Maria Bueno Serrano

Christelle Despas

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

Alain Walcarius

Mesoporous silica thin films for molecular sieving and electrode surface protection against biofouling

Keywords: biofouling, electro-assisted self-assembly, mesoporous silica film, propranolol, haemoglobin adsorption

come

Introduction

The detection of pollutants in real media without sample preparation remains very challenging to the analytical chemist [1]. Fouling and non-specific adsorption on sensor surfaces hinders the deployment of accurate and sensitive sensors for long-term environmental and biomedical monitoring [2]. Indeed; the prolonged use of sensors in real samples for an extended period of time generally results in the formation of an unwanted layer of biomolecules. This fouling often results in the decay of the sensor performance and eventually its failure [3]. A number of strategies were developed in recent years to prevent the fouling of electrochemical sensors [4,5]. Chemical modification of the electrode with molecular monolayers may prevent macromolecules adsorption and hence surface fouling [6,7].

Electrode surface was also modified physically by electrodeposited nanoporous gold [8], which dimensions (< 50 nm) minimised the fouling effect of bovine serum albumin. Although effective for the exclusion of large proteins, nanoporous gold might be ineffective against the fouling of smaller proteins (e.g. cytochrome c and haemoglobin). In recent years, mesoporous silica thin films with pore diameter lower than 10 nm have been developed for electroanalytical, electrocatalytic and other electrochemical applications [9]. MCM-41 and SBA-15 mesoporous silica have been used as molecular sieves [10,11] in the past but never to protect an electrode surface. Mesoporous silica thin films obtained electrochemically with a surfactant template present a high level of order with pores orientation normal to the electrode surface, a feature which improves significantly mass transfer [12,13]. We suggest here their use as a protective surface layer based on the sieving properties at the nanoscale. Indeed, the diameter of pores obtained by electrochemically assisted self-assembly is around 2-3 nm [14], which is large enough to allow diffusion of small redox-active species and at the same time small enough to prevent diffusion of proteins. As a small organic redox-active molecule, we have selected propranolol, a -blocker often used in the treatment of hypertension and which can be detected electrochemically [15][16][17]. As a surface-fouling agent, we have used haemoglobin, whose dimensions are larger than the pore diameter [18]. The electrochemical behaviour of propranolol at bare ITO and at ITO modified with mesoporous silica thin film was investigated. The influence of the presence of haemoglobin on the propranolol oxidation signal was also assessed.

Material and Methods

Chemicals

Tetraethoxysilane (TEOS, 98%, Alfa Aesar), ethanol (95-96%, Merck), NaNO3 (99%, Fluka), HCl (Riedel de Haen, 1 M solution) and cetyltrimethylammonium bromide (CTAB, 99%, Acros) were used for film synthesis. Ruthenium hexamine chloride (Ru(NH3)6 3+ , 98%, Aldrich), NaCl (98%, Prolabo), Na2HPO4 (99%, Prolabo) and propranolol hydrochloride (99%, Sigma) and haemoglobin from bovine blood and bovine serum albumin (lyophilized powder, Sigma-Aldrich) were used for film permeability studies and analytical characterisation. All solutions were prepared with high purity water (18.2 M cm -1 ) obtained from a Purelab Option-Q from ELGA.

Electrochemically assisted self-assembly of mesoporous silica thin films

Mesoporous silica thin films were prepared by electrochemically assisted self-assembly [12]. A sol was prepared daily as follows: ethanol and 0.1 M NaNO3 aqueous solution were mixed in a 1:1 v/v ratio. TEOS and CTAB were added to the mixture to reach 100 mM TEOS and 32 mM CTAB concentrations. After setting the pH at 3 by addition of HCl, the sol was hydrolysed under stirring for 2.5 hours at room temperature. The sol was then ready to be used as the electrodeposition medium.

Indium tin oxide (ITO) plates (surface resistivity, 8-12 , from Delta Technologies) were modified by applying, for 30 s, a potential of -1.3 V versus a silver wire acting as a pseudo-reference electrode. A stainless steel plate was used as a counter electrode. The ITO plates were then rinsed thoroughly with ultrapure water and left overnight at 130°C to ensure a good cross-linking of the silica network. The surfactant template was removed by immersion in a stirred ethanol solution containing 0.1 M HCl for 15 min.

Cyclic voltammetry experiments

All electrochemical experiments were done with a PGSTAT12 (film formation) from Ecochemie (Metrohm, Switzerland) or on an EMStat2 potentiostat (film characterisation and propranolol detection) from Palmsens (Netherlands). A stainless steel rod was used as a counter electrode and the reference electrode was Ag|AgCl|1 M KCl (Metrohm, Switzerland). Modified electrodes were characterised by cyclic voltammetry at 100 mV s -1 in a de-aerated solution of 5 mM Ru(NH3)6 3+ in 0.1 M NaNO3 to assess the defect-free quality of the mesoporous silica thin films. Films presenting defects before surfactant extraction were discarded. Cyclic voltammetry of propranolol was done at 100 mV s -1 in a 0.1 M phosphate buffer (pH 5) either in the absence or the presence of 5 µM haemoglobin, which was used as a model protein inhibiting the detection of propranolol.

Results and Discussion

Propranolol was oxidised irreversibly at a bare ITO electrode at +1.29 V (Figure 1). The peak observed was due to the oxidation of the hydroxyl group to a ketone group, involving two protons and two electrons [19]. The presence of the mesoporous silica thin film on the electrode surface shifted the oxidation potential of propranolol to +1.47 V and the oxidation peak current decreased by 13 %.

Despite a slightly hindered diffusion of propranolol through the mesoporous silica thin film, the electrode surface remained available to propranolol oxidation allowing its detection. The electrochemistry of Hb was investigated at a bare electrode and at a modified electrode (Inset of Figure 1). The absence of Hb reduction at the modified electrode confirmed that the protein could not reach the electrode surface to react and that its size prevented from entering the pores. The oxidation of propranolol in the presence of bovine serum albumin was also investigated at both bare and modified electrodes. At an electrode modified with a mesoporous silica thin film, the signal for propranolol oxidation was not recorded, although it was observed at a bare electrode. Previous studies have shown that albumin and propranolol interact [20]. Propranolol is detected at a bare electrode, which suggests that albumin adsorbs at the electrode surface and bound propranolol can be oxidized at the electrode surface (data not shown). At a modified electrode, no propranolol is detected as albumin might be kept away from the surface by the mesoporous silica thin film (data not shown).

