Corinne F Lepage

Lucie Léonard-Akkari

Stéphanie Guégan

Fabienne Courand

Olivier Couvert

Jean-François Lepage

Corinne Rondeau-Mouro

Noémie Desriac

Anne-Gabrielle Mathot

Ivan Leguérinel

Louis Coroller

Dr Nicolas Decourcelle
email: nicolas.decourcelle@univ-brest.fr

Dispersed phase volume fraction, weak acids and Tween 80 in a model emulsion: Effect on the germination and growth of Bacillus weihenstephanensis KBAB4 spores

Keywords: Bacillus, spoilage, antimicrobial effect, lipophilicity, emulsion, emulsifier Chemical compounds Acetic acid (PubChem CID: 176), Hexadecane (PubChem CID: 11006), Hexanoic acid

Dispersed phase volume fraction, weak acids and Tween 80 in a model emulsion: Effect on the germination and growth of Bacillus weihenstephanensis KBAB4 spores

Introduction

Growth of microorganisms in food emulsions can lead to major economic losses for the food industry through food-poisoning or spoilage due to the development of bacteria, yeasts and molds in food during shelf-life [START_REF] Brocklehurst | The role of lipids in controlling microbial growth[END_REF][START_REF] Zalazar | Effect of stabilizers, oil level and structure on the growth of Zygosaccharomyces bailii and on physical stability of model systems simulating acid sauces[END_REF]. Two forms of emulsions are mostly encountered in food: oil-in-water (such as milk, dairy cream or mayonnaise) and water-in-oil (such as butter or low-fat spread) [START_REF] Brocklehurst | The role of lipids in controlling microbial growth[END_REF]. Emulsions are metastable colloidal systems formed from two immiscible liquids [START_REF] Wilson | Modelling microbial growth in structured foods: towards a unified approach[END_REF]. Due to the large interfacial area between the two immiscible phases, emulsions are thermodynamically unstable systems and flocculation, coalescence and phase separation (e.g. creaming) are frequently observed during processing or storage [START_REF] Mao | Emulsion Design for the Delivery of β-Carotene in Complex Food Systems[END_REF]. They can be stabilized kinetically because of the repulsion forces between neighboring droplets in the systems thanks to various components such as proteins, lipids and emulsifiers [START_REF] Mao | Emulsion Design for the Delivery of β-Carotene in Complex Food Systems[END_REF][START_REF] Wilson | Modelling microbial growth in structured foods: towards a unified approach[END_REF]. In such complex food emulsions, many factors may have effects on the behavior of microorganisms.

In emulsions, the aqueous phase is commonly recognized as a microbial growth location [START_REF] Brocklehurst | The role of lipids in controlling microbial growth[END_REF][START_REF] Wilson | Modelling microbial growth in structured foods: towards a unified approach[END_REF], and the structural features of this phase are relevant to the length scale of the microorganisms that influence microbial growth [START_REF] Wilson | Modelling microbial growth in structured foods: towards a unified approach[END_REF]. Indeed, the food structure may influence both the growth rate and the conditions in which growth is initiated [START_REF] Brocklehurst | The role of lipids in controlling microbial growth[END_REF]. The structure of complex matrices could also involve the formation of a local chemical specific micro-environment around microbial cells, for instance local pH [START_REF] Robins | Food structure and microbial growth[END_REF]. Thus, the surface of bacteria can interact physico-chemically with food matrix components such as proteins, lipids and emulsifiers. Electrostatic, Van Der Waals and Lewis acid-base interactions must also be considered between microbial surface properties and the environment [START_REF] Ly | Importance of bacterial surface properties to control the stability of emulsions[END_REF].

Bacterial cells are micrometer-scale particles bearing at their surface many macromolecules such as proteins, mannoproteins, peptidoglycan, teichoic acid and polysaccharides [START_REF] Ly | Interactions between bacterial surfaces and milk proteins, impact on food emulsions stability[END_REF]. Their surface composition can be modified by environmental chemical characteristics, as well as the adsorption of the oil phase on the bacterial outer surface [START_REF] Naïtali | Effects of pH and oil-in-water emulsions on growth and physicochemical cell surface properties of Listeria monocytogenes: Impact on tolerance to the bactericidal activity of disinfectants[END_REF].

The structural complexity of food emulsions has a substantial impact on the activity of antimicrobial compounds [START_REF] Terjung | Impact of lauric arginate application form on its antimicrobial activity in meat emulsions[END_REF]. Antimicrobial efficacy has been shown to depend on various product characteristics such as concentrations of fats, proteins, carbohydrates, and salt, as well as the pH [START_REF] Castro | Effect of preservatives, tween 20, oil content and emulsion structure on the survival of Lactobacillus fructivorans in model salad dressings[END_REF][START_REF] Jung | Influence of Fat and Emulsifiers on the Efficacy of Nisin in Inhibiting Listeria monocytogenes in Fluid Milk1[END_REF][START_REF] Terjung | Impact of lauric arginate application form on its antimicrobial activity in meat emulsions[END_REF]. However, difficulties in compiling [Texte] the information and discrepancies in the literature remain since emulsion is a complex matrix exhibiting a wide diversity of compositions and physico-chemical characteristics.

The objective of this study was to investigate interactions between microorganisms, antimicrobial compounds and matrices. The model emulsion was composed of nutrient broth, allowing bacterial growth, as the aqueous phase, hexadecane as the dispersed phase and a Tween 80/Span 80 mixture as the emulsifier. These emulsifiers are non-ionic, frequently encountered in food systems [START_REF] Mcclements | Bailey's Industrial Oil and Fat Products: Processing Technologies Edible Oil and Fat Products-Lipid emulsions[END_REF] and listed as authorized additives under European regulations (EU 1129/2011and EC 1333/2008 (https://eur-lex.europa.eu/)).

According to the scientific literature, Tween 80 is particularly commonly used in food emulsions containing microorganisms and its concentration ranges from 0.1 to 10 % (w/w) [START_REF] Buranasuksombat | Influence of emulsion droplet size on antimicrobial properties[END_REF][START_REF] Celiktas | Antimicrobial activities of methanol extracts and essential oils of Rosmarinus officinalis, depending on location and seasonal variations[END_REF][START_REF] Hilbig | Physical and antimicrobial properties of cinnamon bark oil co-nanoemulsified by lauric arginate and Tween 80[END_REF][START_REF] Krogsgard-Nielsen | Antimicrobial effect of emulsion-encapsulated isoeugenol against biofilms of food pathogens and spoilage bacteria[END_REF][START_REF] Landry | Inactivation of Salmonella on Sprouting Seeds Using a Spontaneous Carvacrol Nanoemulsion Acidified with Organic Acids[END_REF][START_REF] Murray | Influence of Inoculum Preparation upon Sensitivity of Common Food Borne Pathogens to Emulsion Based Antimicrobials[END_REF][START_REF] Neves | Antimicrobial Oil-in-Water Nanoemulsions: Synergistic Effect of Nisin and Carvacrol against Bacillus subtilis[END_REF][START_REF] Ribes | Application of cinnamon bark emulsions to protect strawberry jam from fungi[END_REF]. Both emulsifiers were chosen to stabilize the emulsion because of this non-ionic characteristic to limit artefacts. The use of hexadecane prevented any trace of polyunsaturated fatty acid which could affect microorganism behavior (Mvou Lekogo, Coroller, Mathot, Mafart, & Leguérinel, 2010). The emulsion characteristics (pH, weak acid and dispersed phase volume ratio) were modified and the impacts were assessed on several scales, namely Bacillus weihenstephanensis germination and outgrowth, and the interface phenomenon with the emulsifier. Bacillus weihenstephanensis was used in this study because of its capacity i) to contaminate chilled and minimally processed food, ii) to constitute a potential danger for customers' health, and iii) to limit products' shelf life leading to economic losses [START_REF] Antolinos | Modelling the effects of temperature and osmotic shifts on the growth kinetics of Bacillus weihenstephanensis in broth and food products[END_REF][START_REF] Desriac | Sensitivity of Bacillus weihenstephanensis to acidic changes of the medium is not dependant on physiological state[END_REF].

Materials and methods

Model system preparation

The model emulsion was composed of nutrient broth as the aqueous phase, hexadecane as the dispersed phase and a Tween 80/Span 80 mixture as the emulsifier. This emulsion was inoculated with Bacillus weihenstephanensis spores or vegetative cells.

Inoculum preparation

Bacillus weihenstephanensis strain KBAB4 (INRA, Avignon, France) was used in this study.

The strain (in the form of vegetative cells) was stored at -80 °C in Brain Heart Infusion (BHI) (Biokar, Diagnostics, Beauvais, France) mixed with 50 % glycerol (v/v). A 100-mL volume of BHI was inoculated with 1 mL of the stock suspension and incubated for 8 h at 30 °C, the [Texte] optimal growth temperature; then, a 1-mL volume was transferred into 100 mL of BHI for 16 h at 30 °C. Finally, 0.1 mL of the Bacillus weihenstephanensis suspension was added to 100 mL of BHI and was incubated for 6 h. Spores were produced through a two-step sporulation process as described in [START_REF] Baril | The wet-heat resistance of Bacillus weihenstephanensis KBAB4 spores produced in a two-step sporulation process depends on sporulation temperature but not on previous cell history[END_REF]. The final concentration of the bacterial spore suspension was 10 7 spores⋅mL -1 . The number of spores was estimated as the number of cells surviving a 5-min, 70 °C heat treatment.

Emulsion preparation

Hexadecane (H6703, Sigma Aldrich, St-Louis, MO, USA) was mixed with nutrient broth (Biokar, Diagnostics, Beauvais, France) using sonication and stabilized by Tween 80 (P4780, Sigma Aldrich, St-Louis, MO, USA) and Span 80 (S6760, Sigma Aldrich, St-Louis, MO, USA). Emulsions with either 35% (v/v) (dispersed phase volume ratio (ɸ) = 0.35) or 50% (v/v) (ɸ = 0.50) hexadecane content were studied as a function of pH (5.5 and 6.0), adjusted with HCl, with or without weak acid. Three weak acids were studied as antimicrobial compounds: acetic acid (pKa=4.75; logP=0.17) (A6283, Sigma Aldrich, St-Louis, MO, USA), hexanoic acid (pKa=4.88; logP=1.92) (153745, Sigma Aldrich, St-Louis, MO, USA) and lactic acid (pKa=3.90; logP=-0.72) (69775, Sigma Aldrich, St-Louis, MO, USA). The logP values were assessed by [START_REF] Hansch | Exploring Qsar[END_REF]. Acetic acid and lactic acid were studied as food additives. Hexanoic acid was studied because of its higher hydrophobicity (logP=1.92) compared to acetic acid and lactic acid (logP=0.17 and logP=-0.72, respectively).

