6). The wrapping ratio can change among cells, leaving the NP more or less apparent (compare Fig. 6 and When Φ ≤ 50 nm, membrane invaginations (pore like lesions), with well defined spherical edges, were observed randomly distributed on the bacterial surface (Fig. 7a). Even if cell dependent, their depth could reach down to few tens of nanometers (Fig. S6). Two behaviors are associated to these pore like lesions: either (i) the height profile is linked to a phase signature similar to the OM one or (ii) it exhibits a bump linked to a more repulsive signature similar to the NP one (Fig. 7b c).

We here demonstrated that drastic damages induced by SiO 2 NPs - depend on a critical diameter (50 nm < Φ c < 80 nm) above which they were not observed anymore. The true question now is: do NPs induce any bactericidal effect? Besides membrane degradations, the incubation of E. coli cells with SiO 2 NPs -4, 10 and 50 resulted, in some cases, in the collapse of E. coli cells. It is either partial, resulting in a drastic decrease of the bacteria height, or total, and bacteria were drained from their cellular contents and no more exhibited well organized membrane structure (Fig. 8a,b). Moreover, debris were observed on the mica substrate in the vicinity of bacteria aggregates exposed to SiO 2 NPs -4 (Fig. 8c). Such fibrous materials, already observed as the result of cell surface perturbation [START_REF] Alves | Escherichia coli cell surface perturbation and disruption induced by antimicrobial peptides BP100 and pepR[END_REF], are indicative of cellular leakages. They can also correspond to membrane components such as LPS aggregates being expelled due to the stress induced by such NPs.

NPs size > critical diameter Φ c

SiO 2 NPs -80 and 100 did not lead to any of these phenomena. In particular, E. coli cells suffered from no leakage of their intracellular compounds.

SiO 2 NPs charge effect on E. coli cells

To decorrelate between size and charge effects on the interaction between SiO 2 NPs and E. coli, the influence of SiO 2 NPs + 100 (> Φ c ) is SiO 2 NPs + 100 did not affect the CFUs, as for SiO 2 NPs -100. However, AFM observations emphasized the aggregation between such positive NPs and E. coli. Indeed, unlike SiO 2 NPs -100, SiO 2 NPs + 100 tended to exclusively accumulate and stick to, around and on bacteria, none being observed as free isolated or aggregated particles on the substrate (compare Fig. 3 and Fig. 9a). This emphasizes the privileged electrostatic affinity between SiO 2 NPs + and the negatively charged surface of E. coli, as confirmed by zeta potential measurements.

Furthermore, modifying the charge of SiO 2 NPs 100 did not induce any change in E. coli rod shape morphology (Fig. 9b) nor in its OM ripples structure (Fig. 9c). However, unlike SiO 2 NPs -100 which did not lead to any membrane degradations, SiO 2 NPs + 100 induced drastic damages including the formation of (i) extra membrane aggre gates and (ii) membrane invaginations. In the first case, aggregates with heights ranging from 4 to 15 nm surrounded cells (Fig. 10a b). In the second case, pore like lesions, similar to those observed with SiO 2 NPs - smaller than Φ c , exhibited a depth of few tens of nanometer, which are not correlated to the NP size (Fig. 10c d).

Discussion

This work is the first study of exposure to NPs of the strain E. coli MRE 162, which was used, during the 70's, for military purposes. Although thought to be inoffensive, these E. coli were reported to cause diseases in highly susceptible individuals. This emphasizes the necessity of investigating on proper antibacterial agents (harmless for human cells) such as NPs, for commonly found bacteria like E. coli.

Former studies on the toxicity of SiO 2 NPs present contradictory results not only because of different methodologies but also because they do not necessarily address the same size nor the same surface state of SiO 2 NPs, without explicitly mentioning these parameters in some cases. It is thus difficult to compare these results and to potentially demonstrate a size and charge effect tendency. In addition, despite a well known dose dependent cytotoxicity of antibacterial agents such as NPs [START_REF] Napierska | The nanosilica hazard: another variable entity[END_REF], these studies differ in terms of concentration: E. coli growth inhibition was reported for a dose exposure of 2.0 g/L [START_REF] Adams | Comparative eco-toxicity of nanoscale TiO2, SiO 2 , and ZnO water suspensions[END_REF][START_REF] Capeletti | Tailored silica-antibiotic nanoparticles: overcoming bacterial resistance with low cytotoxicity[END_REF] while, in another study, a concentration as low as 0.2 g/L was inefficient [START_REF] Wehling | A critical study: assessment of the effect of silica particles from 15 to 500 nm on bacterial viability[END_REF].

Consequently, we undertook a systematic approach of this issue. We report herein (i) the potential antibacterial activity of SiO 2 NPs -, with diameters varying from 4 to 100 nm, and (ii) the influence of the surface charge for the largest SiO 2 NPs (SiO 2 NPs + 100) on E. coli cells. Based on previous studies and submitted to AFM observation conditions (to correctly visualize both NPs and bacteria), toxicity tests were performed, under static conditions, after a 2 h incubation with NPs at a concentration of 0.1 g/L. Their toxicity was rationalized with regards to their size and charge effects.

Viability experiments revealed the existence of a NP size threshold, 50 nm < Φ c < 80 nm. Above Φ c , SiO 2 NPs -had no influence on CFUs nor on the organization of E. coli population. Below Φ c , SiO 2 NPs -induced an increase in CFUs associated to the easier separation of the initial cell clusters. This phenomenon is not due to an improved ability of cells to divide but rather to the capacity of these SiO 2 NPs -, despite their negative charge, to interact with the LPS layer of the bacteria OM, probably through hydrogen bonding with the LPS hydroxyl groups. These interactions are likely stronger than cell cell ones as they could break down cells clusters. To the best of our knowledge, this splitting effect has never been reported nor correlated to a size dependent action of NPs on cells. Former studies only described the size effect of NPs in terms of CFUs decrease always related to viability losses: for instance, the antibacterial activity decreases with an increase of the size of TiO 2 [START_REF] Simon-Deckers | Size-, composition-and shape-dependent toxicological impact of metal oxide nanoparticles and carbon nanotubes toward bacteria[END_REF] and Ag [START_REF] Martínez-Castañón | Synthesis and antibacterial activity of silver nanoparticles with different sizes[END_REF] NPs. We, here, emphasized that CFUs counting is definitely insufficient by itself to probe toxicity phenomena since it reflects a competition between (i) the number of isolated cells, increased by splitting effects and (ii) the number of viable cells, decreased by potential antibacterial activity (Fig. 2c).