In their studies, Collins et al. demonstrated that albumin adsorbed at the liquid-liquid interface but did not inhibit the propranolol signal due to drug-protein interaction [20]. Oxidation of 100 µM propranolol was then investigated in the presence of 5 µM haemoglobin at both bare and modified ITO electrodes (Figure 2). At a bare electrode, the oxidation signal for propranolol was not observed, hindered by the adsorption of haemoglobin proteins at the surface of the electrode. It is interesting to note that the fouling of the electrode surface occurred at a concentration as low as 5µM. When a mesoporous silica thin film was formed on the electrode surface, the oxidation signal of propranolol at +1.41 V was not hindered by the presence of haemoglobin. The peak current was measured at (7.71±0.48) µA. This value was slightly lower than the oxidation peak current of (8.09±0.29) µA measured for 100 µM propranolol at a modified ITO electrode in the absence of haemoglobin. The oriented silica film enabled the detection of propranolol in the presence of haemoglobin, which was not possible at a bare ITO electrode. The propranolol response should come from diffusion through the pores rather than from eventual defects in the films as demonstrated by the absence of any signal for a redox probe in solution (i.e. Ru(NH3)6 3+ ) before surface extraction. Hence, the homogeneity of the mesoporous silica film needed to be demonstrated (Inset of Figure 2). After template removal, the film became porous to the redox probe as it was shown by the large peak current of Ru(NH3)6 3+ . This indicates that the only access to the electrode surface was through the pores of around 3nm in diameter [12,13].

Small organic molecules (e.g. propranolol) can reach the electrode surface while larger proteins (e.g. haemoglobin with the estimated hydrodynamic diameter of 6.4 nm [18]). are kept away, the mesoporous silica thin film acting as protective layer. Mesoporous silica with larger pores (prepared from Brij56 and Pluronic 123 templates) could accommodate medium size proteins such as myoglobin or cytochrome c while they blocked the transport of the larger bovine serum albumin [21]. Calibration curves were built for propranolol concentrations ranging from 5 to 100 µM at both bare and modified ITO electrodes in the absence and in the presence of haemoglobin (Figure 3). In the absence of haemoglobin, the oxidation peak current increased linearly with the propranolol concentration with approximately the same sensitivity at both bare and modified electrodes. The presence of 5 µM haemoglobin in the solution did not disturb significantly the detection of propranolol at the modified ITO electrode since a sensitivity of (67.4 ± 7.2) mA mol -1 (R 2 = 0.992, N = 7) was obtained (compared to a (72.8 • ± 2.9) mA mol -1 (R 2 = 0.992, N = 7) in the absence of haemoglobin), whereas it totally inhibited the propranolol response at bare ITO. The lowest concentration detected was 5 µM propranolol. A detection limit of this order was expected for simple cyclic voltammetry experiments and it could be improved by the use of square wave or differential pulse voltammetries [16]. These results suggest that electrodes covered with a mesoporous silica thin film could be used for the detection of small organic molecules in fouling media.

Conclusions

We have demonstrated that the electrogeneration of well-ordered and oriented mesoporous silica thin films is very promising for electrode surface protection. Propranolol is detected at the modified electrode in the presence of haemoglobin with a similar sensitivity to the one in the absence of haemoglobin. Protein dimensions are larger than the silica mesopore diameter and cannot diffuse to the electrode surface and foul it. The surface of the modified electrode has therefore remained active for the detection of propranolol in the presence of haemoglobin, while the bare electrode has become inoperative under the same conditions. Unfortunately, the protection was not as effective in the presence of albumin.

Figure 1 :

 1 Figure 1: Cyclic voltammograms in the presence of 100 µM propranolol at (a) a bare electrode and at (b) a modified electrode. Blanks at both bare (c) and modified (d) electrodes are also shown. Inset: Inset: Cyclic voltammetry of Hb at a (a) bare ITO electrode and at a (b) modified electrode.

Figure 2 :

 2 Figure 2: Cyclic voltammograms of 100 µM propranolol in the presence of 5 µM Haemoglobin at a bare ITO electrode (dashed curve) and at an ITO electrode modified with mesoporous silica thin film (solid line). Inset: Cyclic voltammograms of 5 mM Ruthenium hexamine at a bare ITO electrode (a), an ITO modified electrode (b) before and (c) after extraction of surfactant.

Figure 3 :

 3 Figure 3: Calibration curves for propranolol (5 -100 µM) in the absence (a,b) and in the presence (c,d) of 5 µM haemoglobin at bare (a,d) and modified (b,c) ITO electrodes.  = 100 mV s -1 in phosphate buffer (pH 5). Each experiment was done in triplicate.

Acknowledgements

The authors acknowledge Ms Mohana Afsharian for preliminary experiments.


Lukasz Poltorak

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

Alain Walcarius

Electrochemically assisted generation of silica deposits using a surfactant template at liquid/liquid micro-interfaces

Keywords: mesoporous silica, micro-ITIES, ion transfer voltammetry, interfacial electrodeposition, liquid/liquid interface

The electrochemically assisted generation of mesoporous silica deposits at arrays of microscopic liquid/liquid interfaces was investigated. Ion transfer voltammetry was used in order to initiate the formation of silica material by electrochemical transfer of template species (cetyltrimethylammonium, CTA + ), initially present in the organic phase, to the aqueous phase containing the hydrolyzed silica precursors (tetraethoxysilane, TEOS). The deposition mechanism was investigated using cyclic voltammetry, based on the analysis of diffusion layer profiles of CTA + species from the organic side of the interface. The morphology of the deposits varied from hemispherical to almost flat with the potential scan rate, the spacing factor of the micro-interfaces array supporting the liquid/liquid interfaces, or the initial CTA + and TEOS concentrations, as evidenced by scanning electron microscopy and profilometry analyses. The amount of deposited material can be related to the amount of CTA + species passing across the liquid/liquid interfaces.