The aqueous phases were composed of 0.005% iron citrate, 0.1% esculin, 2% nutrient broth, 3% Tween 80 for emulsions with ɸ = 0.50 and 2.3% Tween 80 for emulsions with ɸ = 0.35 (% w/w of the aqueous phase). A weak acid was mixed with the aqueous phase during the preparation process to obtain final concentrations of 14 mM of acetic acid, 1.8 and 4 mM of hexanoic acid, and 82 and 255 mM of lactic acid. Hexadecane and the aqueous phases were sterilized by filtration (0.22 µm, Stericup® Millipore) prior to spore inoculation. Heat-treated spore suspension (5 min.; 70 °C) was added to the aqueous phase at three different concentrations: 100, 20 and 3.5 spores per 100 µL of aqueous phase. The mixtures were sonicated for 1 min at 60 % amplitude using a Branson 450 W-20 kHz digital sonifier equipped with a temperature probe. Thus, for each sample the temperature variation was controlled and rose 40 °C in 1 min of mixing.

Physical characterization of emulsions and microbial cells

Droplet size and stability

[Texte]

The emulsion particle size distributions were measured by laser granulometry with a Malvern MS3000, in 0.1 % SDS dispersant at 2000 rpm. The mean volume diameters D [4,3] and the particle size dispersions were monitored for 10 days with storage at 30 °C which was the temperature used for the microbial assay. The emulsion stability (flocculation, creaming, clarification) was monitored by multiple light scattering with a TurbiScan (Formulaction, France). The destabilization phenomena were monitored for 10 days at 30 °C.

Zeta potential measurements

According to [START_REF] Bundeleva | Zeta potential of anoxygenic phototrophic bacteria and Ca adsorption at the cell surface: Possible implications for cell protection from CaCO3 precipitation in alkaline solutions[END_REF], zeta-potential (ξ-potential) can be used to perform the change in the cell surface potential (mV) of microorganisms as a function of pH. ξ-potential is an important parameter to analyze the stability of particles [START_REF] Mubarakali | An inhibitory action of chitosan nanoparticles against pathogenic bacteria [Texte] and fungi and their potential applications as biocompatible antioxidants[END_REF].

The ξ-potential of vegetative cells and spores was measured using a Zetasizer Nano-ZS (Malvern Instruments, United Kingdom). The analysis was performed at 25 °C between pH 4 and 9; a range covering those used for the microbial assay. Vegetative cells were recovered from a culture in the exponential phase with an optical density of 0.5 at 600 nm and washed twice in tryptone salt medium. The vegetative cell suspension and spore stock suspension were diluted 10-and 1000-fold respectively in 5 mmol⋅L -1 of imidazole/acetate buffer adjusted to the appropriate pH using NaOH or HCl solutions (1 mol⋅L -1 ) prior to measurements. Samples were introduced into capillary cells and measured in automatic mode.

The ξ-potential measurements are reported as the average and standard deviation of the measurements for three freshly prepared samples.

Surface hydrophobicity

The hydrophobicity of vegetative cells and spores was evaluated by Microbial Adhesion to Hexadecane (MATH) [START_REF] Bellon-Fontaine | Microbial adhesion to solvents: a novel method to determine the electron-donor/electron-acceptor or Lewis acid-base properties of microbial cells[END_REF]. Vegetative cells in the exponential phase were harvested by centrifugation at 5000 g for 10 min at 4 °C, washed twice in tryptone salt medium and resuspended to A 600nm = 0.5 (A 0 ) in physiological water. 0.5 mL of hexadecane was added to 3 mL of cell or spore suspension with optical density A 0 .

The two-phase system was mixed by 10 s shaking by hand, then by vortexing 1 min, and finally by 10 s shaking by hand. The system was allowed to separate for 15 min. The absorbance of the aqueous phase at 600 nm (A 1 ) was measured. The percentage of microbial adhesion (Ad%) to solvent was calculated as follows (1): [Texte] Microstructures of the model emulsion containing spores were observed with a Confocal Laser Scanning Microscope (CLSM) Zeiss LSM780 (Carl Zeiss, Oberkochen, Germany). The hexadecane was stained with Nile Red (72485, Sigma Aldrich, St-Louis, MO, USA) (emission/excitation wave length: 488-515/>590 nm) at a final concentration of 16 mg⋅mL -1 .

100 1 % 0 1 ×         - = A A Ad (1)

Microscopy

Spores were labeled with Thioflavin T (T3516, Sigma Aldrich, St-Louis, MO, USA) (emission/excitation wave length: 405/485 nm) at a final concentration of 100 µmol⋅L -1 of spore suspension. Two light sources were used: a diode laser at 405 nm and an Argon laser at 514 nm.

Pulsed Field Gradient-Nuclear Magnetic Resonance (PFG-NMR)

NMR measurements were performed at 30 °C with a 500 MHz Bruker spectrometer, equipped with a field gradient probe (0 to 12 T/m) and using the LED-BP-PFGSTE sequence in which ∆ is the diffusion delay (5.64 to 100 ms) and δ the gradient pulse duration (1 ms). Within a measurement, sequence parameters δ, ∆ and τ (time between the end of each gradient and the next radiofrequency pulse) (1 ms) remained constant and successive and increasing values of the gradient amplitude (g) were applied.

The data was processed using the Dynamics Center (DOSY reconstruction) and the T1/T2 relaxation module (1D processing) developed by Bruker under Topspin 3.2. For 1D processing, the self-diffusion coefficient (D) was determined on the basis of the following equation (2):

      ×       - - ∆ × × × - = D g I I g 4 3 exp 2 2 2 0 τ δ δ γ (2)
(assuming in this case monoexponential decay), where I g is the attenuated signal intensity by application of gradient pulses, I 0 the optimum signal intensity without field gradient, and γ the gyromagnetic ratio). The self-diffusion coefficient (D0) were 4.50 × 10 -11 ± 7.50 × 10 -13 m²⋅s - 1 , 4.15×10 -10 ± 1.0 × 10 -12 m²⋅s -1 , 1.24 ×10 -09 ± 3.0 × 10 -12 m²⋅s -1 and 5.10 × 10 -11 ± 1.63 × 10 - 12 m²⋅s -1 , for pure lactic acid, hexanoic acid, acetic acid and Tween 80, respectively. Then, each weak acid and Tween 80 were diluted in water (ɸ = 0) or prepared in two different emulsions with ɸ = 0.35 and with ɸ = 0.50. For each acid, the self-diffusion coefficient D in water or emulsions was compared to D0 (pure weak acid or Tween 80) by calculating the ratio D/D0. [Texte]

Characterization of bacterial behavior

Assessment of the number of spores able to germinate and to grow in emulsion

The inoculated emulsions were distributed in sterile 96-well microplates. Spores of Bacillus weihenstephanensis KBAB4 able to germinate and to outgrow were detected by the loss of fluorescence of wells due to the esculin metabolism. These spores were then enumerated by the most probable number method (MPN). An estimation of the percentage of spores able to germinate and to give active cells in the emulsion was calculated as follows (3):

100 % 0 ×         = N MPN OutG (3)
with N 0 (Colony Forming Unit, CFU), the initial population of spores determined after a 5min, 70 °C heat treatment and dilution in tryptone salt broth (Biokar, Diagnostics, Beauvais, France) by plating in nutrient agar (Biokar, Diagnostics, Beauvais, France).

Monitoring Bacillus spore germination by flow cytometry

Spore germination and outgrowth in nutrient broth in the presence of Tween 80 were monitored by flow cytometry (FCM) analysis using the fluorescent dye Syto®9 (Invitrogen, Carlsbad, California, United States) to investigate the effect of the emulsifier at different pH values on spores. Syto®9 fluoresces only when bound to either double-stranded DNA or RNA with an emission maximum of 520 nm (green). Dormant refractive spores show only peripheral staining with Syto®9, however, intact germinating spores stain brightly (van Melis, Groot, Tempelaars, Moezelaar, & Abee, 2011). For 1 mL of culture sample, 0.5 µL of Syto®9 was added [START_REF] Van Melis | Characterization of germination and outgrowth of sorbic acid-stressed Bacillus cereus ATCC 14579 spores: phenotype and transcriptome analysis[END_REF]. Flow cytometric analyses were carried out using a CyFlow®Space (Partec, France). Forward (FSC) and side (SSC) scatter signals were collected from 488 nm excitation. Analysis was carried out in true volumetric absolute counting mode (analysis in 200 µL). Samples were diluted with tryptone salt broth prior to analysis in order to respect a sampling rate ranging from 500 to 5000 particles⋅s -1 . Analysis and gating of data were performed using FloMax® software (Partec, France).

Bioscreen® growth experiments

The growth kinetics of Bacillus weihenstephanensis KBAB4 were evaluated in nutrient broth at 30 °C with different concentrations of Tween 80 (0, 1, 5 and 10% (w/w)) to investigate the effect of the emulsifier at different pH values on vegetative cells. Turbidity growth curves were generated with an automatic Bioscreen C reader (Oy Growth Curves Ab Ltd., Helsinki, Finland). From a pre-culture in the end exponential phase, the first line of a plate was inoculated with a 10 5 CFU⋅mL -1 suspension and, to complete the plate, seven serial two-fold [Texte] dilutions were performed in the tested medium. Then the plate was placed in the Bioscreen C reader at 30 °C. The increase in turbidity was monitored at 600 nm. Measurements were taken every 10 min, and the plate was shaken at low intensity for 20 s prior to absorbance measurements. The maximum specific growth rate (µ max ) values corresponding to each Tween 80 concentration were determined by linear regression as previously described [START_REF] Membré | Temperature effect on bacterial growth rate: quantitative microbiology approach including cardinal values and variability estimates to perform growth simulations on/in food[END_REF]. Paired Student's t-tests were performed to compare the maximum specific growth with and without Tween 80. P-values strictly lower than 0.05 were considered significant.

Individual lag times

The distribution of individual lag times was investigated according to a method adapted from [START_REF] Guillier | Influence of stress on individual lag time distributions of Listeria monocytogenes[END_REF]. From end exponential phase cells, serial dilutions were performed in the tested medium in order to obtain a final bacterial suspension at 1.4 CFU⋅mL -1 . Assuming thereafter that the distribution of the cells in the microplate followed a Poisson distribution, the maximum concentration of 0.42 CFU⋅well -1 will correspond to a maximum of 35 % of wells showing growth and 20 % of them, i.e. 7 %, will only have one cell.

Then the plate was placed in the Bioscreen C reader at an incubation temperature of 30 °C.

The increase in turbidity was monitored at 600 nm. Measurements were taken every 10 min, and the plate was shaken at low intensity for 20 s prior to absorbance measurements.

On the assumption that the maximum specific growth rate was constant from one well to another, the detection time (T d ) is related to the lag time (lag) by the following formula [START_REF] Baranyi | Estimating bacterial growth parameters by means of detection times[END_REF]) (4):

( ) ( ) max 0 ln ln µ N N lag T d d - + = (4)
with N d , the bacterial number at T d ; N 0 , the number of cells initiating growth in the considered well, and µ max , the maximum specific growth rate.