From this point of view, AFM proved to provide complementary and necessary information, at the cellular level, on the NPs size dependent antibacterial activity. Firstly, it emphasized the particular ultrastruc ture present on healthy E.coli's surface. This "brain like" organization, here called ripples, is attributed to the conformation of LPS molecules in the OM [START_REF] Gammoudi | Morphological and nanostructural surface changes in Escherichia coli over time, monitored by atomic force microscopy[END_REF]. Then, AFM observations confirmed, at the cellular level, the existence of a NPs size threshold, 50 nm < Φ c < 80 nm. On the one hand, SiO 2 NPs -larger than Φ c do not induce any particular damage on the overall morphology of E. coli nor on its OM structure. Noteworthy is the partial wrapping observed for these NPs trapped in between cells (Fig. 6c). A balance between adhesion and free energies is here assumed for the NP not to translocate through cells, as already observed by Livadaru and Kovalenko in a statistical mechanical approach of membrane invaginations [START_REF] Livadaru | Fundamental mechanism of translocation across liquidlike membranes: toward control over nanoparticle behavior[END_REF]. On the other hand, SiO 2 NPs -smaller than Φ c strongly disturb E. coli cells. The pore like lesions observed are deep enough, as compared to the OM thickness (around 8 nm), for these SiO 2 NPs -to (i) disrupt this OM and induce its invagination or piercing and even to (ii) damage the underlying peptidoglycan layer (Fig. 7c) which is involved not only in cell division processes but also in the preservation of the cell wall uniformity and cell shape. Permeabilization of this layer is also reinforced by the more spherical shape of bacteria. Such invaginations were reported in antibiotic (penicillin and amoxicillin) treatment of E. coli, interfering with enzymes involved in the peptidoglycan synthesis [START_REF] Yang | Atomic force microscopy study of different effects of natural and semisynthetic β-lactam on the cell envelope of Escherichia c oli[END_REF]. In addition, SiO 2 NPs -smaller than Φ c induce a reorganization in E. coli's OM structure from a ripples pattern to more compact and rounded aggregates, typical of weakened bacteria [START_REF] Gammoudi | Morphological and nanostructural surface changes in Escherichia coli over time, monitored by atomic force microscopy[END_REF]. This transition might arise from (i) the spatial reorganization of the LPS molecules from a ripples structure to an aggregated one or (ii) from the transition in their molecular conformation from an extended to a more condensed one. In both cases, this transition is in agreement with the polymer like behavior [START_REF] Leclère | Phase-separated microstructures in "all-acrylic" thermoplastic elastomers[END_REF] of LPS when undergoing an external pressure [START_REF] Abraham | Monolayer film behavior of lipopolysaccharide from Pseudomonas aeruginosa at the air-water interface[END_REF]. It is likely due to small SiO 2 NPs -interfering in the core of the OM and forcing the molecules to reorganize as compression does in the case of Langmuir monolayer experiments (Fig. S3) [START_REF] Abraham | Monolayer film behavior of lipopolysaccharide from Pseudomonas aeruginosa at the air-water interface[END_REF]. It is not excluded either that a strong hydrogen bonding between these SiO 2 NPs -and the multiple hydroxyls groups of LPS molecules does play a role in this disorganization. It induces a break in the crystalline organization of LPS and thus the loss of the brain like structure, this process being much easier with smaller NPs. At some point, especially for the 4 and 10 nm sizes, NPs might penetrate in the grooves of the LPS layer. Finally, observed lysis and consequent leakage of intracellular components, could occur through these pore like lesions or through the complete and direct destruction of the cell inner membrane. Indeed, cell lysis suggests that, when SiO 2 NPs -reach the peptidoglycan layer, they are inter nalized and translocated, and reach the inner membrane leading to potential toxicity. For cells remaining alive (healthy appearance), SiO 2 NPs -might be trapped outside the peptidoglycan layer protecting the inner membrane and thus the cell integrity. Considering these damages, SiO 2 NPs -smaller than Φ c have similar effects as some antimicrobial peptides following the "carpet model" [START_REF] Li | Atomic force microscopy study of the antimicrobial action of Sushi peptides on Gram negative bacteria[END_REF][START_REF] Meincken | Atomic force microscopy study of the effect of antimicrobial peptides on the cell envelope of Escherichia coli[END_REF].

Independently of their sizes, the positive surface charge of SiO 2 NPs generates strong attractive electrostatic interactions with negatively charged E. coli bacteria. This affinity appears to favor antibacterial activity of SiO 2 NPs. Indeed, SiO 2 NPs + 100 lead to drastic membrane invaginations, similar to those observed with SiO 2 NPs -smaller than Φ c and blebbing phenomenon. As Φ > Φ c , the size influence is, here, ruled out, implying a different mechanism for such damages, probably a consequence of the electrostatic field induced by SiO 2 NPs + . This surface charge dependent toxicity confirms previous studies on silver NPs either bare or coated [START_REF] Silva | Particle size, surface charge and concentration dependent ecotoxicity of three organo-coated silver nanoparticles: comparison between general linear model-predicted and observed toxicity[END_REF][START_REF] Badawy | Surface charge-dependent toxicity of silver nanoparticles[END_REF].

Conclusion

We have demonstrated the size and charge dependent toxicity of SiO 2 NPs towards E. coli bacteria, through a systematic approach. Cells were exposed to commercial and home made SiO 2 NPs with different sizes (4, 10, 50, 80 and 100 nm), either bare (SiO 2 NPs -) or functio nalized (SiO 2 NPs + ) and their toxicity emphasized by viability tests and AFM observations. We demonstrated the existence of a critical dia meter, 50 nm < Φ c < 80 nm, under which cell damages were ob served: OM invaginations and reorganization (from ripples for healthy cells to aggregates for weakened and damaged ones). We reported, for the first time, the splitting effects of these SiO 2 NPs -on cells and its potential interaction with toxicity phenomena. We also showed that SiO 2 NPs larger than Φ c did not affect E. coli cells unless they were positively charged, SiO 2 NPs + 100 inducing similar damage as the smallest negative SiO 2 NPs -. Finally, the particular membrane reorga nization emphasized the importance of LPS structure in E. coli in the resistance to external stress and the need for further studies on its nanomechanical properties and its interaction with NPs.

Materials and methods

For convenient reasons, we will use thereafter short names for all used NPs. For instance, negatively charged silica NPs of 4 nm in diameter will stand as SiO 2 NPs -4. Similar expressions will be used for all NPs.

Materials

Commercial SiO 2 NPs -10, 50 and 100 were purchased from Biovalley (Marne la Vallée, France) and SiO 2 NPs -4 from Alfa Aesar (Schiltigheim, France). Gram negative E. coli bacteria (MRE 162 strain) were a kind gift from the Centre d'Etudes du Bouchet, DGA (Direction Générale de l'Armement, France). Mica, purchased from Electron Microscopy Sciences (Hatfield, United States), was used as support for all AFM measurements. Luria Broth (LB) medium was used as nutritive medium and purchased from Fisher Scientific (Waltham, Massachusetts, United States). L Arginine and tetraethylorthosilicate (TEOS), used for silica seeds synthesis, were purchased from Sigma Aldrich (Saint Louis, Missouri, United States) as well as aminopropyl triethoxysilane (APS), used for NPs functionalization. Ethanol and ammonium hydroxide, used for SiO 2 NPs growth, were also purchased from Sigma Aldrich. Millipore ultrapure water (pH 5.5, resistivity > 18.2 MΩ cm) was used for NPs cleaning and bacteria suspensions preparation.