Confocal Raman spectroscopy was used to confirm the presence of surfactant-templated silica deposits and to analyze the effectiveness of calcination in removing the organic molecules filling the interior of the pores. After template removal, the mesoporous network became accessible to external reagents, as checked by interfacial alkylammonium cations transfer, suggesting a possible analytical interest of such modified micro liquid/liquid interfaces.

Introduction

Determination of ionic analytes at the ITIES ranges from inorganic molecules [START_REF] Stockmann | Determination of Alkali Metal Ion Transfers at Liquid|liquid Interfaces Stabilized by a Micropipette[END_REF][START_REF] Bingol | Facilitated Transfer of Alkali and Alkaline-Earth Metal Ions by a Calix[4]arene Derivative Across Water/1,2-Dichloroethane Microinterface: Amperometric Detection of Ca2+[END_REF] to organic compounds and even biochemically important derivatives. [START_REF] Arrigan | Applications of Electrochemistry at Liquid-Liquid Microinterfaces[END_REF] Despite unique attractive features (e.g., detection not restricted to electron transfer reactions [START_REF] Samec | Electrochemistry at the Interface between Two Immiscible Electrolyte Solutions[END_REF] or defect-free 'working' liquid/liquid interface [START_REF] Edel | Self-Assembly of Nanoparticle Arrays for Use as Mirrors, Sensors, and Antennas[END_REF] ), electroanalysis at the ITIES still suffers from a lack of selectivity. This drawback can be somewhat overcome by introducing ionophores to the organic phase, which selectively transfer target ions, [START_REF] Arrigan | Bioanalytical Detection Based on Electrochemistry at Interfaces between Immiscible Liquids[END_REF] or by interfacial modification. [START_REF] Dryfe | Zeolitic Rectification of Electrochemical Ion Transfer[END_REF][START_REF] Lillie | Zeolite-Membrane Modulation of Simple and Facilitated Ion Transfer[END_REF] The latter was reported by Dryfe et al. in a series of papers, where the liquid/liquid interface was modified ex-situ with a zeolite membrane initially grown on a mercury surface. The molecular sieving properties of the zeolite allowed selective transfer of small cations in the presence of larger ones (i.e., respectively smaller and larger than the size of pinholes) and considerably increased the potential window when using an appropriate organic electrolyte. [START_REF] Dryfe | Zeolitic Rectification of Electrochemical Ion Transfer[END_REF][START_REF] Lillie | Zeolite-Membrane Modulation of Simple and Facilitated Ion Transfer[END_REF] When sodium zeolite-Y was employed to modify the ITIES, size selectivity was completed with charge selectivity (anions exclusion). [START_REF] Senthilkumar | Size-Selective Voltammetry: Modification of the Interface between Two Immiscible Electrolyte Solutions by Zeolite Y[END_REF] However, zeolite-based ITIES membranes still suffer from limited film porosity control (restricted to the microporous domain) and from the impossibility of further functionalization (e.g., with organic groups), which could be basically achieved with silica-based mesoporous materials, for instance.

Mesoporous materials play a crucial role in electrochemistry, [START_REF] Walcarius | Analytical Applications of Silica-Modified Electrodes -A Comprehensive Review[END_REF] but they have been almost exclusively restricted to solid electrodes to date. Mesoporous silica particles exhibiting high specific surface area and an ordered three-dimensional structure made of highly open spaces (as typically prepared by the sol-gel technology in the presence of surfactant templates [START_REF] Wan | On the Controllable Soft-Templating Approach to Mesoporous Silicates[END_REF] ), have been exploited in carbon paste electrodes for sensing applications. [START_REF] Walcarius | Electroanalytical Applications of Microporous Zeolites and Mesoporous (Organo)Silicas: Recent Trends[END_REF] Continuous thin films of surfactant-templated mesoporous silica can be also generated onto solid electrode surfaces and used for electroanalytical purposes. [START_REF] Walcarius | Electroanalytical Applications of Microporous Zeolites and Mesoporous (Organo)Silicas: Recent Trends[END_REF][START_REF] Walcarius | Template-Directed Porous Electrodes in Electroanalysis[END_REF] In this respect, the combination between sol-gel chemistry together with electrochemical methods has emerged as an easy and straightforward way to generate mesoporous silica thin films on conducting supports with pores oriented perpendicular to the surface by electrochemically assisted self-assembly method. [START_REF] Walcarius | Electrochemically Assisted Self-Assembly of Mesoporous Silica Thin Films[END_REF][START_REF] Goux | Oriented Mesoporous Silica Films Obtained by Electro-Assisted Self-Assembly (EASA)[END_REF] This method can be extended to the deposition on ultramicroelectrodes, [START_REF] Herzog | Electrochemically Assisted Self-Assembly of Ordered and Functionalized Mesoporous Silica Films: Impact of the Electrode Geometry and Size on Film Formation and Properties[END_REF] and to the generation of functionalized and oriented thin films exhibiting promising properties for electrochemical sensing. [START_REF] Etienne | Oriented Mesoporous Organosilica Films on Electrode: A New Class of Nanomaterials for Sensing[END_REF][START_REF] Herzog | Platinum Ultramicroelectrodes Modified with Electrogenerated Surfactant-Templated Mesoporous Organosilica Films: Effect of Film Formation Conditions on Its Performance in Preconcentration Electroanalysis[END_REF][START_REF] Rafiee | Hydroquinone Functionalized Oriented MCM-41 Mesochannels at the Electrode Surface[END_REF][START_REF] Rafiee | Electrochemical Fabrication of Electroactive Ordered Mesoporous Electrode[END_REF] To date, only very few examples are available, dealing with sol-gel electrodeposition at ITIES, [START_REF] Mareček | Electrochemically Controlled Formation of a Silicate Membrane at a Liquid|liquid Interface[END_REF][START_REF] Niedziolka | Electrochemically Assisted Sol-gel Process at a Three Phase Junction[END_REF][START_REF] Poltorak | In-Situ Formation of Mesoporous Silica Films Controlled by Ion Transfer Voltammetry at the Polarized Liquid-liquid Interface[END_REF] as discussed hereafter.