The Kolmogorov-Smirnov statistical test was applied to compare the distributions of latency times with and without Tween 80 at one pH value (XLSTAT software, 2017 version).

Results

Physical characteristics of emulsion constituents

Droplet size

The dispersion was studied to characterize the spatial organization and locate the different components. Measurements by laser granulometry showed that emulsions with ɸ = 0.35 and ɸ = 0.50 were monodispersed. Indeed, an emulsion is considered concentrated when it reaches [Texte] a concentration 64 % of monodisperse droplets, which represents the random-close-packing volume fraction [START_REF] Leal-Calderon | Emulsion science: basic principles[END_REF]. The droplets were agitated by a Brownian motion. They presented a monomodal distribution of hexadecane droplets with, respectively, a D [4,3] of 0.484 ± 0.005 µm and 0.437 ± 0.006 µm. During a 10-day period at 30 °C, neither the creaming nor the stability of the emulsions was modified with or without organic acid and whatever the pH. As observed in the emulsions using confocal microscopy (Figure 1), the droplets of the dispersed phase appeared to be smaller than the spores (~1 µm) [START_REF] Carrera | Difference between the spore sizes of Bacillus anthracis and other Bacillus species[END_REF]. In our systems, microbial growth seemed to occur in the aqueous phase as is commonly recognized [START_REF] Brocklehurst | The role of lipids in controlling microbial growth[END_REF][START_REF] Wilson | Modelling microbial growth in structured foods: towards a unified approach[END_REF].

Spore characteristics

The surface of the spores and vegetative cells were characterized by MATH [START_REF] Bellon-Fontaine | Microbial adhesion to solvents: a novel method to determine the electron-donor/electron-acceptor or Lewis acid-base properties of microbial cells[END_REF]. We found that the spores had a hydrophobic surface contrary to vegetative cells. The ξ-potential values of spores and vegetative cells, i.e. net charges on the surface, were also determined in phosphate buffer ranging from pH 4 to 9. The electrophoretic motilities of the spores and vegetative cells indicated an electronegative character with ξpotential ranging, respectively, from -9.8 to -41.0 mV and from -11.2 to -18.7 mV.

Diffusion of emulsion constituents by NMR

In order to highlight the effect of emulsion structure on the diffusion coefficients of weak acids, the D/D 0 ratio was reported as a function of pH for each hexadecane/water ratio (Figure 2). The D/D0 ratio variations were significant as showed by the calculated standard error for each ratio. Figure 2 shows that for each weak acid, whatever the pH, D/D 0 tended to decrease when the hexadecane/water ratio increased. This indicates that a fraction of each weak acid may be trapped in the hexadecane, and/or the emulsion structure impacts on its diffusion coefficient. Discrepancies of D/D 0 values among acids were likely due to two effects: D/D 0 > 1 may be due to a lower viscosity in water or in emulsion in comparison to bulk acid, and D/D 0 < 1 may be due to a structure effect in emulsion either by molecule trapping or interactions between weak acids and droplets. These results were obtained by assuming that the total amount of weak acid was diffused in the emulsion in a unique way (mono-exponential decrease of the NMR signal). In contrast to lactic acid and acetic acid, D/D 0 for hexanoic acid tends to decrease with pH (Figure 2.a). This discrepancy could account for both the higher pKa and the higher logP for hexanoic acid, favoring its affinity for the dispersed hexadecane phase.

[Texte]

For Tween 80, the self-diffusion coefficient was around 10 -11 m 2 ⋅s -1 in water and around 10 -10 m 2 ⋅s -1 in emulsions, whatever the pH value and hexadecane volume fraction. These two different values might be due to the spatial arrangement of Tween 80 in the form of micelles in water and at the emulsion interfaces [START_REF] Henry | Emulsification mechanism and storage instabilities of hydrocarbon-in-water sub-micron emulsions stabilised with Tweens (20 and 80), Brij 96v and sucrose monoesters[END_REF]. First of all, the percentage of germination and outgrowth in the nutrient broth with HCl at pH 5.5 was 1.5-fold lower compared to pH 6 (Table 1). The percentages of germination and outgrowth obtained with the weak acids was 0 to 1.2-fold lower than with HCl at pH 6.0 and 1.3 to 2.8-fold lower at pH 5.5.

Comparison

Secondly, the percentages of germination and outgrowth in the aqueous phase (nutrient broth with 3 % (w/w) Tween 80 (ɸ = 0)) used for the emulsion with HCl at pH 5.5 were lower than those at pH 6, also reflecting the influence of pH on B. weihenstephanensis KBAB4 (Table 1). Yet, we noticed that the aqueous phase (ɸ = 0) in the presence of the weak acids and HCl at pH 5.5 revealed that less than 0.03 % of spores outgrew compared to 22 to 61 % in the nutrient broth. This highlighted an antimicrobial synergistic effect between Tween 80 and weak acid depending on the pH value.

Moreover, the percentages of outgrowth observed in all ɸ = 0.35 emulsions as well as those observed in the ɸ = 0.50 emulsions with lactic acid and acetic acid at pH 5.5 and pH 6 did not differ from the aqueous phase (Table 1). Surprisingly, data in Table 1 show that the ɸ = 0.50 emulsions with HCl at pH 5.5 revealed a higher percentage of outgrowth compared to the aqueous phase or the ɸ = 0.35 emulsions with HCl at the same pH value. In the same way, these data also show that the ɸ = 0.50 emulsions with hexanoic acid at pH 6 revealed a higher percentage of outgrowth compared to the aqueous phase or the ɸ = 0.35 emulsions with hexanoic acid at the same pH value.

These results confirm the influence of pH and of the weak acids as hurdles preventing the germination and outgrowth of B. weihenstephanensis KBAB4 in the studied systems. Yet, Tween 80, used as the emulsifier in the emulsions, also acted as an additional hurdle.

Furthermore, the ability of spores to recover and grow was modulated by the interactions [Texte] among Tween 80, hexanoic acid and their environment. This illustrated the complexity of controlling the bacterial behavior through physico-chemical factors in complex matrices.

Effect of Tween 80 on the growth of vegetative cells (µ max , lag times)

In order to characterize the antimicrobial effect of Tween 80 on the growth of KBAB4 cells, Bioscreen® growth experiments were carried out to determine growth parameters in the presence or absence of Tween 80 at pH 5.5 and pH 6.0 (Figure 3). At pH 6.0 with a Tween 80 concentration of 5 % (w/w) and more, µ max was significantly lower than the control without Tween 80. At pH 5.5 with a Tween 80 concentration of 2% and more, µ max was significantly lower than without Tween 80 (Student's t-test, p < 0.05). Tween 80 seemed to have an antimicrobial effect on the growth of vegetative cells depending on the pH value.

Minimum inhibitory concentrations were determined by a linear regression: at pH 6.0, MIC was 64.8 % (w/w) and at pH 5.5, MIC was 19.9 % (w/w).

The distribution of individual lag times of vegetative cells with and without Tween 80 at pH 6.0 and 5.5 were assessed (Figure 4). The presence of Tween 80 in the growth medium increased the individual lag times. At pH 6, the frequency distribution was maximal for the 10-11 h lag time interval without Tween 80, and for the 10-12 h interval with Tween 80. At pH 5.5, the frequency distribution was maximal for the 12-13 h lag time interval without Tween 80, and for the 16-18 h interval with Tween 80. The effect of Tween 80 on the growth delay was more marked at acidic pH (higher D statistic at pH 5.5 (Kolmogorov-Smirnov statistical test)). The frequency distributions without Tween 80 and with Tween 80 were significantly different at both pH values (Kolmogorov-Smirnov statistical test). As shown by these results, at pH 5.5 and with Tween 80, the variability of lag times was higher than for any other combination tested, which indicates that, at single cell level, the adaptation period, and therefore the lag times, had a much greater variability than in the other conditions tested. This indicates that the combination has an important inhibitory effect on cells.

Effect of Tween 80 on spore germination (kinetics determined by flow cytometry)

To determine whether Tween 80 induced a delay in germination, spores were exposed to different concentrations of Tween 80 in nutrient broth at pH 5.5 and their germination was monitored by flow cytometry (Figure 5). According to [START_REF] Van Melis | Characterization of germination and outgrowth of sorbic acid-stressed Bacillus cereus ATCC 14579 spores: phenotype and transcriptome analysis[END_REF], dormant refractive spores are slightly stained (Figure 5, quadrant I). Upon germination, spore permeability to Syto®9 and the accessibility of DNA increased, leading to an increase in the [Texte] fluorescence level (Figure 5, quadrant II). The size increase of the outgrowing spores could then be monitored by an increase in the forward-scatter signal (Figure 5, quadrant III).

After 4 h, for all the conditions, the spores were still dormant. Within the following four hours, the spore populations became permeable to Syto®9 (quadrant II, germinated spores).

Under unstressed conditions (without Tween 80), the majority of the cell population was able to outgrow (located in quadrant III), while for stress conditions, a substantial part of the spore population remained dormant (quadrant I) until 10 h of incubation. Without Tween 80, 4.59 % of spores were still refractive spores (quadrant I) after 10 h of incubation, while with 5 % and 10 % of Tween 80 in nutrient broth, the percentage of dormant spores was respectively 12.04 % and 54.59 %. It can also been seen from Figure 5C that after 8h and 10h germinated spores and growth (quadrant II and quadrant III, respectively) decreased more sharply than for other conditions. Thus, increasing the concentration of Tween 80 drastically decreased germination and growth.

Discussion

First of all, H + ion concentrations had an effect on spore germination and outgrowth as expected. H + ions will move into the cytoplasm decreasing internal pH. Drastic variations in cytoplasmic pH can harm bacteria by disrupting the plasma membrane or inhibiting enzyme activities and membrane transport proteins. Changes in the external pH might also alter the ionization of nutrient molecules and thus reduce their availability to the organism. In our model emulsion, the effect of pH was detected at pH 5.5 regardless of the dispersed phase volume fraction. Nevertheless, at pH 5.5 less than 0.03 % germination and outgrowth may occur. The minimum growth pH value is 4.35 and the estimated optimal recovery pH is 7.80 for B. weihenstephanensis KBAB4 [START_REF] Trunet | Modeling the recovery of heat-treated Bacillus licheniformis Ad978 and Bacillus weihenstephanensis KBAB4 spores at suboptimal temperature and pH using growth limits[END_REF]. This result suggested a synergistic effect between pH value and matrix composition.

The role of undissociated weak acid molecules was also important. Weak acids are the most commonly used chemical preservatives in the food industry to control both pH and microbial growth [START_REF] Van Melis | Characterization of germination and outgrowth of sorbic acid-stressed Bacillus cereus ATCC 14579 spores: phenotype and transcriptome analysis[END_REF]. It is generally accepted that that bacterial growth inhibition by weak acids is based on the rapid diffusion of undissociated molecules through the plasma membrane, followed by immediate dissociation and acidification of the cytoplasm.