Preparation and characterization of SiO 2 NPs solutions

To obtain a large range of NPs sizes, we used both commercial and home synthesized SiO 2 NPs. Home synthesis was also used to functio nalize the negatively charged NPs (SiO 2 NPs -) and get a positive surface charge (SiO 2 NPs + ).

Commercial SiO 2 NPs -were centrifuged three times at 9000g for 45 min (20 min were enough for SiO 2 NPs -100), cleaned and redis persed in ultrapure water. SiO 2 NPs -were then placed in a sonication bath for approximately 1 h to ensure minimal aggregation in the solution.

Synthesis of home made SiO 2 NPs -was carried out following synthesis developed by Hartlen et al. [START_REF] Hartlen | Facile preparation of highly monodisperse small silica spheres (15 to > 200 nm) suitable for colloidal templating and formation of ordered arrays[END_REF]. The first step consisted in synthesizing SiO 2 seeds. 100 mL of a 6 mM L arginine water solution was introduced in a vial at 60 °C, under magnetic stirring (150 rpm). After temperature stabilisation, 10 mL of TEOS were slowly added so as to respect the interface. Seeds of about 30 nm in diameter were obtained after complete consumption of TEOS. The second step consisted in the growth of these seeds. Typically, 10 mL of SiO 2 seeds were introduced in a mixture of 455 mL of ethanol and 35 mL of ammonium hydroxide in water solution ([NH 3 ] = 1 M), under stirring (700 rpm). TEOS was then introduced in the amount calculated to get the targeted diameter, at a rate of 0.5 mL/h [START_REF] Gaudon | Thermochromic phase transition on CuMo 0.9 W 0.1 O 4 @SiO 2 core shell particles[END_REF]. Surface modification was performed by adding APS (six equivalents assuming a rate of 2 3 μmol/m 2 of active OH groups at the surface of the silica particles) to the SiO 2 NPs -solution; the mixture was then stirred for 48 h in a water bath at room temperature, and then for 2 h at 100 °C. Final SiO 2 NPs + were washed using ultrapure water and ethanol and 3 cycles of centrifugation (13,000 rpm for 20 min.) Dynamic Light Scattering (DLS) experiments were carried out on a Malvern Zetasizer Nano ZS setup (Orsay, France) to determine the size and polydispersity of NPs. This method provides information on both the hydrodynamic diameter of the SiO 2 NPs in solution and their polydispersity. Transmission Electron Microscopy (TEM) with a JEOL JEM 1400 Plus and AFM observations were also used to investigate not only the size and morphology of SiO 2 NPs but also their organization on a substrate. Consequently, while TEM images give the silica NPs diameter (i.e. its core), DLS measurements give the hydrodynamic diameter of the NPs including the first sphere of coordination of the solvent. Zeta potential measurements were carried out on a Wallis Zeta potential analyzer from Cordouan (Pessac, France). For these three characterizations, SiO 2 NPs were suspended in ultrapure water to obtain a final concentration of 0.1 g/L and sonicated before any experiments.

Bactericidal susceptibility test

A fresh E. coli culture was obtained by inoculating 50 mL of LB medium with 250 μL of an overnight culture. The bacterial growth was followed by measuring the optical density (OD) at 600 nm, using a Helios gamma spectrophotometer (Thermo Scientific), until it reached 0.60. The suspension was further centrifuged (8000 rpm for 10 min. at 4 °C), and the cell pellet was re diluted in ultrapure water to obtain an OD of 0.01. Finally, NPs were introduced in the suspension at the desired concentration (1 g/L) and left to interact for 2 h, under static conditions, at room temperature. At the end of the incubation period, ten fold serial dilutions in ultrapure and sterile water of the suspensions were made and 100 μL of each dilution were spread onto LB agar plates. Bactericidal effects were evaluated by counting the number of colonies (CFU for colony forming units) developed on plates after 16 h at 37 °C. Average values of three independent experiments are reported there after.

Dark field microscopy observations

For dark field microscopy observations, E. coli cells were grown in LB medium until the culture reached an OD = 0.60 at 600 nm. The bacterial cells were centrifuged (8000 rpm for 10 min at 4 °C) and re suspended in ultrapure, sterile water to obtain an OD = 0.60 at 600 nm before being incubated in the presence of the different NPs at 1 g/L for 2 h at room temperature under static conditions. A microscope Eclipse Ni (Nikon) equipped with a dark field condenser (oil 1.463 1.20) and an objective Achromat 40×/0.65 both from Nikon was used for dark field image acquisition. Images were acquired with a Nikon DS Qi1Mc camera and NIS Elements software. Representative images are provided thereafter.

Preparation of NPs E. coli suspensions for AFM experiments

E. coli cells were grown on LB agar plates and incubated for 16 h at 37 °C. After scraping, bacteria were suspended in ultrapure water and their concentration was adjusted to 10 6 cells/mL. An equivalent volume of E. coli cells and SiO 2 NPs suspensions at a concentration of 0.1 g/L, were mixed and gently manually shaken (4 times every 20 min), then left to interact at room temperature for 2 h. Five microliters of each mixed suspension was deposited on freshly cleaved mica, left to dry in a desiccator and imaged by AFM the following day.

AFM experiments

Briefly, AFM consists of a cantilever with a nanoprobe at its end which, along with a piezo scanner, scans the sample surface. In close proximity, small (attractive and repulsive) forces between the probe and the surface lead to the deflection of the cantilever, which is recorded. This technique gives information on morphological and mechanical properties (adhesion, visco elasticity) of a surface, at the nanometer level. In this work, AFM experiments were carried out using a Bioscope II mounted on an IX71 Olympus inverted optical microscope operating with the NanoScope V controller and also with a Multimode AFM (Veeco Brucker, Santa Barbara, USA).

Though, imaging in liquid is preferred for cells hydration, air conditions were privileged for a better resolution [START_REF] Bolshakova | Microbial surfaces investigated using atomic force microscopy[END_REF][START_REF] Bolshakova | Comparative studies of bacteria with an atomic force microscopy operating in different modes[END_REF] (due to tip bacteria interactions), notably on the topographical and nanomechani cal contrasts on the surface structure of bacteria. It also allows to work in dynamic tapping mode and, consequently, to take advantage of the harmonic oscillator behavior of the cantilever. Thus, very small forces were used, preventing any damages to the samples. Tapping mode images were obtained with commercial cantilevers with a spring constant around 40 N/m, at a scan rate of 0.5 to 1.0 Hz and with a resolution of 512 × 512 pixels. For each experiment, height, amplitude and trace phase images were recorded. Height images stand for topographical relief of the sample. Phase images show the phase difference between exciting and response signals, arising from the sample nanomechanical properties. Thus, they are linked to (i) dissipa tion processes between the tip and the sample and (ii) surface hardness. For instance, a homogenous surface (composition and organization) will not show any significant contrast in phase. Amplitude images (error signal) provide additional information on the topography and morphology of very fine details which might be saturated in height images.