Silica in-situ deposition ator in the vicinity ofthe ITIES has been achieved at the interface between water and dichloroethane by electrochemical transfer of trimethyloctadecylammonium cations from the organic phase to an aqueous solution containing a silica precursor. [START_REF] Mareček | Electrochemically Controlled Formation of a Silicate Membrane at a Liquid|liquid Interface[END_REF] A second example includes the use of a nitrobenzene/water interface supported at solid electrode (so-called three phase junction) where electrochemical generation of protons was made to catalyze the condensation of n-octyltriethoxysilane present in nitrobenzene, resulting in the deposition of a silica film in the vicinity of three junction phase from aqueous side of the liquid/liquid interface. [START_REF] Niedziolka | Electrochemically Assisted Sol-gel Process at a Three Phase Junction[END_REF] Recently, our group has proposed a simple route for the generation of porous silica at macroscopic liquid-liquid interfaces, based on the electrochemical transfer of cetyltrimethylammonium cations (CTA + ) from an organic phase to an aqueous solution of hydrolyzed tetraethoxysilane (TEOS), and the characteristics of the silica deposits were shown to depend on the initial experimental conditions (template to precursor concentration ratio, composition of organic phase, etc.). [START_REF] Poltorak | In-Situ Formation of Mesoporous Silica Films Controlled by Ion Transfer Voltammetry at the Polarized Liquid-liquid Interface[END_REF] In this report, we modified liquid-liquid interfaces of microscopic dimensions (µITIES) by the ion transfer voltammetry method previously reported. [START_REF] Mareček | Electrochemically Controlled Formation of a Silicate Membrane at a Liquid|liquid Interface[END_REF][START_REF] Poltorak | In-Situ Formation of Mesoporous Silica Films Controlled by Ion Transfer Voltammetry at the Polarized Liquid-liquid Interface[END_REF] Modification of µITIES offers the advantage of stabilizing mechanically the deposits for further analysis by physical chemistry techniques (scanning electron and Raman confocal microscopies, shear force profilometry and cyclic voltammetry). Scanning electron microscopy and shear force profilometry characterizations were correlated to the electrochemical measurements to investigate the impact of CTA + transfer on the deposit morphology. The silica deposits were analysed by Raman confocal microscopy to identify the different species present. Finally, template removal was performed by calcination and its effect on the film properties was studied by ion transfer voltammetry of tetraalkylammonium cations and confocal Raman spectroscopy.

Materials and methods

Chemicals and reagents

Most chemicals used here were commercially available: Bis(triphenylphosphoranyldiene) ammonium chloride, (BTPPA + Cl -, 97%, Aldrich), potassium tetrakis(4-chlorophenylborate), (K + TPBCl -, ≥98%, Fluka), sodium chloride, (NaCl, ≥98%, Prolabo), cetyltrimethylammonium bromide (CTAB, ≥99%, Acros Organics), tetraethoxysilane (TEOS, 98%, Alfa Aesar), lithium chloride (LiCl, ≥99%, Aldrich), tetramethylammonium chloride (TMA + Cl -, ≥99%, Fluka), tetrabuthylammonium chloride (TBA + Cl -, ≥99%, Fluka), ferric chloride hexahydrate (FeCl3•6H2O, 99 -102%, Fluka), HCl (1M, volumetric solution, Riedel-de Haen), NaOH (pellets, pure, Riedel-de Haen), and 1.2-dichloroethane (DCE, ≥99%, Sigma-Aldrich). All these chemicals were used as purchased. BTPPA + Cl -and K + TPBCl -were used to prepare the organic electrolyte by metathesis reaction (procedure of preparation reported elsewhere). [START_REF] Poltorak | In-Situ Formation of Mesoporous Silica Films Controlled by Ion Transfer Voltammetry at the Polarized Liquid-liquid Interface[END_REF] NaCl and LiCl were used as aqueous electrolytes, CTAB and K + TPBCl -were used to prepare the template salt (protocol of preparation reported elsewhere, [START_REF] Poltorak | In-Situ Formation of Mesoporous Silica Films Controlled by Ion Transfer Voltammetry at the Polarized Liquid-liquid Interface[END_REF] ) and TEOS was used as silica precursor. Moreover HCl (1M) and NaOH (1M) were used to tune the pH of the aqueous phase to allow hydrolysis (pH = 3) and condensation (pH = 9.5) of TEOS. The high density organic phase was a 1.2-dichloroethane (DCE) whereas the low density phase was H2O of high purity obtained from a Millipore milli-Q water purification system.

The permeability of the µITIES before and after calcination was tested by ion transfer voltammetry using TMA + and TBA + as cationic probes.

Electrochemical setup

The electrochemical cell was custom made and is presented in Fig. 1. The cell consists of a glass vessel covered by a lid with three holes. The side places were occupied by Ag/AgCl reference and platinum mesh counter aqueous electrodes. We are using a 'four electrode' set-up where the organic reference electrodes also acts a counter electrode. This is possible because the surface area of the interface is relatively small compared to the size of the reference electrode. The Ag/AgCl reference electrode was prepared by oxidation of silver wire in oversaturated solution of FeCl3. The center hole was occupied by glassy capillary at the bottom of which a wafer supporting µITIES was fixed with silicone acetate sealant (Rubson® company). Before immersing the glassy capillary to the aqueous phase, it was filled with the organic phase and left for around one minute in order to impregnate the array of pores with the organic solution. Then, a silver wire was placed inside the capillary and the upper hole was covered with ParaFilm ® . The membrane used to support the µITIES is a 4 × 4 mm 2 membrane fabricated from a silicon wafer. The pores were patterned by UVphotolithography and pierced by a combination of wet and DRIE etches as described elsewhere. [START_REF] Zazpe | Ion-Transfer Voltammetry at Silicon Membrane-Based Arrays of Micro-Liquid-Liquid Interfaces[END_REF] Figure 1 decribes schematically one of these membranes. The pore depth (d) and the pore diameter (⌀) were maintained constant in this study and were 100 µm and 10 µm respectively. Four macroporous membranes with various pore center-to-center distances (spacing factor) and numbers of pores have been used, as listed in Table 1. The cell 1 configuration was used for silica material deposition at µITIES. The various TEOS and CTA + concentrations used in the above mentioned cells were in the ranges 50 mM ≤ x ≤ 300 mM and 1.5 mM ≤ y ≤ 14 mM, respectively. Silica deposition was mainly achieved by cyclic voltammetry but constant potential chronoamperometry (Eappl = -0.15V) was also used. µITIES modified with silica deposits were then tested in cell 2 via ion transfer voltammetry with interfacial active model ions (i.e., TMA + and TBA + ). The fabrication process of the macroporous membranes leads to hydrophobic pore walls. [START_REF] Zazpe | Ion-Transfer Voltammetry at Silicon Membrane-Based Arrays of Micro-Liquid-Liquid Interfaces[END_REF] The pores are therefore filled with organic phase, presenting an array of inlaid liquid-liquid micro-interface.