Concerning their effects on bacterial spores, van Melis et al. ( 2012) formulated two possibilities. Inhibition could be due, on one hand, to the accumulation of acids in the spore [Texte] inner membrane or, on the other hand, to the accumulation of foreign compounds in the inner membrane. As we have shown, such effects may occur in emulsions and may be affected by the dispersed phase volume fraction and the presence of Tween 80. These relevant effects should be highlighted and characterized, as they may have important safety implications in foods based on emulsions.

There was no influence of the dispersed phase volume fraction in the presence of acetic acid and lactic acid, whereas hexanoic acid was less effective in ɸ = 0.50 emulsions. However, results contrary to ours are described in the literature. [START_REF] Prachaiyo | Oil-in-Water Emulsion as a Model System to Study the Growth of Escherichia coli O157: H7 in a Heterogeneous Food System[END_REF] showed an increase in the generation time of bacteria as the hexadecane concentration increased. This is likely to be due to space limitation, nutrient limitation and/or the concentration of metabolic products. In the same way, [START_REF] Zalazar | Effect of stabilizers, oil level and structure on the growth of Zygosaccharomyces bailii and on physical stability of model systems simulating acid sauces[END_REF] described how an increase in the oil level promoted a decrease in the specific growth rate. On the other hand, [START_REF] Dong | Inactivation of Bacillus amyloliquefaciens spores by continuous high-pressure-assisted thermal sterilization in an oil-in-water (o/w) emulsion with 10% soybean oil[END_REF] reported a comparable inactivation of Bacillus amyloliquefaciens spores by thermal and high hydrostatic pressure treatments in an oil/water emulsion with 20 % and 10 % soybean oil. As the dispersed phase droplets in our study were smaller than the B.

weihenstephanensis KBAB4 spores, the cells seemed to evolve in the aqueous phase following a Brownian motion and the increase in hexadecane concentration did not have an effect on the germination and growth percentage.

In addition, our results showed a decline in efficiency of hexanoic acid with the increase in hexadecane concentration. This could be related to the lipophilic character of this weak acid.

Indeed, according to the NMR results, the impact of hexadecane/water ratio impacted the hexanoic acid diffusion coefficient more, which decreased with the pH. This could mean that a fraction of hexanoic acid may be trapped in the dispersed phase limiting its access to cells that were in the aqueous phase. Similarly, [START_REF] Brocklehurst | The role of lipids in controlling microbial growth[END_REF] wrote that the weak acid components interact with the lipid phase, and the effect was a decrease in the preservation of the products. For example, acetic acid had a low partition coefficient: just a small proportion of this acid was dissolved in the lipid phase and therefore predominantly available in the water phase as a preservative. This was not the case for sorbic acid or benzoic acid which had respectively a logP-value of 1.33 and 1.87. Furthermore, in most emulsified food, emulsifiers are above their critical micelle concentration at which aggregates known as micelles are formed. The non-polar inner regions of micelles present an environment closely similar to that created by an organic solvent, where solutes such as weak acid will tend to partition. For this reason, the preservative effectiveness of a weak acid will be associated with its distribution in the different phases of the food emulsion, considering the interior of the [Texte] micelles as a third phase [START_REF] Castro | Effect of preservatives, tween 20, oil content and emulsion structure on the survival of Lactobacillus fructivorans in model salad dressings[END_REF]). In our system, the lipophilicity of the biocide, hexanoic acid, seemed to have had an impact on the distribution of the weak acid between the dispersed phase and the aqueous phase and thus, on the antimicrobial activity.

Moreover, the structure and composition of the model emulsion seemed to affect antimicrobial activity. The loss of microbial inhibitory activity when the dispersed phase volume fraction increased could be due to phenomena at the interfaces. Factors such as hydrophobicity, surface charge, and cell density influence the attachment of microorganisms to surfaces and the behavior at interfaces [START_REF] Seale | Recovery of spores from thermophilic dairy bacilli and effects of their surface characteristics on attachment to different surfaces[END_REF]. The main result that emerged from the work of [START_REF] Naïtali | Effects of pH and oil-in-water emulsions on growth and physicochemical cell surface properties of Listeria monocytogenes: Impact on tolerance to the bactericidal activity of disinfectants[END_REF] was that the cell surface hydrophobicity of Listeria monocytogenes increased after growth in emulsion. This may have been due to modifications to the cell surface composition, as well as to the adsorption of the oil phase on the outer surface of the bacteria [START_REF] Naïtali | Effects of pH and oil-in-water emulsions on growth and physicochemical cell surface properties of Listeria monocytogenes: Impact on tolerance to the bactericidal activity of disinfectants[END_REF]. The hydrophobicity of various species of the B. cereus group spores varies from moderate to high [START_REF] Faille | Morphology and physico-chemical properties of Bacillus spores surrounded or not with an exosporium: consequences on their ability to adhere to stainless steel[END_REF]. This hydrophobic character it thought to be linked to the presence of an exosporium on the outer spore membrane composed of proteins, polysaccharides, lipids and ash [START_REF] Faille | Morphology and physico-chemical properties of Bacillus spores surrounded or not with an exosporium: consequences on their ability to adhere to stainless steel[END_REF][START_REF] Sagong | Combined effects of ultrasound and surfactants to reduce Bacillus cereus spores on lettuce and carrots[END_REF]. Spores are more hydrophobic than vegetative cells. It has been suggested that the increased hydrophobicity of the spores may be due to a relative abundance of protein in the outer coats and exosporium compared with the peptidoglycan on vegetative cell surfaces [START_REF] Parkar | Factors influencing attachment of thermophilic bacilli to stainless steel[END_REF]. Previous works have shown that a lower ξ-potential, i.e. net charge on the spore surface, may be correlated with increased surface attachment [START_REF] Husmark | Forces involved in adhesion of Bacillus cereus spores to solid surfaces under different environmental conditions[END_REF], while others did not identify a correlation [START_REF] Parkar | Factors influencing attachment of thermophilic bacilli to stainless steel[END_REF]. These different observations reflect the fact that the surface attachment or the location of these bacteria in a complex system is a multifactorial process, combining physical factors such as surface appendages, chemical signaling between cells and chemical interactions.

Furthermore, the component linked to interfaces, namely the emulsifier, could also interact with cells and affect germination and/or growth. Tween 80 is commonly used as an emulsion stabilizer in a wide range of food and personal care products [START_REF] Henry | Emulsification mechanism and storage instabilities of hydrocarbon-in-water sub-micron emulsions stabilised with Tweens (20 and 80), Brij 96v and sucrose monoesters[END_REF].

Tween-type emulsifiers possess a branched hydrophilic region of three polyoxyethylene chains substituted to a sorbitan ring. The results of the present study revealed an antimicrobial effect of Tween 80 on B. weihenstephanensis KBAB4 spores and on vegetative cells which depended on pH. An effect of Tween 80 on the action of antimicrobial compounds has [Texte] (permeability, hydrophobicity/hydrophilicity). Moreover, the synergistic effect between Tween 80/pH and Tween 80/organic acid shows that Tween 80 could destabilize the membrane, facilitating the action of pH or organic acid. Concerning this effect on germination, spore surfaces were hydrophobic, so interactions could have been possible on the surface of the spores with Tween 80, inhibiting germination mechanisms. Tween 80 is a non-ionic emulsifier and would therefore not be expected to have any charge, although previous studies have shown that droplets stabilized by non-ionic emulsifiers may be charged due to the presence of ionizable surface active impurities (such as free fatty acids) or due to preferential adsorption of hydroxyl ions at high pH or hydrogen ions at low pH to the droplet surfaces [START_REF] Yang | Formation and stability of emulsions using a natural small molecule surfactant: Quillaja saponin (Q-Naturale®)[END_REF].

These interactions between the surface of the spores and Tween 80 could explain in some cases the fact that germination and outgrowth were lower in ɸ = 0.35 emulsions than in ɸ = 0.50 emulsions. The interfacial region plays a crucial role in controlling the distribution of molecules in the emulsified system (Pastoriza-Gallego, Losada-Barreiro, & Bravo-Diaz, 2012). Amphiphilic molecules are primarily located in the interfacial region; meanwhile nonpolar molecules tend to be mainly distributed between the oil and interfacial regions and polar molecules are distributed between the aqueous and interfacial regions [START_REF] Pastoriza-Gallego | Effects of acidity and emulsifier concentration on the distribution of Vitamin C in a model food emulsion[END_REF]. It seems that the effect of the dispersed phase volume fraction on antimicrobial activity observed in our system was due to the distribution of the emulsifier between the aqueous phase and the interface. According to [START_REF] Henry | Emulsification mechanism and storage instabilities of hydrocarbon-in-water sub-micron emulsions stabilised with Tweens (20 and 80), Brij 96v and sucrose monoesters[END_REF], Tween 80 had a head group size of 0.78 nm 2 (average of 3 values in the literature). With the dispersed phase volume fraction, the dispersed phase droplet volume and the concentration of emulsifier in the emulsion, it was possible to calculate the proportion of each emulsifier required to give the surface coverage of dispersed phase droplets for the emulsions produced. In ɸ = 0.50 emulsions, with the concentration of Tween 80 used (1.5 % (w/w) in final emulsions), all molecules were at the interfaces: there were no free Tween 80 molecules. They were not in contact with the cells, hence the antimicrobial activity due to Tween 80 was limited. On the contrary, in ɸ = 0.35 emulsions, Tween 80 molecules were in excess to cover all droplets, so a share of the molecules remained free. They were therefore in contact with the cells in the aqueous phase and the antimicrobial activity of Tween 80 was higher. [Texte] http://eur-lex.europa.eu/homepage.html?locale=en

Conclusions

  of the percentage of outgrown B. weihenstephanensis spores in aqueous phases and model emulsions An estimation of the percentage of outgrown B. weihenstephanensis KBAB4 spores (i) in the nutrient broth, (ii) in the aqueous phase (nutrient broth with 3 % (w/w) Tween 80 (ɸ = 0)), (iii) in ɸ = 0.35 emulsions and (iv) in ɸ = 0.50 emulsions was calculated from an enumeration by a most probable number (MPN) assay.

Fig. 1 .

 1 Fig. 1. Confocal microscopy observations of inoculated ɸ = 0.50 emulsions with labeling of Bacillus spores with Thioflavin T (green) and the dispersed phase with Nile Red (red). (a, b, c: 2D observations; d: 3D observations). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3 .

 3 Fig. 3. Maximal growth rate (μmax) at pH 6.0 and 5.5 in nutrient broth in the presence of Tween at different concentrations from 0% to 10% (*,**: μmax significantly different from μmax obtained without Tween 80 following a Student t-test (P < 0.05)).

Fig. 4 .

 4 Fig. 4. Frequency distribution of individual lag times during growth of B. weihenstephanensis KBAB4 in nutrient broth without Tween 80 (black histogram) and with 5% (w/w) of Tween 80 (gray shaped histogram): a-at a pH 6.0; b-at a pH 5.5.