Hereafter, samples were observed, in tapping mode, in the day following their preparation to prevent any interference, like ageing of E. coli biofilm [START_REF] Gammoudi | Morphological and nanostructural surface changes in Escherichia coli over time, monitored by atomic force microscopy[END_REF], with the potential damage caused by SiO 2 NPs. Different areas were systematically scanned and images shown there after are representative of the cells in a given set of colonies and of all the tested samples.

Fig. 1 .

 1 Fig. 1. a) Potential antibacterial activity of SiO 2 -NPs (1 g/L) against E. coli cells (OD = 0.01). b) Optical microscopy (dark field) observations of representative samples, with and without SiO 2 -NPs , revealing modifications in cells organization and morphology (scale bar: 10 μm). c) Scheme of the potentially entangled effects of SiO 2 -NPs with Φ < Φ c and their consequences on CFUs.

Fig

  Fig.S5).When Φ ≤ 50 nm, membrane invaginations (pore like lesions), with well defined spherical edges, were observed randomly distributed on the bacterial surface (Fig.7a). Even if cell dependent, their depth could reach down to few tens of nanometers (Fig.S6). Two behaviors are associated to these pore like lesions: either (i) the height profile is linked to a phase signature similar to the OM one or (ii) it exhibits a

Fig. 2 .

 2 Fig. 2. Characterization of E. coli cells morphology and rheology by AFM observations. a) Height image of the rod bumped shape of a single cell. In some cases, despite their fragility, flagella and pili are still attached to the cell (white arrow). A zoom on its irregular surface reveals (b) a typical relief and (c) a phase separation pattern corresponding to the lamellae organization of the cell OM.

Fig. 3 .

 3 Fig. 3. Height (a-b) and phase (c-d) AFM images showing the organization of E. coli cells treated with (a) SiO 2 -NPs 100, (b) SiO 2 -NPs 4, (c) SiO 2 -NPs 80 and (d) SiO 2 -NPs 10. NPs are either deposited on and around cells or free on the substrate (white arrows).

2. 5 .

 5 Bactericidal effect of SiO 2 NPs - 2.5.1. NPs size < critical diameter Φ c

Fig. 4 .Fig. 5 .

 45 Fig. 4. Height AFM images, emphasizing no change in the morphology of E. coli cells after treatment with (a) SiO 2 -NPs 100, (b) SiO 2 -NPs 80, and aspect ratio modification after treatment with (c) SiO 2 -NPs 50, (d) SiO 2 -NPs 10 and (e) SiO 2 -NPs 4.

Fig. 6 .

 6 Fig. 6. a) Height AFM image showing the wrapping of SiO 2 -NPs 100 in between two neighbor cells. b) A closer view of this phenomenon in phase image. c) Profile section of a SiO 2 -NPs 100 showed by the dashed line in (a). d) Proposed scheme of the OM deformation, leading to the formation of a "cocoon" around the NP.

Fig. 7 .Fig. 8 .

 78 Fig. 7. a) AFM amplitude image showing membrane invaginations (white arrows) when E. coli cells are exposed to SiO 2 -NPs 4. b) AFM phase image of a closer view of membrane invaginations with SiO 2 -NPs 50 and (c) corresponding height profiles of the pore-like lesions pointed by red and blue lines in (b). Proposed scheme of the OM deformation is also shown in (c). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 9 .

 9 Fig. 9. a) AFM height images of E. coli cells population with SiO 2 -NPs + 100, aggregated on cells (white arrow). AFM height (b) and phase (c) images of E. coli incubated with SiO 2 -NPs + 100 showing its unchanged morphology and OM structure.

Fig. 10 .

 10 Fig. 10. E. coli membrane damages induced by SiO 2 -NPs + 100, observed on AFM phase (a) and amplitude (c) images, and pointed by full arrows. a) Formation of extra-aggregates, with (b) height profiles representative of diverse aggregates. c) Formation of membrane invaginations with (d) height profiles representative of diverse invaginations.

Table 1

 1 Characterization of SiO 2 -NPs, both commercial and home-made, by DLS and zeta potential measurements and statistics on TEM pictures.

	Name	Diameter (nm)			Zeta potential
					(mV)
		Manufacturer	TEM	DLS
	SiO 2 -NPs 4	4 (large distribution) aggregated 3	53.3
	SiO 2 -NPs 10	10 ± 5	12 ± 4	7
	SiO 2 -NPs 50	50 ± 10	53 ± 12	68
	SiO 2 -NPs 80	Home made	81 ± 4	90
	SiO 2 -NPs	100 ± 30	113 ± 11	124
	100
	SiO 2 -NPs +	Home made	94 ± 4	135	+ 45.4
	100

Acknowledgments

The authors thank the Région Aquitaine and CNRS (France) for supporting this work through the equipment of the NanoSpectroImagerie (NSI LOMA) platform used in this work (CPER COLA2). They are grateful to the Direction Générale de l'Armement (DGA, Ministère de la Défense France, grant number : 2014017) and the Région Aquitaine (France) for their financial support through the Ph.D. grant of M. Mathelié Guinlet. Finally, authors thank NSI LOMA plat form for technical help and J.P Chapel for its helpful advises and discussions about DLS measurements.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cis.2017.04.012.


Jacques Simonet
email: jacques.simonet@univ-rennes1.fr

Thin porous palladium layers modified by cathodically doped graphene and organic donors

Keywords: Doped palladium metal, Palladium-graphene electrode, Metal-carbonaceous donor composites

Thin porous layers of palladium deposited onto gold or Pd bulk metal treated by cathodic reduction in the presence of CsI under super-dry conditions were tested to establish their reactivity toward electrolytes. Specifically, an iono-metallic layer [Pd 4 -, TMA + {TMABF 4 }] was found to be generated in tetra-alkylammonium salts, especially TMA + , BF 4 -. This material is then taken as a starting substrate for in situ exchange between BF 4 -and many electrogenerated organic acceptor anions. Importantly, the list includes doped graphene and electro-active conducting organic polymers in their basic form. Thus, for example, an intriguing Pd-graphene layer can be created as a relatively thick interface, and may be either doped or un-doped, depending on the applied potential. The method was also shown to work with many organic acceptors (coronene, anthraquinone, etc.) immobilized in their reduced state. The proposed method suggests a new strategy for building stable composite materials of considerable interest for use as electrodes and/or in 3D redox applications.