Instrumentation

All electrochemical measurements were performed with a PGSTAT 302N with the four electrode cell described above. SEM images were obtained using a Philips XL30 microscope (with acceleration voltage equal to 1 kV), without any sample metallization. The initial observation of modified µITIES was made with an optical microscope NIKON 883315. Glass tips for profilometry based on shear force measurements were fabricated from borosilicate glass capillaries (O.D. 1.5 mm, I.D. 0.9 mm) using vertical capillary puller. Prior to pulling, the capillaries were heated in the center and closed under vacuum. The tip diameter oscillates around 500 nm. The software allowing the control of piezo positioning was custom written. Confocal Raman spectroscopy measurements were carried out with a Jobin Yvon T64000 spectrometer with green light laser (514 nm).

Results and discussion

Silica formation at µITIES

Electrochemically assisted assembly of surfactant-templated silica

The silica deposits were formed at the µITIES by cyclic voltammetry (CV) and a typical curve is shown in Fig. 2. Cyclic voltammogram marked with solid line from Fig. 2 was recorded in cell 1

for [CTA + ] = 14 mM and [TEOS] = 50 mM, and corresponds to interfacial silica deposits formation at the µITIES design number 3 (Table 1). The dotted line on the same figure represents a blank curve recorded in cell 2 in the absence of precursor and template. Since the cationic template initially dissolved in the organic phase should be transferred to the aqueous phase to initiate the self-assembly condensation process, the interface was polarized from + 0.6 Vto -0.1 V during forward scan. We assumed that the interface at the beginning of polarization is covered with a monolayer of surfactant molecules (Fig. 2, scheme 1)

and the small resulting current is only due to electrical double layer charging. At around +0.1 V, CTA + species start to transfer from the organic to the aqueous phase. The local concentration of CTA + transferred to the aqueous phase should exceed its critical micellar concentration (CMC), which is 1.4 mM for CTAB in aqueous medium, [START_REF] Sepulveda | Ionization Degrees and Critical Micelle Concentrations of Hexadecyltrimethylammonium and Tetradecyltrimethylammonium Micelles with Different Counterions[END_REF] to enable the formation of charged micelles. [START_REF] Poltorak | In-Situ Formation of Mesoporous Silica Films Controlled by Ion Transfer Voltammetry at the Polarized Liquid-liquid Interface[END_REF] The presence of these micelles in the aqueous phase accelerates the condensation of the TEOS precursor around the surfactant template, resulting in interfacial silica material formation (Fig. 2, scheme 2). The negative signal of the CV curve is of sigmoidal shape (Fig. 2), which may suggest that the transfer of surfactant species across ITIES is not limited by linear diffusion (the origin of the curve shape will be discussed later). Forward scan was stopped at -0.1 V because further negative polarization causes current increase due to background ion transfer (Cl -transfer from aqueous to organic phase or BTPPA + transfer from organic to aqueous). On scan reversal, the inversed polarization of the interface induces the back transfer of CTA + species to the organic phase, resulting in a characteristic positive peak located at around +0.26 V and terminated with an abrupt drop of current (i.e., no more CTA + species available for back transfer to the organic phase).

This characteristic shape supports the presence of CTA + in the aqueous medium and within the silica gel network formed during the forward scan, and clearly indicates that the back transfer is not a diffusion-limited process. The µITIES is expected to be completely covered with the silica material after one cycle, as illustrated in Fig. 2 (scheme 3). This will be confirmed later on by SEM (see section 3.2).

Factors affecting the electrochemical deposition process

Both the limiting currents and the shape of current-potential curves are highly affected by the pore center-to-center distance, S, of the membrane support (Fig. 3). Actually, tuning the spacing factor (pores center-to-center distance) allows the control of planar and radial diffusion contributions governing the interfacial transfer of the active molecules. Davies and Compton [START_REF] Davies | The Cyclic and Linear Sweep Voltammetry of Regular and Random Arrays of Microdisc Electrodes: Theory[END_REF] divided microdisc arrays into four main groups based on their geometrical size with respect to the size of diffusion zones: (i) δ < r, δ < S; (ii) δ > r, δ < S; (iii) δ > r, δ > S and (iv) δ > r, δ >> S, where r is the pore radius, S is the spacing factor and δ is the diffusion zone radius. For each group, they propose current response characteristics, which are as follows: (i) clear peak, (ii) steady state wave, (iii) slight peak to clear peak and (iv) clear peak, from which only group (ii) does not follow scan rate dependency. [START_REF] Davies | The Cyclic and Linear Sweep Voltammetry of Regular and Random Arrays of Microdisc Electrodes: Theory[END_REF] This terminology will be helpful to the description of CV data obtained here for the electrogeneration of silica films at µITIES. For the purpose of this study, we employed four different µITIES supporting membranes with constant pore radius (5 µm) but various pore numbers and spacing factors (Table 1). Two distinct cases are reported in Fig. 3 where CV curves have been presented as current density versus potential variations to take into account the fact that the surface area of the array of pores supporting µITIES varies among each silicon wafer design. As shown, the increase in spacing from 20 µm (solid line) up to 100 µm (dashed line) led to dramatic increase in current densities along with significant change in the shape of the forward negative response, shifting from peak-like to wave-shaped. This can be explained by distinct diffusional regimes.