Acknowledgments

The authors gratefully acknowledge the "Conseil Départemental du Finistère" for their financial support, Pr. Y. Grohens and Dr. B. Saulnier from the "Institut de Recherche Dupuy de Lôme" (IRDL) for their support concerning the ξ-potential measurements, the PIMM-DRX platform of the University of Western Brittany ("Université de Bretagne Occidentale, UBO") for the technical support for microscopic observations and the PRISM platform for access to high-field NMR facilities. [Texte]

.

previously been mentioned in the context of interactions in food systems [START_REF] Castro | Effect of preservatives, tween 20, oil content and emulsion structure on the survival of Lactobacillus fructivorans in model salad dressings[END_REF][START_REF] Jung | Influence of Fat and Emulsifiers on the Efficacy of Nisin in Inhibiting Listeria monocytogenes in Fluid Milk1[END_REF][START_REF] Krogsgard-Nielsen | Antimicrobial effect of emulsion-encapsulated isoeugenol against biofilms of food pathogens and spoilage bacteria[END_REF]. Tween 80 may have hampered the antimicrobial properties of isoeugenol in an emulsion [START_REF] Krogsgard-Nielsen | Antimicrobial effect of emulsion-encapsulated isoeugenol against biofilms of food pathogens and spoilage bacteria[END_REF]. The solubilization capacity of the emulsifier on isoeugenol was so strong that it prevented the antimicrobial compound from penetrating the cell membrane. Alternatively, Tween 80 might affect the biofilm or individual cells in a way that makes them less susceptible to isoeugenol, by interacting with membrane proteins which made them more hydrophilic and therefore less prone to interaction with the essential oil [START_REF] Krogsgard-Nielsen | Antimicrobial effect of emulsion-encapsulated isoeugenol against biofilms of food pathogens and spoilage bacteria[END_REF]. This remark might also have implications for the use of other hydrophobic substances: [START_REF] Krsta | Bacterial fatty acid synthesis: effect of tween 80 on antibiotic potency against Streptococcus agalactiae and methicillin-resistant Staphylococcus aureus[END_REF] showed that Tween 80 reduced the effects of the antibiotic triclosan on Streptococcus agalactiae and S. aureus. In a model salad dressing, the Tween effect was entirely dependent on oil content when nisin was present: (i) at 23 % oil, Tween had an antagonistic effect with nisin; (ii) at 11 % oil, the presence of Tween increased the preservative effect; (iii) at 46 %, Tween had no effect [START_REF] Castro | Effect of preservatives, tween 20, oil content and emulsion structure on the survival of Lactobacillus fructivorans in model salad dressings[END_REF]. In the same way, [START_REF] Jung | Influence of Fat and Emulsifiers on the Efficacy of Nisin in Inhibiting Listeria monocytogenes in Fluid Milk1[END_REF] demonstrated interactions between nisin, milk and Tween: the addition of Tween 80 has been shown to significantly increase the activity of nisin on L. monocytogenes in milk regardless of fat content. The authors suggested that nisin may adsorb to milk fat globules, which may render it unavailable to destroy microbial cells. Tween, which has the ability to displace proteins from the milk fat globule, may increase nisin availability. These results highlighted the importance of considering ingredient interactions when evaluating the microbial stability of food systems. Nevertheless, only a few studies have mentioned the testing of the antimicrobial effect of an emulsifier as a function of pH and its concentrations. In our case, Tween 80 seemed to stress spores and vegetative cells, slowing down germination and growth. In previous works, Tween 80 appears to have affected cell permeability in certain microorganisms to promote both uptake and exit of compounds from the cell though modifications of plasma membrane permeability [START_REF] Brown | Effect of polysorbate 80 on cell leakage and viability of Pseudomonas aeruginosa exposed to rapid changes of pH, temperature and tonicity[END_REF][START_REF] Noh | Incorporation of Cholesterol into the Cellular Membrane of Lactobacillus acidophilus ATCC 431211[END_REF][START_REF] Sattler | The effect of "Tween 80," bovine albumin, glycerol, and glucose on the growth of Mycobacterium tuberculosis var. hominis (H37Rv)[END_REF]. [START_REF] Noh | Incorporation of Cholesterol into the Cellular Membrane of Lactobacillus acidophilus ATCC 431211[END_REF] showed that the influence of Tween 80 was related to alterations in the cellular membrane of the organism on Lactobacillus acidophilus. On Pseudomonas aeruginosa, 0.125 % polysorbate led to the greatest uptake of a dye showing cell membrane permeability [START_REF] Brown | Effect of polysorbate 80 on cell leakage and viability of Pseudomonas aeruginosa exposed to rapid changes of pH, temperature and tonicity[END_REF]. Non-ionics had little denaturing action on proteins, the uptake of polysorbate was presumably via hydrogen bonding. Flow cytometry on vegetative cells in the presence of Tween 80 in the medium did not determine whether Tween 80 permeabilized the membrane of B. weihenstephanensis KBAB4 cells. Thus, the reduction in growth rate could be due to a modification of the cellular membrane and its properties [Texte] The present study shows that the inhibition of spore germination and growth in the model emulsion depended on several factors: pH value and the dispersed phase volume fraction.

Antimicrobial activity resulted in a reduction in the number of cells capable of germination and outgrowth, a reduction in maximal specific growth rate and an increase in lag times. The effect of the dispersed phase volume fraction was due to a combination of:

(i) the lipophilicity of the biocide, hexanoic acid, that may have had an impact on the distribution of organic acid between hexadecane and the aqueous phases;

(ii) the antimicrobial activity of the emulsifier Tween 80 detected at the acidic pH value.

The effect of Tween 80 has rarely been described in the literature and this study highlights the caution with which this emulsifier must be used. Furthermore, the addition of Tween 80 in a formulation may improve the antimicrobial activity of preservatives and this model emulsion could be used as a surface treatment for example. The interface phenomena seemed to have a great influence. Future work will focus on the exploration of these phenomena at the interface by the use of biocides with other physical-chemical characteristics (lipophilicity, antimicrobial mechanism of action, hydrophobicity, electrical charge) and of bacterial cells with other surface characteristics (hydrophobicity, electrical charge).


Martha Collins

Marc Hébrant

Grégoire Herzog
email: gregoire.herzog@univ-lorraine.fr

Ion transfer at polarised liquid-liquid interfaces modified with adsorbed silica nanoparticles

published or not. The documents may come

Ion transfer at polarised liquid-liquid interfaces modified

with adsorbed silica nanoparticles

Introduction

The interface formed between two immiscible electrolyte solutions (ITIES) is a highly reproducible, defect-free surface that allows the assembly of a large variety of nanoscale objects [START_REF] Dryfe | Modifying the liquid/liquid interface: pores, particles and deposition[END_REF][START_REF] Booth | Assembly of Nanoscale Objects at the Liquid/Liquid Interface[END_REF][START_REF] Poltorak | Decorating soft electrified interfaces: From molecular assemblies to nano-objects[END_REF][START_REF] Scanlon | Gold Nanofilms at Liquid-Liquid Interfaces: An Emerging Platform for Redox Electrocatalysis, Nanoplasmonic Sensors, and Electrovariable Optics[END_REF]. The adsorption of nano-objects at a liquid-liquid interface is governed by their dimensions, the organic-aqueous interfacial tension and the wettability of the material [START_REF] Booth | Assembly of Nanoscale Objects at the Liquid/Liquid Interface[END_REF][START_REF] Duan | Directing self-assembly of nanoparticles at water/oil interfaces[END_REF][START_REF] Binder | Supramolecular assembly of nanoparticles at liquid-liquid interfaces[END_REF][START_REF] Böker | Self-assembly of nanoparticles at interfaces[END_REF][START_REF] Hu | Oil-water interfacial self-assembly: a novel strategy for nanofilm and nanodevice fabrication[END_REF].

The ITIES allows the application of an external electric field that can drive the adsorption of nanoparticles (Au or TiO2) to the interface [START_REF] Su | Reversible Voltage-Induced Assembly of Au Nanoparticles at Liquid|Liquid Interfaces[END_REF][START_REF] Jensen | Organization and Reactivity of Nanoparticles at Molecular Interfaces. Part I. Photoelectrochemical Responses Involving TiO 2 Nanoparticles Assembled at Polarizable Water|1,2-Dichloroethane Junctions[END_REF][START_REF] Fermín | Organisation and Reactivity of Nanoparticles at Molecular Interfaces. Part II. Dye Sensitisation of TiO2 Nanoparticles Assembled at the Water|1,2-Dichloroethane Interface[END_REF]. Carbon nanomaterials (e.g. carbon nanotubes, graphene and graphite flakes) [START_REF] Biswas | A novel approach to create a highly ordered monolayer film of graphene nanosheets at the liquid-liquid interface[END_REF][START_REF] Gudarzi | Self assembly of graphene oxide at the liquid-liquid interface: A new route to the fabrication of graphene based composites[END_REF][START_REF] Bian | Floating conductive catalytic nano-rafts at soft interfaces for hydrogen evolution[END_REF][START_REF] Ge | Hydrogen evolution across nano-Schottky junctions at carbon supported MoS2 catalysts in biphasic liquid systems[END_REF] and gold nanoparticles [START_REF] Smirnov | Interfacial Redox Catalysis on Gold Nanofilms at Soft Interfaces[END_REF][START_REF] Smirnov | Gold Nanofilm Redox Catalysis for Oxygen Reduction at Soft Interfaces[END_REF] adsorbed at the ITIES improve electron transfer kinetics between electron donors and acceptors distributed on either sides of the interface [START_REF] Bian | Floating conductive catalytic nano-rafts at soft interfaces for hydrogen evolution[END_REF][START_REF] Ge | Hydrogen evolution across nano-Schottky junctions at carbon supported MoS2 catalysts in biphasic liquid systems[END_REF][START_REF] Smirnov | Interfacial Redox Catalysis on Gold Nanofilms at Soft Interfaces[END_REF][START_REF] Smirnov | Gold Nanofilm Redox Catalysis for Oxygen Reduction at Soft Interfaces[END_REF]. The nano-objects assembled at the interface act as conductive rafts floating at the interface catalysing electron transfer through Fermi level equilibration [START_REF] Scanlon | Charging and discharging at the nanoscale: Fermi level equilibration of metallic nanoparticles[END_REF][START_REF] Peljo | Redox Electrocatalysis of Floating Nanoparticles: Determining Electrocatalytic Properties without the Influence of Solid Supports[END_REF]. Gold nanoparticles have also been injected onto the interface between a mixture of heptane and 1,2-dichloroethane and water to form a self-assembled monolayer, which exhibits reflectance and electrical conductance, opening the way to the development of electrovariable optical materials [START_REF] Fang | Conductive gold nanoparticle mirrors at liquid/liquid interfaces[END_REF]. Despite the abundant literature on silica nanoparticles [START_REF] Böker | Self-assembly of nanoparticles at interfaces[END_REF][START_REF] Binks | Silica Particle-Stabilized Emulsions of Silicone Oil and Water: Aspects of Emulsification[END_REF][START_REF] Niu | Synthesis of Nano/Microstructures at Fluid Interfaces[END_REF][START_REF] Calzolari | Nanoscale structure of surfactant-induced nanoparticle monolayers at the oil-water interface[END_REF][START_REF] Manga | Measurements of Submicron Particle Adsorption and Particle Film Elasticity at Oil-Water Interfaces[END_REF][START_REF] Hua | Competitive Adsorption between Nanoparticles and Surface Active Ions for the Oil-Water Interface[END_REF], their assembly at the ITIES has not been investigated yet. Nevertheless, silica sphere structures have been self-assembled at non-polarised liquid-liquid interfaces to fabricate 2 or 3D ordered structures with potential applications in optical, magnetic and electronic devices as photonic band gap materials [START_REF] He | Selfassembly of modified silica nanospheres at the liquid/liquid interface[END_REF]. Li et al modified the surfaces of silica spheres and then selfassembled them at the liquid-liquid interface in order to fabricate 2D/3D film structures [START_REF] He | Selfassembly of modified silica nanospheres at the liquid/liquid interface[END_REF].