Introduction

It is usual to consider precious metals like platinum and palladium to be totally unreactive toward electrons and ions. Most researchers working in organic electrochemistry or organometallic chemistry (whatever the overall conditions concerning solvents and salts) therefore still consider these materials as ideal electrodes, free of side reactions with other reagents. However, recent work [START_REF] Simonet | On the cathodic behaviour of tetraalkylammonium cations at a platinum electrode[END_REF][START_REF] Simonet | On the cathodic corrosion of platinum in the presence of iodides in dry aprotic solvents[END_REF][START_REF] Cougnon | The cathodic insertion of tetraalkylammonium iodides into palladium[END_REF][START_REF] Cougnon | Réactivité cathodique du platine et du palladium en présence d'électrolytes en milieu superanhydre[END_REF][START_REF] Simonet | Cathodic reactivity of platinum interface in the presence of tetramethylammonium salts. A pro-base cathode material?[END_REF][START_REF] Simonet | The platinized platinum interface under cathodic polarisation[END_REF] on the behaviour of Pt used as a cathode in super-dry aprotic polar solvents showed high reactivity when applied potentials are < -2 V vs. Ag/AgCl. Furthermore, as well as reduction in poly-anions (similarly to Zintl metals), the cathodic insertion of electrolytes (alkaline metal and tetra-alkylammonium salts) has been observed [START_REF] Cougnon | Réactivité cathodique du platine et du palladium en présence d'électrolytes en milieu superanhydre[END_REF]. This means that both Pt and Pd could be considered as cathodic materials under

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specific conditions, leading to the emergence of a new generation of modified interfaces with applications in the field of electrocatalysis [START_REF] Cougnon | Are tetraalkylammonium cations inserted into palladium cathodes? Formation of new palladium phases involving tetraalkylammonium halides[END_REF][START_REF] Simonet | The platinized platinum interface in super-dry solvents: Cathodic reversible reactivity and morphology modifications in the presence of tetramethylammonium salts[END_REF].

Quite intriguingly, these recent observations revealed the generation of poorly conducting "iono-metallic" layers which totally cover the concerned metal until a quasi overall inhibition of electron transfer. The thickness of these modified layers depends on the electrolytes used and might reach several tens of microns. The exact stoichiometry of the iono-metallic complexes has been determined: with palladium, the general surface reaction was found [START_REF] Cougnon | Réactivité cathodique du platine et du palladium en présence d'électrolytes en milieu superanhydre[END_REF] to correspond to the following equation:

4 Pd + e -+ 2 {MX} {Pd 4 -M + , MX } + X -(1)
where M + is an alkaline metal cation or TAA + cation. A variety of anions could be employed

(for example BF 4 - , PF 6 - , ClO 4 -
). Halides I -and Br -could also be used, but in this case the layer stoichiometry is changed [START_REF] Cougnon | Réactivité cathodique du platine et du palladium en présence d'électrolytes en milieu superanhydre[END_REF]. The generation and stability of iono-metallic structures depend crucially on the nature of the inorganic anion. In particular, inorganic anions X - selected for their electrochemical stability are likely to be exchanged with carbanions, principally those electrogenerated in situ at the cathode surface and within the metallic bulk in the course of successive scans. The reactivity of electrogenerated anion-radicals and dianions (mainly from organic π-acceptors) formed at potentials less negative than the threshold of the Pd cathodic step (corresponding to reaction (1)) resulted in significant modification of the iono-metallic layer through inorganic anion ↔ organic anion or dianion exchange. This has been demonstrated [START_REF] Cougnon | Réactivité cathodique du platine et du palladium en présence d'électrolytes en milieu superanhydre[END_REF][START_REF] Cougnon | Are tetraalkylammonium cations inserted into palladium cathodes? Formation of new palladium phases involving tetraalkylammonium halides[END_REF] for platinum in the presence of π-acceptors under super-dry conditions (electrolytes TBABF 4 or CsI in DMF in the presence of activated neutral alumina). The highly intriguing insertion of organic species in their reduced form(s) can be obtained under very simple conditions (a potential of about -2 V is applied to the precious metal cathode) assuming that the organic compound is electroactive at less negative potentials than the one permitting the metal doping. Additionally, one may imagine that the irreversible formation of mono-anions -as in the case of carbanions derived from the cathodic cleavage of carbon-heteroatom bonds -could also lead to insertion, as well as the cathodic doping of organic conducting polymers (e.g. electrogenerated polyfluorenes, owing to the relative acidity of the fluorene C-H function in position 9 [START_REF] Rault-Berthelot | The anodic oxidation of fluorine and some of its derivatives. Conditions to the formation of a new conducting polymer[END_REF][START_REF] Rault-Berthelot | The polyfluorenes: a family of versatile electroactive polymers. II Poly(9,9-disubstituted) fluorenes and existence of reversible p and n doping[END_REF]). Basic forms of polymeric substances such as these could be formed simply at the interface of electrogenerated bases (EGBs) obtained by the reduction of electroactive impurities and therefore acting as base catalysts. Importantly, the cathodic doping of certain allotropic forms

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of carbon (graphene, C 60 , fullerenes) reactive under electron transfer to their poly-anionic transients is of great interest in generating porous dual metal-carbon materials expected to show high 3D reactivity.

The present preliminary work is focused on porous palladium layers generated by cathodic treatment of Pd in the presence of CsI, or very thin galvanostatic Pd deposits on gold electrodes (gold does not afford iono-metallic transients [START_REF] Ghilane | Reactivity of platinum metal with organic radical anions from metal to negative oxidation states[END_REF][START_REF] Hapiot | Reduction of platinum under superdry conditions: an electrochemical approach[END_REF]). Thanks to the exclusive use of TMA + salts that appear to eliminate the Hofmann degradation (known to generate protons and then alter the neutrality of the media towards electrogenerated organic anions), the redox potentials of some sample iono-metallic layers modified by π-acceptors are measured.

Additionally, the reactivity of the iono-metallic layer towards the electrogenerated anionic forms (derived from the scission of RX type compounds or doping of graphene) is clearly demonstrated. In all cases, the experimental conditions are specified in the figure captions.

Results

The In fact many organic acceptors were found to be suitable candidates to be embedded in the bulk metal (depending on the total amount of electricity injected into the system).

Using this method, coronene, pyrene, dinitrobenzene, anthraquinone, acenaphthenoquinone, and acenaphthylene (Scheme 1) were efficiently inserted in palladium, probably through exclusive exchange with BF 4 -. After electrolysis, complexes {palladium, TMA + , organic acceptor reduced forms} were found to be poorly oxidized by air in the course of the workup. Such chemically modified interfaces used as electrodes may involve a new process of

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redox catalysis in which the catalyst is not immobilized by a conventional method (e.g. via radical grafting), but through efficient and easy embedding of organic anions (for example, via the two-electron scission of the carbon-heteroatom bond).

It is also important to consider the cathodic capability of palladium to directly permit insertion of acceptors not through exchange, but under electron transfer during a concomitant reduction. Thus, organic anions and electrogenerated polymeric anionic forms (possibly under the action of electrogenerated bases) may also react with graphene -precharged at -2 V -as depicted in Scheme 1.