Using the nomenclature proposed above, and based on shapes of current densitypotential curves, one can attribute membrane number 1 to group (iii), whereas membrane number 3 belongs to borderline from groups (ii) and (iii). Since the small spacing (Fig. 3,c) give rise to slight peak, the condition δ > S has to be met. On the contrary, the steady-state wave indicates that δ < S in case of larger spacing (Fig. 3,b). As shown in supplementary information (Fig. SI1 a), membrane design number 2 gave rise to a wave with a slight-peak negative response, which means that δ is still larger than pore center-to-center distance. As expected, the CV response of membrane design number 4 was similar as membrane 3 (steady-state wave, as S >> δ, Fig. SI1 b). The unaffected shape of the positive peak on scan reversal indicates a non-dependence on the type of diffusional regime, confirming that the reverse scan signals are not due to diffusion controlled processes. In addition, the larger current densities observed for wider spacing are explained by a radial diffusion process (i.e., as also observed for arrays of ultramicroelectrodes) [START_REF] Morf | Performance of Amperometric Sensors Based on Multiple Microelectrode Arrays[END_REF] whereas almost total overlap happens in case of pore center-to-center distances becoming of the same order of magnitude as the pore size and the resulting linear diffusion controlled processes led to lower currents passing across ITIES. For the highest scan rate -10 mV s -1the diffusion layer thickness, δl, is less than the membrane thicknessd = 100 µm -(see scheme b in Fig. 4) and hence the transfer inside the pore is limited only by linear diffusion [START_REF] Wilke | Diffusion Effects at Microhole Supported Liquid/liquid Interfaces[END_REF] (resulting in peak like response). In this case, not enough CTA + species are transferred from the organic to the aqueous phase and no silica film formation was observed (as checked by profilometry based on shear force measurements). All the amount of CTA + species transferred to the aqueous phase on forward scan are transferred back to the organic medium on scan reversal, as supported by forward and reverse charge transfer of similar magnitude (Figure SI2). Decreasing potential scan rates down to 5 and 1 mV s -1 led to diffusion layer thicknesses increasing from slightly larger (i.e., 90 -130 µm for 5 mV s -1 ) to much larger (i.e., 200 -300 µm for 1 mV s -1 ) than the membrane thickness, leading to sigmoidal-shaped current responses on forward scan (yet with an additional residual and very slight peak at 5 mV s -1 ). Such wave found its origin from the prolongation in diffusion layers profiles (forming hemispherical diffusion zones at the pores entrance due to fulfilling of S/2 > δnl conditions, see schemes c and d in Fig. 4). In addition, the local changes in the interfacial properties as a result of growing silica deposit also play a role in the evolution of CV curves, as one can expect some additional resistance to mass transport through the interface in the presence of a silica film. This is notably the case of 1 mV s -1 scan rate (see scheme d in Fig. 4) for which long experimental time would cause an increase in δnl exceeding half of pore center-to-center distance (S/2) on the basis of the estimated δ values (i.e., 200 -300 µm in this case), leading to an expected overlap of radial diffusion zones, which should have given rise to a peak-like response. The fact that a sigmoidal response was obtained here is attributed to the restricted transfer of CTA + across the interfacial synthesized silica material (apparent diffusion values for the overall process lower than those in solution at an unmodified liquid-liquid interface).

Actually, much longer experimental times were necessary (for instance, that corresponding to a potential scan rate as low as 0.1 mV s -1 ) to get conditions of total diffusion overlap (see scheme e in Fig. 4). In this case, the 𝐶𝑇𝐴 𝑜𝑟𝑔→𝑎𝑞 + transfer is limited by linear diffusion above the pores, which becomes the limiting factor, leading to a peak-shaped voltammogram. The large difference between the charge passed through the µITIES during forward and reverse scan indicates that a significant part of CTA + species transferred to the aqueous phase has been involved in the formation of silica deposits, as trapped inside the mesostructure, and thus no more available for back transfer to the organic phase on scan reversal (Figure SI2).

The interfacial silica formation is possible only when CTA + is present in the organic phase and hydrolyzed TEOS in the aqueous phase. The driving force of the process is the electrochemicallyassisted transfer of CTA + species from the organic to the aqueous medium where self-assembly condensation occurs. Therefore, the composition of both media is likely to affect the deposition process. To check this, we have chosen deposition conditions corresponding to pores behaving independently of each other (i.e. avoiding overlap of radial diffusion layers), which is achieved for membrane design number 3 (100 µm pore center-to-center distance) and a moderate potential scan rate (5 mV/s). The results are depicted in Supporting Information (Fig. SI3). When maintaining

[TEOS] at a constant value in the aqueous phase and varying [CTA + ] from 1.5 mM up to 14 mM in the organic phase, continuous increase in negative currents was observed due to enhanced

𝐶𝑇𝐴 𝑜𝑟𝑔→𝑎𝑞 +
transfer. This reaction is the rate-determining step. On scan reversal, the backward transfer peak increased first with [CTA + ] (up to 3 mM) and then tended to level off for higher CTA + concentrations (see graph a in Fig. SI3). This trend can be explained by the formation of the surfactant-templated silica deposits at the interface: for low [CTA + ], most CTA + species are incorporated into the film and are thus no more available for back transfer, whereas higher [CTA + ] values led to the formation of much thicker silica deposits which starts to act as a physical barrier to the back transfer reaction. On the other hand, no significant changes neither in current intensity nor in CV curve shape was noticed when keeping [CTA + ] constant and rising [TEOS] from 50 to 300 mM (see graph b in Fig. SI3). The only difference was a potential shift of the whole CV curve to the positive direction, indicating an easier 𝐶𝑇𝐴 𝑜𝑟𝑔→𝑎𝑞 + transfer, which is explained by enhanced interactions between the transferring CTA + cations with the more concentrated negatively-charged silica species in the aqueous phase. These results confirm that the driving force (and limiting factor) of the deposition process are governed by the amount of CTA + species transferring to the aqueous phase where film formation occurs, and that TEOS concentration does not constitute a ratedetermining factor, on the contrary to what was reported for electro-assisted self-assembly of mesoporous silica films on solid electrodes, [START_REF] Goux | Oriented Mesoporous Silica Films Obtained by Electro-Assisted Self-Assembly (EASA)[END_REF][START_REF] Walcarius | Electrochemically Assisted Self-Assembly of Mesoporous Silica Thin Films[END_REF] because of different deposition mechanism. This qualitative discussion is based on a rather simple micro-interface array model and is supported by the following morphological study.