The ability to generate low-dimensional nanostructures is by far the most popular application of self-assembled silica nanoparticles at non-polarised liquid-liquid interfaces. The film obtained at the interface after spontaneous adsorption of the silica nanoparticles has low resistance to lateral motion and so is transferrable, allowing it to be used for other applications [START_REF] Fan | Centrifugation-assisted Assembly of Colloidal Silica into Crack-Free and Transferrable Films with Tunable Crystalline Structures[END_REF]. The growing interest for the use of silica-based materials at the liquid-liquid interface is also due to their favourable adsorption kinetics and selectivity [START_REF] Dai | Hierarchically Imprinted Sorbents for the Separation of Metal Ions[END_REF], which could be harnessed to the development of new sensing methods [START_REF] Herzog | Recent developments in electrochemistry at the interface between two immiscible electrolyte solutions for ion sensing[END_REF][START_REF] Yan | Vertically ordered silica mesochannel films: electrochemistry and analytical applications[END_REF]. ITIES was modified by the electrogeneration of mesoporous silica deposits [START_REF] Poltorak | In-situ formation of mesoporous silica films controlled by ion transfer voltammetry at the polarized liquid-liquid interface[END_REF][START_REF] Poltorak | Electrochemically Assisted Generation of Silica Deposits Using a Surfactant Template at Liquid/Liquid Microinterfaces[END_REF][START_REF] Poltorak | Electrochemical characterization of liquid-liquid micro-interfaces modified with mesoporous silica[END_REF][START_REF] Liu | Visualization of Diffusion within Nanoarrays[END_REF][START_REF] Holzinger | Investigation of modified Nanopore Arrays by FIB/SEM Tomography[END_REF], which were the result of an electrochemically driven ion transfer followed by interfacial precipitation. Mesoporous silica was generated at the ITIES by the transfer of a cationic surfactant (e.g. trimethylhexadecylammonium) from the organic phase to the aqueous phase containing hydrolysed tetra(alkoxy)silanes, catalysing the condensation reactions of the sol-gel process.

Micro-ITIES arrays were also modified with mesoporous silica channels prepared ex-situ by a Stöber-like method [START_REF] Teng | Highly Ordered Mesoporous Silica Films with Perpendicular Mesochannels by a Simple Stöber-Solution Growth Approach[END_REF]. The vertically aligned channels obtained have demonstrated their potential for analytical applications [START_REF] Li | Differential voltammetry detection of dopamine and ascorbic acid by permselective silica mesochannels vertically attached to the electrode surface[END_REF][START_REF] Huang | Permselective Ion Transport Across the Nanoscopic Liquid/Liquid Interface Array[END_REF][START_REF] Huang | Detection of Metoprolol in Human Biofluids and Pharmaceuticals via Ion-Transfer Voltammetry at the Nanoscopic Liquid/Liquid Interface Array[END_REF][START_REF] Xie | Portable Sensor for the Detection of Choline and Its Derivatives Based on Silica Isoporous Membrane and Gellified Nanointerfaces[END_REF]. We investigated here the adsorption of silica nanoparticles onto the ITIES and their impact on the electrochemical transfer behaviour of ions (e.g. methylene blue, eosin B and tetraethylammonium) in the view of developing new analytical methods. Since silica nanoparticles themselves do not have any electrochemical signal at the ITIES they can serve as an aid to enhance the selectivity and preconcentration of target ionic species at the interface and thus their transfer across the ITIES. This ability to detect ionic species is of major benefit in both environmental monitoring and clinical diagnosis. The sensing of synthetic organic molecules, such as drug molecules and pollutants, using silica nanoparticles adsorbed at the ITIES to improve sensitivity is of significant interest.

Experimental section

Chemicals & Reagents

The high density organic phase electrolyte was 1,2-dichloroethane (1,2-DCE, ≥99% Sigma-Aldrich) containing 10 mM Bis (triphenylphosphoranyldiene) ammonium tetrakis (4chlorophenylborate) (BTPPA + TPBCl -). The organic salt was prepared by metathesis of Bis (triphenylphosphoranyldiene) ammonium chloride (BTPPA + Cl -, 97% Aldrich) and Potassium tetrakis (4-chlorophenylborate) (K + TPBCl -, ≥ 98% Fluka) [START_REF] Poltorak | In-situ formation of mesoporous silica films controlled by ion transfer voltammetry at the polarized liquid-liquid interface[END_REF]. Lithium Chloride (LiCl, ≥99% Aldrich) was used as the aqueous phase electrolyte. Solutions of the interfacial active ions: Tetraethylammonium chloride (TEA + Cl -, ≥ 99% Fluka), Methylene Blue (> 82% Fluka) and Eosin B (> 85% Sigma-Aldrich), were prepared in the aqueous phase electrolyte. The Ludox® HS-40 colloidal silica (∅ 𝑚𝑒𝑎𝑛 = 12 nm, 10.6 < ø < 13.7 nm) was purchased from Aldrich. Ferric chloride hexahydrate (FeCl3.6H2O, ≥ 99% Fluka) was used to prepare silver/silver chloride reference electrodes.

Experimental methods

The custom-made electrochemical cell (Ainterface = 1.13 cm 2 ) consisted of 2 sets of platinum mesh and silver/silver chloride wires as counter and reference electrodes respectively for both aqueous and organic phases. Different electrochemical cells were used and their compositions were as follows:

Electrochemical cell 1: The formal transfer potential of TEA + , ∆ 𝑜 𝑤  𝑇𝐸𝐴 + 0′ = 0.049 V [START_REF] Abraham | Solubility of electrolytes in 1,2-dichloroethane and 1,1-dichloroethane, and derived free energies of transfer[END_REF], was used to calibrate the potential window. TEA + was added to the aqueous phase of the electrochemical cell at the end of a series of experiments for potential calibration purposes. For CV experiments, the transfers of Li + and Cl -from the aqueous phase to the DCE phase establish the positive and negative limits of the potential window, respectively. In the presence of SiO2, the potential was calibrated using the limits of the potential window as previous studies have shown that the transfer of both Li + and Cl -is not hindered by the presence of electrogenerated silica [START_REF] Poltorak | Electrochemical characterization of liquid-liquid micro-interfaces modified with mesoporous silica[END_REF]. Scans were run at a scan rate of 5 mVs -1 . AC voltammetry experiments were run using GPES software with an Autolab PGSTAT-12. Parameters for the AC experiments were as follows -modulation time: 0.33 s, interval time: 0.73 s, frequency: 6 Hz, step potential: 5 mV, amplitude: 5 mV RMS. The potential region was scanned at phase angles of 0° and 90°. In this frequency range, the impedance response of the cell is determined by a single RC component associated with the uncompensated resistance and the double-layer capacitance [START_REF] Jensen | Organization and Reactivity of Nanoparticles at Molecular Interfaces. Part I. Photoelectrochemical Responses Involving TiO 2 Nanoparticles Assembled at Polarizable Water|1,2-Dichloroethane Junctions[END_REF][START_REF] Herzog | Interactions of proteins with small ionised molecules: electrochemical adsorption and facilitated ion transfer voltammetry of haemoglobin at the liquid|liquid interface[END_REF].

UV/Vis absorption spectroscopy experiments were carried out using an Agilent Technologies Cary 60 UV-Vis spectrometer. Solutions analysed were prepared in 10 mM LiCl and poured in standard plastic cuvettes with a path length of 1 cm. For the determination of the association constant and the total number of adsorption sites by UV-vis absorption spectroscopy, Solutions of 50 µM of MB + in 10 mM LiCl with varying concentrations of SiO2 nanoparticles (final silica nanoparticle concentrations were between 0.040 and 0.867 g L -1 , total volume was 10 mL) were poured in a stirred ultrafiltration cell (25 mm diameter, Amicon Model 8010, Millipore, Carrigtwohill, Ireland) equipped with a regenerated cellulose membrane (PLGC, 10 kDa, Millipore). The filtrate was collected and analysed by UV-vis absorption spectroscopy. Preconcentration experiments were prepared with 200 mL of a 25 µM MB + solution containing 0.05 g L -1 of SiO2 nanoparticles. The solutions were mixed for 5 min and then centrifuged at 10,000 rpm for 15 min. The supernatant was removed and 3 mL of a 10 mM LiCl solution was added to the nanoparticle-MB + precipitate, which is then used as the aqueous phase in the electrochemical cell.