This response is obtained with many π-acceptors (triphenylene, dinitrobenzene, anthraquinone, chloranile, bi-benzyle, diacethylbenzene, etc.). Astonishingly, the peak potentials assigned to the presence of acceptor reduced forms (now considered as donors towards the iono-metallic palladium phases), are all significantly shifted to a more positive potential range (up to ΔV ≈ +1.0 V). Figure 2A shows the response of anthraquinone trapped in the iono-metallic layer. The double redox response is quite different to that of free anthraquinone in the same solution at a platinum or smooth gold electrode (potential shifts > 1 V), strongly supporting the hypothesis of a structural change in the palladium surface through addition of the acceptor to the surface. The first step (I) in Figure 2A may be assigned to the anodic response of the organic radical anion fully inserted as a donor species into the metallic phase, while step (II) corresponds to the occurrence of the dianion in the bulk Pd. In the course of scans towards negative potentials, peak (II) is larger; however, in the course of the reverse scan, peak (I) (associated with the presence of the anion radical) turns to be fairly huge, which supports a prior dianion oxidation. Significantly, the increase in steps (I) and (II) when re-doping along a short scan up to the Pd charge threshold may regenerate reducing centres located at (or very close to) the electrode surface after modification.

Moreover, Figure 2B shows that acenaphthenoquinone also reacts with the electrochemically generated iono-palladium intermediate, leading to a deep blue layer which is totally stable when in contact with di-oxygen after work-up.

Furthermore, the as-produced surfaces were shown to favour redox catalyses at a potential < -1 V, possibly due to the occurrence of reduced forms (radical anion of the acceptor) at the Pd interface. (For example, 1-iodopropane can be indirectly reduced under these conditions with a potential gain of 0.5 V.) The method should be seen as a new process

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for chemical modification of metallic interfaces, with a limited amount of organic species easily activated electrochemically by mean of a cathodic pulse.

Charge-discharge of palladium-graphene layers

Since graphene can be electrochemically reduced in aprotic conditions (a poly-anion is formed after successive electron transfers onto graphene micro-flakes), it was interesting to consider whether such reduced materials could react with charged palladium.

Figure 3 depicts the cathodic charge (according to the double peak C1) of the Pdgraphene layer. The specific reduction of palladium (only observed in super-dry solvents) is superimposed on that of the graphene already described under rather similar conditions [START_REF] Poizot | Facile route to gold-graphene electrodes by exfoliation of natural graphite under electrochemical conditions[END_REF][START_REF] Simonet | Electron transfer at platinum and palladium interfaces in super-dry electrolytes. Generation of iono-metallic layers. A mini-review[END_REF]. Interestingly, multi-step C1 increases during the scans while the anodic peaks A1

(discharge of Pd) and A2 (assigned to implication of the graphene poly-anion in the palladium layer) behave differently. A charge with pre-functionalized graphene was also successfully obtained. The resemblance with acceptor immobilization can be seen (see Scheme 1). The use of marked graphene (e.g. by means of 1,4-anthraquinone or ferrocene)

clearly confirms the selective reactivity of graphene toward doped palladium by simple analysis of the resulting sonicated Pd electrodes (as clarified in the graphical abstract).

Conclusions

This communication reports the unexpected insertion of reduced acceptors and/or poly-anionic forms, possibly electrogenerated, in metals such as palladium matrixes.

Preliminary work on platinum reduction and the concomitant insertion of π-acceptors is strongly improved with palladium as a host for several reasons: (i) a very sharp and clear discharge of the reduced iono-metallic layer is obtained with this metal; (ii) clear redox steps can be obtained with many π-acceptors (comparison between curves B and C in Figure 1);

(iii) there is evidence of a palladium complex (probably including the anion-radical of the acceptor), stable in the presence of air, that allows further studies of the exact stoichiometry, provided total insertion within the layer is obtained. Additionally, the present report stresses the exclusive use of TMA + , which strongly facilitates the formation of the iono-metallic complex and the exchange with acceptors. This is probably due to the stability of TMA + in contact with electrogenerated bases as a result of the much weaker impact of the Hofmann degradation of the salt.

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The other main point stressed in this report is that reduced poly-anionic forms of graphite (and graphene as an exfoliation product) can be reactive towards palladium and form a stabilized metal-graphene material (already described with Ag and Au [START_REF] Poizot | Facile route to gold-graphene electrodes by exfoliation of natural graphite under electrochemical conditions[END_REF]), which is reported to be very reactive both chemically and electrochemically.

The method is -for the moment -exclusively based on the exchange of the anion of the electrolyte involved during the cathodic doping of the metal. Importantly, the insertion relies on a preliminary treatment that implies the n-doping of the metal under unusual conditions (exclusive use of super-dry electrolytes [START_REF] Simonet | Electron transfer at platinum and palladium interfaces in super-dry electrolytes. Generation of iono-metallic layers. A mini-review[END_REF]). Preliminary experiments emphasize that insertion of inorganic and organic anions can also be achieved by direct mixed electrolyses that involve both the metal and the pro-base. It is hoped that use of this technique will make it possible to design new polyvalent 3D materials based on Pd. Figure 3. Electrochemical multi-charge of a thin layer of graphene deposited onto a Pd microelectrode (area: 0.8 mm 2 ). Electrolyte: DMF/TMABF 4 . Scan rate: 50 mV s -1 . Notice that the reduced graphene (possibly obtained by exfoliation of deposited natural graphite), occurring in C1 (successive steps 1, 2, and 3) for generating a poly-anion acting as a polynucleophile.

Scheme 1. Concomitant charge of palladium (according to reaction (1) in super-dry conditions, electrolyte A -,M + ) together with reduction of organic acceptors or contact with a carbonaceous charged material. Reactivity of electrogenerated anionic species from 1,4dinitrobenzene and that of deposited graphene micro-flakes (implication of poly-anion obtained by a multi-electron transfer occurring at the Pd charge potential at <-2 V). The alternative use of electrochemically marked graphenes (e.g. through use of redox markers such as anthraquinone (AQ) or ferrocene (Fc)) allows the integration of certain reduced carbonaceous products within the superficial palladium space to be confirmed, despite the fact that a sonication rinsing is carried out.

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Graphical abstract

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Highlights

• Electrochemical reduction of porous Pd layers used as electrodes incorporate anionic forms of doped carbonaceous derivatives. • Graphite, graphene, polyarenes reduced as poly-anions may react with doped Pd.

• Graphene (or graphite via an in situ exfoliation process) through mixed electrolyses with Pd leads to Pd-graphene stable composite materials. • Organic acceptors in contact with doped Pd produce quite stable redox systems.

  use of a palladium electrode in super-dry conditions appears to open new avenues of research in the study of conducting organometallic surfaces, layers, and/or materials that can possibly be modified by electrogenerated bases. As an example, Figure1shows that the electrochemical insertion of π-acceptor reduced entities is real. Curves A correspond to the charge (> -2 V) and discharge (≈ -0.5 V) of palladium. Addition of 1,4-diacetylbenzene (DAB) to the solution and further reduction at a pre-modified surface allows the deposition/insertion of redox species for which the electrochemical response in a solution free of DAB resembles that of DAB (two reversible steps) but strongly shifted towards more anodic potentials (ΔE > +1.2 V). Repetitive cycling results in the progressive disappearance of signals, presumably due to the gradual oxidation of the deposit when scans up to + 0.5 V are made. Importantly, slow rate scans up to -2 V result in the regeneration of the original signal (through a new reductive generation of the transient organo-iono-metallic layer, possibly limited by the diffusion rate of TMA + through the bulk metal).