Morphological study

Morphology of the silica deposits generated at µITIES under various conditions was analyzed uniformly on all pores, and their crack-free appearance after surfactant removal (images were recorded after calcination at 400°C) suggests a good mechanical stability. When using the same support (µITIES number 2), keeping constant the potential scan rate (at 5 mV s -1 ), but increasing the number of cycles from half scan (first column) to 3 scans (second column), one can notice an increase in both the deposit height (from 1.7 µm to 3.4 µm) and the deposit diameter (from 14 µm to 15.7 µm), with some more pronounced curvature of the top surface of the deposits. Also, there was no significant difference in the deposit diameter between samples prepared from half and whole potential cycle, showing only an increase in the deposit height from 1.7 µm up to 2.7 µm, consistent with longer duration of negative potentials application. All these results confirm that the electrochemically-induced 𝐶𝑇𝐴 𝑜𝑟𝑔→𝑎𝑞 + transfer is indeed at the origin of the surfactant-templated silica self-assembly process. Control experiments showed that the condensation of silica does not occur in the absence of surfactant. 23 (2) deposits prepared by three successive CV scans at 5 mV s -1 using µITIES number 2; and (3) deposits prepared by one CV scan at 0.1 mV s -1 using µITIES number 3.

However, the deposit height growth was not directly proportional to the number of CV cycles, most probably due to enhanced resistance to 𝐶𝑇𝐴 𝑜𝑟𝑔→𝑎𝑞 + transfer in the presence of much thicker deposits. A more effective way to get massive silica material deposition at the µITIES is to slow down the potential scan rate (which also requires the use of a silicon membrane with larger pore spacing to avoid overlap of the radial diffusion profiles). This is shown on part 3 of Fig. 5 for the modification of membrane design number 3 by one scan at 0.1 mV s -1 , leading to much thicker deposits (13.1 µm height and 33.1 µm diameter). Since the generated deposits are massive comparing to other experiments, they are likely to be more affected by calcination and can suffer from some losses (see, e.g., one deposit missing on the bottom left of part 3b in Fig. 5).

Another way to control the generation of silica deposits is the use of constant-potential amperometry (i.e., chronoamperometry) instead of linear sweep or cyclic voltammetry. Too short experiment times (5-10 s) did not lead to any silica material formation. Then, the amount of deposited material was found, as expected, to increase with the amperometry duration (for example 50 s deposition time led to deposits with 3.7 µm in height and 14.9 µm in diameter), while extending the electrosynthesis up to 200 s enabled the formation of massive deposits, with 8.4 µm in height and 18.6 µm in diameter. As for deposits prepared by cyclic voltammetry, their shape was also found to change from rather flat to hemispherical when increasing the charge passing across the interface contributing thereby to larger amounts of silica material deposited at the µITIES.

Finally, the variation in shape of the silica deposits resembles to an image of the evolution of the diffusion layer profiles: short deposition times corresponds to mainly linear diffusion limitations, yet with some radial contribution at the macropore edges, while longer experiments are governed by radial diffusion control (Figure 6), where the deposit height, h, was plotted as function of the difference between deposit and pore radii, rd-rp. In the beginning of the deposition process, h  rdrp, corresponding to a rather flat morphology, while subsequent growing led to more hemispherical shape with h values likely to rise up to 2×(rd-rp). Finally, more massive deposits were characterized by preferential lateral growth (h < 2×(rd-rp)). Under the conditions presented here, the displacement of liquid/liquid micro-interfaces under charge transfer [START_REF] Dale | Polarised Liquid/liquid Micro-Interfaces Move during Charge Transfer[END_REF] does not seem to be an issue. Indeed, SEM images for detached membranes (see Fig. SI4 in Supporting Information) suggest that the silica deposits are filled inside and rather flat at the bottom. Another point, yet minor, to mention from the morphological analysis is the presence of cubes on some silica deposits (see, e.g., image 3a in Fig. 5), which are NaCl crystals arising from residual background electrolyte back transfer from organic to aqueous phase. This phenomenon is similar to that reported by Silver et al. [START_REF] Silver | Protein Crystallization at Oil/water Interfaces[END_REF] , on the basis of protein crystallization experiments at liquid/liquid interfaces. We thus anticipate that the present electro-assisted generation of silica material could be also suitable to the entrapment of proteins in the silica matrix (sol-gel silica is indeed known to enable encapsulation of proteins [START_REF] Gupta | Entrapment of Biomolecules in Sol-Gel Matrix for Applications in Biosensors: Problems and Future Prospects[END_REF] ) as an effective way to immobilize biomolecules at liquid/liquid interfaces. Finally, a very thin additional silica layer covering the whole silicon membrane + silica deposits can be also evidenced from SEM pictures (see micrographs 1a and 2a in Fig. 5). This arises most probably from some evaporation induced condensation of TEOS and silica deposition even if each electrosynthesis experiment was followed by careful rinsing with distilled water.

Spectroscopic and electrochemical characterization of the deposits

Confocal Raman spectroscopy was first used to show the formation of a silica network at µITIES. It has been performed after thermal treatment of the deposits (24h storage at 130°C followed by 30 min at 400°C) to be able to detect the characteristic lines of silica without being disturbed by huge signals arising from the organic matter expected to be present in the same spectral region (see a typical spectrum on Fig. SI5 in Supporting Information). In addition to the narrow and intense band at 520 cm -1 due to the vibration mode characteristic of the silicon wafer support, one can notice a broad band from 375 cm -1 to 500 cm -1 attributed to the Si-O-Si vibrational mode, and a single band at 793 cm -1 suggesting the presence of terminal Si-OH groups.