Results and discussion

Silica nanoparticles adsorption at the ITIES

Previous studies on the behaviour of nanoparticles at the liquid-liquid interface have shown that their adsorption is the result of the presence of a promoter or a modifier that weakens the electrostatic repulsion between the nanoparticles in suspension and allows the assembly of nanoparticles at the interface [START_REF] Xu | A Method for Promoting Assembly of Metallic and Nonmetallic Nanoparticles into Interfacial Monolayer Films[END_REF]. An alternative to this chemically induced assembly is the application of an interfacial potential difference that causes the assembly [START_REF] Su | Reversible Voltage-Induced Assembly of Au Nanoparticles at Liquid|Liquid Interfaces[END_REF]. The adsorption of silica nanoparticles was investigated here by both cyclic and AC voltammetries at the ITIES (Figure 1). After the addition of 0.5 g L -1 silica nanoparticles to the aqueous phase, there was no significant changes to the CV, suggesting that no ion transfer is taking place in the potential window due the presence of the silica nanoparticles. Silica nanoparticles did not transfer across the interface and remained on the aqueous side of the interface throughout the potential window. AC voltammetry is more sensitive to interfacial phenomena than cyclic voltammetry and was then further used for the characterisation of the interface modified with silica nanoparticles. Even with quantities of silica nanoparticles as low as 0.05 g L -1 , significant changes were visible on the AC voltammograms recorded in the potential region where no background salt ion transfer was observed in cyclic voltammetry (Figure 1B). The differential capacitance was calculated from the AC voltammograms (see Experimental section). The potential of zero charge was taken as the potential of minimum of capacitance. In the absence of silica nanoparticles, it was measured at -0.06 V. Upon addition of silica nanoparticles to the aqueous phase, the capacitance value at -0.145 V increased from 14 (0 g L -1 ) to 19 µF cm -2 (0.1 g L -1 ) indicating that the quantity of charge located at the interface grew in the presence of silica nanoparticles. This behaviour can be attributed to the adsorption of negatively charged silica nanoparticles accumulating at the ITIES. This result is in agreement with that previously obtained for the interfacial assembly of TiO2 nanoparticles [START_REF] Jensen | Organization and Reactivity of Nanoparticles at Molecular Interfaces. Part I. Photoelectrochemical Responses Involving TiO 2 Nanoparticles Assembled at Polarizable Water|1,2-Dichloroethane Junctions[END_REF] or Au nanoparticles [START_REF] Su | Reversible Voltage-Induced Assembly of Au Nanoparticles at Liquid|Liquid Interfaces[END_REF] upon polarization at the liquid-liquid interface. Positively charged TiO2 particles (at pH 3.5) caused the capacitive current to increase at positive Galvani potentials, whereas at pH 11, TiO2 nanoparticles are negatively charged due to ionisation of the oxide surface and so the interfacial excess of the particles is increased at negative potentials. The potential value for the minimum capacitance shifted to higher potential values with the increase of silica nanoparticles amount in suspension (from -0.062 V in the absence of silica nanoparticles to -0.013 V for 0.1 g L -1 ). This observation agrees with previous studies on the behaviour of haemoglobin at the liquid-liquid interface where positively charged haemoglobin caused a shift to lower potential values [START_REF] Herzog | Interactions of proteins with small ionised molecules: electrochemical adsorption and facilitated ion transfer voltammetry of haemoglobin at the liquid|liquid interface[END_REF]. At a higher ionic strength, I, of the aqueous phase (I = 100 mM), the potential for the lowest capacitance value was slightly shifted from + 0.020 to + 0.035 V when the concentration of silica nanoparticles increased from 0 to 0.1 g L -1 (Figure 1C). The capacitance variation was very limited due to the charge screening of higher electrolyte concentration. When the pH was decreased to 2 (10 mM HCl) no significant potential shift or increase in capacitance values was observed (Figure 1C). At this pH, silica nanoparticles are expected to be nearly neutral [START_REF] Kosmulski | Positive Electrokinetic Charge of Silica in the Presence of Chlorides[END_REF] explaining why changes in the potential of zero charge or in the capacitance were not observed. In the previous experiments, the electrochemical cell was prepared with an organic phase denser than water (the organic solvent was 1,2-dichloroethane, d = 1.25 g mL -1 ). To prove that silica nanoparticles assembled at the liquid-liquid interface, and did not just sediment because of gravity, an organic phase less dense than water was used (Figure 2). Recent studies have shown that a mixture of 5-nonanone and 1,2-dichloroethane is lighter than water and it can be used for electrochemistry at the ITIES [START_REF] Toth | Novel organic solvents for electrochemistry at the liquid/liquid interface[END_REF]. We recorded AC voltammetry at such an electrochemical cell in the absence and in the presence of 0.5 g L -1 silica nanoparticles in the aqueous phase. The potential of zero charge shifted to higher potential values and the capacitance increased at -0.195 V from 26 to 32 µF cm -2 in the presence of silica nanoparticles. This observation confirmed that silica nanoparticles adsorbed at the ITIES.

Silica nanoparticles interactions studies in aqueous solutions

Interactions between the negatively charged silica nanoparticles and charged organic dyes (cationic methylene blue, MB + ; anionic Eosin B, EB -) were studied in a monophasic aqueous system. The organic dye was dissolved in the aqueous phase at 50 µM and a UV-vis absorption spectrum was recorded before and after a silica nanoparticle suspension was added to reach a concentration of 0.5 g L -1 . In the absence of silica nanoparticles, two bands were observed in the UV-vis. spectrum of MB + (Figure 3). The main band at λ = 664 nm is attributed to the n-* transition whereas the shoulder at λ = 614 nm is attributed to the vibronic transition [START_REF] Párkányi | A quantitative study of the effect of solvent on the electronic absorption and fluorescence spectra of substituted phenothiazines: evaluation of their ground and excited singlet-state dipole moments[END_REF][START_REF] Heger | Aggregation of methylene blue in frozen aqueous solutions studied by absorption spectroscopy[END_REF][START_REF] Yu | Controllable preparation, characterization and performance of Cu2O thin film and photocatalytic degradation of methylene blue using response surface methodology[END_REF]. In the presence of silica nanoparticles, the UV-vis. spectrum looks quite different with the largest band at λ = 590 nm and a smaller at λ = 664 nm, suggesting that MB + and silica nanoparticles interact with one another. This blue shift is attributed to the aggregation of MB + to form dimers, trimers… [START_REF] Morgounova | Photoacoustic lifetime contrast between methylene blue monomers and self-quenched dimers as a model for dual-labeled activatable probes[END_REF] on the silica nanoparticles surface and is in good agreement with spectra reported in the literature for MB + adsorbed onto silica-coated magnetic nanoparticles [START_REF] Tada | Methylene Blue-Containing Silica-Coated Magnetic Particles: A Potential Magnetic Carrier for Photodynamic Therapy[END_REF]. There was no difference between the EB -spectra in the absence or in the presence of silica nanoparticles, showing that there was no interaction between both negatively charged species (data not shown).

Figure 3: UV/Vis absorption spectroscopy of 50 µM MB + free in solution (solid line) and adsorbed on 0.5 g L -1 of silica nanoparticles (dashed line). Both solutions contained 10 mM LiCl.

We aimed at determining the total number of sites, ST, available for MB + adsorption on silica nanoparticles and the association constant, 𝐾 𝑎𝑑𝑠 𝑎𝑞 , of adsorption of MB + onto silica nanoparticles.

The adsorption of free MB + in solution onto silica free sites, SF, can be described by the following equilibrium.

𝑀𝐵 𝑓𝑟𝑒𝑒 + + 𝑆 𝐹 = 𝑀𝐵 𝑎𝑑𝑠 + ( 1
)
𝐾 𝑎𝑑𝑠 𝑎𝑞 = [𝑀𝐵 + ] 𝑎𝑑𝑠 [𝑀𝐵 + ] 𝑓𝑟𝑒𝑒 [𝑆 𝐹 ] (2)
The total number of sites is equal to the sum of the occupied sites, SO, and of the free sites, SF.

Similarly, the total number of MB + is equal to the sum of adsorbed MB + and of MB + free in solution. Since we worked at constant volume, equations 3 and 4 are valid.

[𝑆 𝑇 ] = [𝑆 𝑂 ] + [𝑆 𝐹 ] (3) [𝑀𝐵 + ] 𝑇 = [𝑀𝐵 + ] 𝑎𝑑𝑠 + [𝑀𝐵 + ] 𝑓𝑟𝑒𝑒 (4)
We assume that the number of occupied sites is equal to the number of MB + molecules adsorbed, equation (2) becomes:

𝐾 𝑎𝑑𝑠 𝑎𝑞 = [𝑀𝐵 + ] 𝑎𝑑𝑠 ([𝑀𝐵 + ] 𝑇 -[𝑀𝐵 + ] 𝑎𝑑𝑠 )([𝑆 𝑇 ]-[𝑀𝐵 + ] 𝑎𝑑𝑠 ) (5)
Experimentally, we investigated the adsorption of MB + onto silica by UV-vis spectroscopy adsorption. We maintained the initial amount in solution of MB + constant, [MB + ]T = 50 µM, throughout the series of experiments and we varied the silica concentration from 0.040 to 0.867 g L -1 . Silica nanoparticles and MB + were mixed together and the solution was then filtered through a membrane. The filtrate was collected and analysed by UV/Vis absorption spectroscopy, which allowed the determination of the amount of MB + present in the filtrate, [MB + ]free deduced from the optical density variation at 650 nm. [MB + ]ads were then deduced from equation ( 4) and the results were plotted as the function of silica concentration and fitted with a Langmuir isotherm to determine (a) the SiO2 surface area per MB + (i.e. equivalent to the total site concentration) and (b) the adsorption constant (𝐾 𝑎𝑑𝑠 𝑎𝑞 ). These two parameters were left as adjustable parameters [START_REF] Russo | Design of an adsorption column for methylene blue abatement over silica: From batch to continuous modeling[END_REF]. The best fit was obtained for 𝐾 𝑎𝑑𝑠 𝑎𝑞 = 1.66 10 5 and a = 1 nm 2 / MB + . This represents approximately 900 adsorption sites per nanoparticle. 𝐾 𝑎𝑑𝑠 𝑎𝑞 can be related to the free Gibbs energy of adsorption:

∆𝐺 𝑆𝑖𝑂 2 -𝑀𝐵 + 0 = -𝑅𝑇 ln 𝐾 𝑎𝑑𝑠 𝑎𝑞 (6)
Where T is the temperature (T = 293 K) and R is the universal gas constant (R = 8.314 J mol -1 K -1 ). The value of -29.3 kJ mol -1 for the free Gibbs energy of adsorption testified of the spontaneous nature of the adsorption of MB + onto silica nanoparticles. These results are in agreement with the existing literature, where spontaneous adsorption of MB + on mesoporous silica formed as a thin film on an electrode was reported [START_REF] He | Vertically ordered silica mesochannels as preconcentration materials for the electrochemical detection of methylene blue[END_REF] with similar free Gibbs energy of adsorption. Based on the fitting of Figure 4, the surface area per MB + was calculated at 1 nm 2 , which is lower than the results of the adsorption of one MB + molecule for every 3 nm 2 of silica reported in the literature for a different type of silica and experimental conditions [START_REF] Su | Reversible Voltage-Induced Assembly of Au Nanoparticles at Liquid|Liquid Interfaces[END_REF].

Figure 4: Amount of adsorbed MB + as a function of the amount of silica nanoparticles. Experimental data represented by the black squares were fitted using a Langmuir isotherm. Inset shows the UV-vis absorption spectra of free MB + in the filtrate after mixing with concentrations of silica nanoparticles (between 0.040 and 0.867 g L -1 ) and filtration.