Figure 1 .

 1 Figure 1. Cathodic reduction (and insertion) of diacethylbenzene (DAB: 10 mM L -1 ) in DMF/TMABF 4 under super-dry conditions. (A) Pd-Au electrode. Scan rate 50 mV s -1 .(B)The same solution at a smooth gold electrode (0.8 mm 2 ). (C) Electrode (A) after the two scans, rinsed and dried, and then cycled in DMF + 0.1 M TMABF 4 .

Figure 2 .

 2 Figure 2. Charge of a palladium sheet (total surface area ≈ 4 cm 2 ) in super-dry conditions: (A) modification of a Au-Pd electrode by TMABF 4 and then insertion of anthraquinone. Response of the electrode in DMF after rinsing. Scan rate: 50 mV s -1 . (B) Deposition at Pd reduced in CsI 0.1 mol L -1 in DMF. Palladium sheet (total surface area ≈ 4 cm 2 ). Total charge 10 C followed by a contact with a super-dry solution of acenaphthenoquinone 10 mmol L -1 in DMF for 5 minutes. The stable blue coloured zone (on the left side) is assigned to acenaphthenoquinone dianion trapped within the reduced Pd phase.

Figure 1 A

 1 Figure 1


Jochen Fick

Bertrand Ménaert

Julien Zaccaro

Paul Moretti

Hemisphere mline spectroscopy and its application to birefringent KTiOPO 4 planar waveguides

Keywords: codes: 42, 70, Mp: Nonlinear optical crystals 42, 82, Et: Waveguides couplers 42, 87, d: Optical testing techniques 48, 25, Lc: Birefringence

A modified mline technique, based on the use of a high index hemisphere instead of a coupling prism is presented. It allows fast characterization of the optical properties as a function of the propagation direction inside a planar waveguide. Results of an ionimplanted KTP waveguide are presented and compared to prism measurements. It is shown that this techniques enhances the precision of the waveguide parameters determination and allows to study advanced features such as strong mode hybridization.

Introduction

The classic mline spectroscopy, based on a coupling prism, is a widely used technique for the characterization of planar optical waveguides, allowing to test for waveguiding, determine the main waveguide parameters and to get an estimation of the waveguide losses. It allows fast characterization without any sample preparation, such as endface polishing in the case of butt coupling. The measurement is made on a small waveguide region, allowing to test for homogeneity and/ or to work with mediocre quality samples during waveguide fabrication optimization. The m line measurement does not cause any damage, allowing further sample processing. Automatized m line systems are commercially available for some years. The mline spectroscopy was developed in the seventies by P.K. Tien and R. Ulrich. They complete the experimental observation of waveguide modes in semiconductor thin films [1] by the theoretical treatment [2] of a prism coupler using a plain wave approach. Later, this theory was extended to non linear distributed couplers [3,[START_REF] Nevière | Electromagnetic Resonances in Nonlinear Optics[END_REF] allowing to simulate dynamical and non linear effects in prism and grating couplers [START_REF] Fick | [END_REF]. The mlines are mostly used for the isotropic linear waveguides. However, a non linear versions of the mlines was developed to study the optical Kerr effect of thin film waveguides [68]. Further on, mlines were used for the characterization of anisotropic waveguides by measuring the waveguide modes as a function of the propagation direction in the waveguide plane. This method was used for the investigation of induced anisotropy of protonexchanged lithium niobate waveguides [9], KTP ionimplanted waveguides [10], liquid crystal films [START_REF] Lindgren | Proc. SPIE[END_REF] or the anisotropy of sputtered titanium or hafnium oxide films [START_REF] Flory | [END_REF]13]. The variation of the propagation direction was obtained by demounting the sample and remounting it slightly tilted. This is a awkward and time consuming procedure. Moreover, it is difficult to reproduce exactly the same coupling conditions at the same spot of the planar waveguide and the determination of the exact propagation direction is difficult. These problems can be overcome by replacing the coupling prism by a hemisphere and adding a rotation stage to allow rotation of the sample around the waveguide normal. In this paper, the hemisphere mline setup will be presented. It will be used for the investigation of the TETM mode hybridization in an ionimplanted KTP waveguide.

Experimental setup

The hemispherical mline setup is depicted in Fig. 1. A laser diode emitting at 980 nm is used as light source followed by a ball lens (f = 4 mm) for beam collimation. A Glan prism polarizer and a λ/2 plate allows to control the beam polarization by turning the λ/2 plate. No shift of the incident beam was observed when changing from TE to TM polarization. The main complication when using the hemisphere instead of the coupling prism is the fact that the hemisphere acts as a lens with a short focal length (f hs ≅ 4 mm). To obtain a well collimated laser beam at the base of the hemisphere, the beam has to be focused near the hemisphere surface. Gaussian beam theory was used to simulate the beam propagation. A telescope with f 1 = 80 mm and f 2 = 100 mm leads to a beam waist of ω = 0.2 mm and a Rayleigh length of z r = 130 mm. This high z r value assures a well collimated incident beam and allows noncritical alignment. The sample is mounted in the center of a theta/2theta two circle goniometer actuated by two step motors. The reflected intensity is recorded using a silicon photodiode. The aperture of the detector is enlarged by a lens to enhance the capture of the reflected beam. The waveguide is pressed onto the hemisphere by means of a micrometer screw. A index matching liquid with n λ=589 nm = 1.58 is used to lower the effective thickness of the gap between the hemisphere and the sample. A manual rotation stage allows the rotation around the waveguide normal. The hemisphere is obtained by polishing a commercial 8 mm ball lens made of SLAH79 high index glass from Ohara. The glass refractive index is n hs = 1.9703 at 980 nm, which is well adapted for KTP waveguides. A coupling prism of the same glass was used to test the performance of the hemisphere coupler.

KTP waveguide fabrication

A planar waveguide was fabricated by He + ion implantation of a xcut KTP substrate from Cristal Laser. Two subsequent implantations were applied at energies of 1.5 and 1.45 MeV with a fluence of 0.75•10 16 ions/cm 2 each. The estimated waveguide depth was 4 µm. A post irradiation annealing at 250°C for one hour was applied to cure eventual electronic defects in the waveguide. Details on the waveguide fabrication process can be found in Ref. [10]. The propagation direction inside the plane waveguide is described by the angle ϕ. For ϕ = 0°, the propagation is parallel to the KTP zaxis. In this case the TE modes are related to the n y component of the KTP refractive index tensor. For ϕ = 90°, propagation is parallel the KTP yaxis and the TE modes are related to n z . In both configurations, the TM modes are related to n x .

Mode propagation in birefringent waveguides

The m-line technique gives the effective indexes of the waveguide modes. Some theoretical considerations are necessary to deduce the waveguide geometry form the set of effective indexes. The index profile of ion-implanted waveguides can be approximated by a Fermi-type function. This type of index profile is best reconstructed using an inverse WKB method. In this method, a depth or turning point is attributed to each measured effective index [14], thus reconstructing the index profile (Fig. 2). In general, a few modes are sufficient to obtain the effective depth of the waveguide. The total index change is however difficult to calculate as the distinction between guided and leaky modes is not straightforward. This problem can be solved by supposing a step index profile (SIP), allowing to reconstruct the index profile with only two modes. In Fig. 2 the results of the two methods are compared. The index profiles are similar, although the SIP underestimates the waveguide depth, whereas the IWKB overestimates the surface refractive index. The effective indexes calculated from SIP (lines on the right of Fig. 2.) are in very good agreement to the experimental ones, and the TE 3 modes is clearly identified as leaky mode. In conclusion, the ion-implanted waveguides can be reasonably well approximated by a step index profile, facilitating the investigation of anisotropic waveguides. Different approaches were applied to study the waveguiding properties of anisotropic planar waveguides, including the direct solution of the wave-propagation equation [15,16] the finiteelement method [17], the zigzag ray model [18,19], approaches based on the separation of the waveguide modes into ordinary and extraordinary waves [20,21] and the transfer matrix model [22][23][24]. It was found that for "off-axis" propagation the waveguide modes are of hybrid nature containing contributions of TE-and TM-type modes. Further on, for angles ϕ greater than a critical angle, the dominantly TM-type modes are lossy modes with energy leakage into the substrate. The guided to leaky mode transition in uniaxial materials were identified near the ordinary or extraordinary cut-off for negative and positive birefringent materials respectively [25,26]. We developed a numerical simulation tool based on a transfer matrix approach [23] and using a step index profile. Our model allows the calculation of the effective indexes of the guided modes for any propagation direction in birefringent planar waveguides. The non linear Lenvenberg Marquardt method was used for fitting the waveguide parameters to the experimental results. The free Scilab environment was used for the calculations [27]. As already mentioned, pure TE and TM polarized modes only exist for propagation parallel to a dielectric axis of the material. For all other propagation directions the waveguide modes are hybrid modes containing TE and TM contributions. In most cases one contribution is, however, dominant. For simplicity, the labels TE' and TM' will be used for modes with dominant TE or TM contribution.

Results and Discussion

A typical mline curve of ion implanted KTP waveguides is shown in Fig. 3 (dashed line). For convenience, the experimental incident beam angle was converted to the effective index using simple algebra. Two different phenomena are observed: three distinct mlines related to the waveguide modes and a slow intensity decrease for decreasing effective indexes corresponding to light tunneling trough the low index barrier. The overlap of these two phenomena results in a slight shift of the minimum position of the mlines and a decrease of their visibility. The pure mline contribution are extracted by subtracting the envelop function which corresponds to the light tunneling (full line Fig. 3). The mlines obtained using prism and hemisphere couplers are compared in Fig. 4. Typical curves for TE and TM polarization are shown at ϕ = 0°. The minimal positions are in good agreement for the first two modes and excepting the first order mode, the contrast is equivalent. The hemisphere can thus be used without significant decrease of experimental precision. The evolution of the mlines curves with changing propagation direction is shown on Fig 5 . As predicted by the theory, the TM' modes remains nearly unchanged, whereas the TE' modes are shifting towards higher effective indexes with increasing values of ϕ. The contrast of the mlines does not change significantly with ϕ. Thus, the measurements with TE and TM polarized incident light beams allows the distinction of the hybrid TE' and TM' waveguide modes. The effective indexes as a function of the propagation angle ϕ are shown on Fig 6 . All values were obtained using the hemisphere except for ϕ = 90°, where the prism was used. The experimental points were used to determine the waveguide structure by fitting to our theoretical model. A very good agreement between experiment and theory is observed up to the third order TE' or TM' modes. The TE' 4 mode could be resolved only for some angles. The good agreement of the higher order modes is surprising considering the large width of the respective mlines (Fig. 5). The substrate indexes, index change, and thickness deduced from the theoretical fit are : n x = 1.7419, n y = 1.7521, n z = 1.8343, d = 4.3 µm, and ∆n = 0.025, respectively. No significant anisotropy of the index change could be observed. Compared to the values obtained by fitting only the TE modes at ϕ = 90° (Fig. 2), the index and index change are identical to the SIP model. The waveguide depth is, however, closer to the value of the IWKB model, which defines this parameter with better precision than the SIP model. In general, the precise determination of the waveguide parameters requires more than two fully guided modes in order to have a high ratio between the number of experimental points and the number of free parameters of the numerical fitting problem. Our results shows that a sufficient number of experimental points can also be obtained by varying the propagation direction in birefringent waveguides. The good angular resolution of the hemisphere mlines allows the investigation of the strong mode hybridization near the mode crossing in the n eff (ϕ) diagram (inset of Fig. 6). The theoretical model predicts that strong mode coupling is only possible for modes with opposite parity, e.g. TM'2 and TE'3. In this case the mode hybridization is total, resulting in the opening of a local effective index gap. In the case of equal parity modes (e.g. TM'1 and TE'3), the modes do not couple and no index gap can be observed. The experimental results seems to confirm this theoretical prediction. The precision of the presented results is actually not limited by the experimental method, but by the investigated waveguide. Narrower mlines, and thus a better resolution, can be obtained by improving the light confinement inside the waveguide. This feature can be realized by increasing the index barrier height and width.

Conclusions

In conclusion we have shown that the hemisphere mline spectroscopy is a useful tool for the characterization of birefringent planar waveguides. It allows the fast measurements of the effective indexes of the guided modes as a function of the propagation direction. The precision of the deduced waveguide parameters is enhanced and more advanced features such as the mode hybridization can be investigated. The presented results are only an example of possible applications of this technique.

d=4.2 µm TE3

Figure 1 :

 1 Figure 1: Experimental hemisphere mline setup.

Figure 2 :

 2 Figure 2: Reconstruction of the waveguide geometry using the measured TE modes for ϕ = 90°. The full line corresponds to the step index calculation and the dashed line and squares to the IWKB method. At the right of the figure are shown the effective indexes values calculated from the step index profile.

Figure 3 :

 3 Figure 3: TE mline curve at ϕ = 0°: dashed line (left yaxis) original curve, continuous line (right yaxis) curve after envelope subtraction.

Figure 4 :

 4 Figure 4: Comparison between mline curves obtained with coupling prism (continuous) and the hemisphere curves (dashed) for TE and TM polarization at ϕ = 0°.

Figure 5 :

 5 Figure 5: Evolution of mlines for ϕ in between 0 and 40° for TE (continuous) and TM(dashed) polarized incident beam.

Figure 6 :

 6 Figure 6: Evolution of the effective indexes as a function of propagation angle ϕ The circles corresponds to the experimental points (full : TM', empty : TE'), the lines to the theoretical model. The different modes are indexed at ϕ = 90°. The inset shows a zoom of the ϕ = 10° to 30° region.

Figure 1

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Figure 5

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Figure 6

 6 Figure 6