Ion transfer at the ITIES modified with silica nanoparticles

As mentioned in the previous section, UV-vis absorption spectroscopy experiments showed a strong affinity between the MB + and silica nanoparticles in an aqueous solution. Cyclic voltammetry was used to study the effect of the interaction between silica nanoparticles and ions on the ion transfer at the ITIES. In the absence of silica nanoparticles, the standard transfer potential of MB + was measured at ∆ 𝑜 𝑤  𝑀𝐵 + 0 ′ = -0.223 V (Figure 5), which was only 0.034 V

larger than the open circuit potential of electrochemical cell 1 (∆ 𝑜 𝑤  𝑜𝑐𝑝 = -0.257 V). The concentration ratio of MB + in both phases at open circuit potential can be calculated using the Nernst equation:

[𝑀𝐵 + ] 𝑜𝑟𝑔 [𝑀𝐵 + ] 𝑎𝑞 = 𝑒 (∆ 𝑜 𝑤  𝑜𝑐𝑝 -∆ 𝑜 𝑤  𝑀𝐵 + 0 ′ ) 𝑧𝐹 𝑅𝑇 (7)
∆ 𝑜 𝑤  𝑀𝐵 + 0 ′
is the formal transfer potential of MB + , z is the MB + charge and F is the Faraday

constant. At open circuit potential, the ratio , calculated with Eq (7), was 0.007. Less than 1 % of MB + transfers spontaneously across the interface modified with silica nanoparticles. The shift of transfer potential allows the estimation of the Gibbs free energy of ion association between the MB + and the silica nanoparticle adsorbed at the ITIES [START_REF] Nagatani | Encapsulation of Anilinonaphthalenesulfonates in Carboxylate-Terminated PAMAM Dendrimer at the Polarized Water|1,2-Dichloroethane Interface[END_REF][START_REF] Sakae | Spectroelectrochemical characterization of dendrimer-porphyrin associates at polarized liquid|liquid interfaces[END_REF][START_REF] Osakai | Potential-modulated fluorescence spectroscopy of zwitterionic and dicationic membrane-potential-sensitive dyes at the 1,2-dichloroethane/water interface[END_REF].

∆𝐺 𝑆𝑖𝑂 2 -𝑀𝐵 + 0,𝑖𝑛𝑡𝑒𝑟𝑓𝑎𝑐𝑒 = 𝑧𝐹 (∆ 𝑜 𝑤  𝑀𝐵 + 0 ′ -∆ 𝑜 𝑤  𝑀𝐵 + 1/2 ) (8)
∆𝐺 𝑆𝑖𝑂 2 -𝑀𝐵 + 0,𝑖𝑛𝑡𝑒𝑟𝑓𝑎𝑐𝑒 was -8.9 kJ mol -1 , which is lower than the Gibbs free energy of adsorption,

∆𝐺 𝑆𝑖𝑂 2 -𝑀𝐵 + 0
, previously determined in the aqueous phase by UV-vis spectroscopy (Figure 4).

The difference between the two energies can be explained by the amphiphilic nature of MB + [START_REF] Da Silva | Effect of pH and dye concentration on the n-octanol/water distribution ratio of phenothiazine dyes: a microelectrode voltammetry study[END_REF]. Indeed, the presence of the organic solvent at the interface lowers the adsorption constant of MB + onto silica nanoparticles. We also investigated the influence of the presence of silica nanoparticles on anionic EB -and cationic TEA + . The transfer potential of both EB -(+0.011 V) and TEA + (+0.017 V) increased slightly after addition to the silica nanoparticles to the aqueous phase, suggesting that the transfer for these two ions is hindered by the presence of the nanoparticles. Thermodynamic values determined from the cyclic voltammograms for the three different ions are summarised in Table 1. The interfacial mechanism of MB + transfer in the presence of silica nanoparticles is summarised in Figure 6. MB + have shown a strong affinity constant, 𝐾 𝑎𝑑𝑠 𝑎𝑞 , for the silica nanoparticles. When silica nanoparticles are adsorbed on the interface, MB + transfer remained possible, provided that a sufficient excess of energy is provided: ∆𝐺 𝑆𝑖𝑂 2 -𝑀𝐵 + 0,𝑖𝑛𝑡𝑒𝑟𝑓𝑎𝑐𝑒 . Based on this mechanism, we can harness the affinity between MB + and silica nanoparticles and the adsorption of silica nanoparticles at the ITIES to accumulate MB + ions at the ITIES and increase locally the MB + concentration.

Figure 6: Schematic of the interfacial mechanism for the transfer of MB + in the presence of silica nanoparticles.

To demonstrate the accumulation of MB + at ITIES modified with silica nanoparticles, a solution of 25 µM MB + was mixed with 0.05 g L -1 of silica nanoparticles. The solution was centrifuged and the supernatant removed. The nanoparticles were then re-suspended in a 10 mM LiCl solution, which was used to prepare an electrochemical cell. Cyclic voltammograms obtained with such an electrochemical cell showed MB + transfer, whereas no transfer was observed for a 25 µM MB + solution in the absence of silica nanoparticles (Figure 7). Such an experiment demonstrated that MB + ions were accumulated at the ITIES with the help of the silica nanoparticles. Interactions between the negatively charged silica nanoparticles and charged organic dyes (cationic methylene blue, MB + ; anionic Eosin B, EB -) were studied in a monophasic aqueous system. A change in the shape of the UV-vis spectrum for MB + in the presence of silica nanoparticles suggested that MB + and silica nanoparticles interact with one another. The variation of [MB + ]ads was plotted as the function of silica concentration. Experimental results

were fitted with a Langmuir isotherm to determine the SiO2 surface area per MB + (i.e.

equivalent to the total site concentration) to be 1 nm 2 / MB + and the adsorption constant (𝐾 𝑎𝑑𝑠 𝑎𝑞 ) equal to 1.66 10 5 , indicating a high affinity of MB + for the silica nanoparticle surface. The strong affinity for cationic MB + on the negatively charged silica nanoparticles was also confirmed by cyclic voltammetry, which monitored the transfer potential of the ions at the ITIES. In the presence of silica nanoparticles the transfer potential of MB + shifts to higher potential. The strong affinity of MB + for silica nanoparticles and their adsorption at the ITIES showed that MB + can be accumulated at the interface. As a result, the transfer of MB+ in the presence of silica nanoparticle was monitored at MB + concentrations that were not detected in the absence of silica nanoparticles.

  Ag(s) | AgCl(s) | 10 mM LiCl(aq) x mM interfacial active ion(aq) + X g L -1 silica nanoparticles || 10 mM BTPPA + TPBCl - (org) in 1,2-DCE | Saturated BTPPA + Cl -in 10 mM LiCl in H2O | AgCl(s) | Ag(s); Electrochemical cell 2: Ag(s) | AgCl(s) | 100 mM LiCl(aq) + X g L -1 silica nanoparticles || 10 mM BTPPA + TPBCl - (org) in 1,2-DCE | Saturated BTPPA + Cl -in 10 mM LiCl in H2O | AgCl(s) | Ag(s); Electrochemical cell 3: Ag(s) | AgCl(s) | 10 mM HCl(aq) + X g L -1 silica nanoparticles || 10 mM BTPPA + TPBCl - (org) in 1,2-DCE | Saturated BTPPA + Cl -in 10 mM LiCl in H2O | AgCl(s) | Ag(s); Electrochemical cell 4: Ag(s) | AgCl(s) | Saturated BTPPA + Cl -in 10 mM LiCl in H2O | 10mM BTPPA + TPBCl - (org) in 40:60 1,2-DCE:5-nonanone (v/v) || 10mM LiCl(aq) x mM interfacial active ion(aq) + X g L -1 silica nanoparticles | AgCl(s) | Ag(s). Two electrochemical techniques were used during these experiments -Alternating Current (AC) Voltammetry and Cyclic Voltammetry (CV). Cyclic voltammetry experiments were run with an Autolab 302N potentiostat using Nova 1.10.1.9 software by Metrohm Autolab B.V.

Figure 1 :

 1 Figure 1: (A) Cyclic voltammetry in the absence (black solid line) and in the presence (red dashed line) of 0.5 g L -1 of silica nanoparticles in the aqueous phase. Electrochemical cell 1,  = 5 mV s -1 ; (B) Potential dependence of the interfacial capacitance for the blank cell (red solid line) and for (▲) 0.05 and () 0.1 g L -1 of silica nanoparticles. Electrochemical cell 1. (C) Potential dependence of the interfacial capacitance for the blank cell (solid line) and for () 0.1 g L -1 of silica nanoparticles with 100 mM LiCl (Electrochemical cell 2) or 10 mM HCl (Electrochemical cell 3) as an aqueous electrolyte solution.

Figure 2 :

 2 Figure 2: Potential dependence of the interfacial capacitance for the blank cell (solid line) and for () 0.5 g L -1 of silica nanoparticles with a denser aqueous phase. Electrochemical cell 4.

[

  𝑀𝐵 + ] 𝑜𝑟𝑔 [𝑀𝐵 + ] 𝑎𝑞 was 0.26 in the absence of silica nanoparticles at the interface, confirming the spontaneous MB + transfer from the aqueous to the organic phase. The spontaneous transfer was observed experimentally at open circuit potential with a blue coloration of the organic solution. However, in the presence of silica nanoparticles, the transfer potential of MB + , ∆ 𝑜 𝑤  𝑀𝐵 + 1/2 , shifts to a potential of -0.131 V, confirming the strong affinity between silica nanoparticles and MB + . ∆ 𝑜 𝑤  𝑀𝐵 + 1/2 is calculated as the half-wave potential of MB+ transfer in the presence of silica nanoparticles adsorbed at the ITIES. At open circuit potential and in the presence of silica nanoparticles, the ratio [𝑀𝐵 + ] 𝑜𝑟𝑔 [𝑀𝐵 + ] 𝑎𝑞

Figure 5 :

 5 Figure 5: Cyclic voltammetry of (A) 600 µM of MB + , (B) 300 µM of EB -and (C) 100 µM of TEA + in the absence (solid line) and in the presence (dashed line) of (A,B) 0.867 g L -1 and (C)

Figure 7 :

 7 Figure 7: Cyclic voltammetry of 25 µM of MB + in the absence of silica nanoparticles (solid line) and in the presence of silica nanoparticles and MB + after centrifugation and re-suspension with 3 mL of 10 mM LiCl. Initial concentrations were 25 µM MB + and 0.05 g L -1 of silica nanoparticles. Electrochemical cell 1.  = 5 mV s -1 .

Table 1 : Thermodynamics parameters determined experimentally

 1

	Ion	∆ 𝒐 𝒘  𝒊𝒐𝒏 𝟎 ′	/ V	∆𝑮 𝒊𝒐𝒏 𝟎 / kJ mol -1	∆ 𝒐 𝒘  𝒊𝒐𝒏 𝟏/𝟐 / V	∆𝑮 𝑺𝒊𝑶 𝟐 -𝒊𝒐𝒏 𝟎,𝒊𝒏𝒕𝒆𝒓𝒇𝒂𝒄𝒆 / kJ mol -1
	MB +	-0.223	-20.9	-0.131	-8.9
	EB -	-0.204	-19.7	-0.193	-1.1
	TEA +	+0.049	+4.7	+0.066	-1.6

Acknowledgements

The authors are grateful to the Agence Nationale pour la Recherche for the funding of the HYPERION project (grant number: ANR-14-CE14-0002-01). MC is grateful to SESAMES Doctoral school for PhD funding.