This strongly supports the formation of silica. Raman spectroscopy can be also used to characterize the organic species that have been incorporated into the silica material during the deposits formation, as well as to check the effectiveness of their removal upon calcination. This is shown on Fig. 7b, where the broad and intense signal located in the region 2760 -3020 cm -1 region, corresponding to the -CH2stretching mode of the long alkyl chain of CTA + species, [START_REF] Ho | Alkyl Chain Conformation of Octadecylsilane Stationary Phases by Raman Spectroscopy . 1 . Temperature Dependence the Effect of Temperature on the Conformational Order of[END_REF] confirms the presence of the surfactant template in the material. This signal almost disappears after heat treatment, indicating the successful removal of the organic template. From Fig. 7b, one can also notice a signal of small to medium intensity at 3060 cm -1 , which can be ascribed to the C-H stretching mode of aromatic rings, which are present in the organic electrolyte (BTPPA + TPBCl -), suggesting that some of these ions have been co-encapsulated in the silica material in addition to the surfactant template. This is best shown on Fig. 7a, where the typical signature of BTPPA + TPBCl -is seen via the bands located in the 600 to 1800 cm -1 region [START_REF] Booth | Electrochemical Modulation of SERS at the Liquid/liquid Interface[END_REF] (for instance the peak at around 1000 cm -1 is due to the vibration of the aromatic ring [START_REF] González-Henríquez | Theoretical and Experimental Vibrational Spectroscopic Investigation of Two R(1)R(2)-Diphenylsilyl-Containing Monomers and Their Optically Active Derivative Polymer[END_REF] ). After calcination, all these bands disappeared, which indicates the complete removal of this organic electrolyte from the deposits. One can also notice from Fig. 7a two broad peaks of weak intensity in the region from 1250 to 1750 cm -1 , which appeared upon calcination, representing actually the D and G bands of traces of amorphous carbon arising from thermal decomposition of the organic molecules. [START_REF] Veres | Surface Enhanced Raman Scattering (SERS) Investigation of Amorphous Carbon[END_REF] These results support that the mesopore channels have been liberated from most of their organic content, even if the quantitative analysis of the porosity by the classical gas adsorption method was not possible due to the too low amount of available material (too small deposits). For the same reason, the observation of the mesostructure by transmission electron microcopy was not achieved, but one can reasonably assumed that it corresponds to the same wormlike structure as that reported for similar electroassisted deposition of larger amounts of material at macroscopic liquid/liquid interfaces. 23 The deposits were then characterized by cyclic voltammetry data (Fig. 8). Silica deposits employed here were obtained by CV in the following conditions: [TEOS]aq = 50 mM; [CTA + ]org = 14 mM; potential scan rate 5 mV s -1 ; 2 subsequent cycles at the membrane design number 3, and then, calcinated at 400°C after 24h storage at 130°C. The cyclic voltammogram recorded in a blank electrolyte for µITIES with silica deposits before calcination (Fig. 8Aa) is characterized by a high capacitive contribution, which is significantly lower after heat treatment (Fig. 8Ab), suggesting an effect of the removal of organic species from the silica material. This is also evidenced by the appearance of a broad signal in the region of 𝐶𝑙 𝑎𝑞→𝑜𝑟𝑔 -

38

, indicating possible interfacial transfer through the silica film after template removal. In order to better evaluate the permeability of µITIES modified with silica deposits before and after calcination, we employed model interfacial active cations. As shown with TMA + (Fig. 8B), the transfer of 𝑇𝑀𝐴 𝑎𝑞→𝑜𝑟𝑔 + through the µITIES modified with the silica deposits was clearly observed after removal of the surfactant template (Fig. 8Bb), confirming good permeability of the mesoporous film, whereas TMA + transfer was either negligible or masked by the large capacitive current, which occurred before calcination (Fig. 8Ba). The corresponding CV curve was characterized by a sigmoidal wave when transferring from aqueous to organic phase and a peak-like response on scan reversal (due to the restricted amount of TMA + for back transfer). The absence of significant 𝑇𝑀𝐴 𝑎𝑞→𝑜𝑟𝑔 + transfer prior to template removal also confirms the good quality of the deposits and the fact that allor the greatest part ofthe macropores of the silicon membrane have been successfully modified with the silica deposits.

Although a bulky model cations such as TBA + (hydrodynamic radii equal to 0.48 nm [START_REF] Ishimatsu | Ion-Selective Permeability of an Ultrathin Nanoporous Silicon Membrane as Probed by Scanning Electrochemical Microscopy Using Micropipet-Supported ITIES Tips[END_REF] ) is used, apparent high rates of mass transport through the mesoporous silica films are obtained after template removal. This is shown in Fig. 9 for the µITIES design number 4 (i.e., with the largest macropores spacing). The macropores coverage with silica deposits was 100% (i.e., no pores without silica), as controlled by optical microscopy. Fig. 9 (a) compares the transfer of TBA + across the bare µITIES and the silica modified membrane, as recorded in cell 2 containing 56.8 µM TBA + .

As shown, the transfer reaction 𝑇𝐵𝐴 𝑎𝑞→𝑜𝑟𝑔 + was characterized by an positive wave in both cases, with a small drop (by ca. 10%) which was noticed in the presence of silica deposits with respect to the unmodified µITIES, while the half-wave potential shifted from +0.015 V to +0.025 V. The very small decrease in intensity indicates very few resistance to mass transport of TBA + species through the mesoporous silica material deposited at the µITIES, which is also consistent with the rather fast mass transport rates reported for solid electrodes modified with mesoporous silica particles [START_REF] Walcarius | Electrochemical Probing of Mass Transfer Rates in Mesoporous Silica-Based Organic-inorganic Hybrids[END_REF] or thin films. [START_REF] Walcarius | Electrochemically Assisted Self-Assembly of Mesoporous Silica Thin Films[END_REF][START_REF] Etienne | Evaporation Induced Self-Assembly of Templated Silica and Organosilica Thin Films on Various Electrode Surfaces[END_REF] The above observations of possible and fast ion transfer across µITIES modified with mesoporous silica are promising for analytical applications, by taking advantage of the high sensitivity of the microscopic interfaces combined to fast transport of the analytes through the silica film modifier. Moreover, the selectivity of the detection could be manipulated by tuning the interactions between the analyte and the mesoporous silica deposits, which would be achievable by suitable functionalization of the silica walls (for instance by tuning the hydrophobic-to-hydrophilic balance [START_REF] Guillemin | Electrogeneration of Highly Methylated Mesoporous Silica Thin Films with Vertically-Aligned Mesochannels and Electrochemical Monitoring of Mass Transport Issues[END_REF] or by attaching selected organo-functional groups [START_REF] Etienne | Evaporation Induced Self-Assembly of Templated Silica and Organosilica Thin Films on Various Electrode Surfaces[END_REF][START_REF] Yantasee | Simultaneous Detection of Cadmium, Copper, and Lead Using a Carbon Paste Electrode Modified with Carbamoylphosphonic Acid Self-Assembled Monolayer on Mesoporous Silica (SAMMS)[END_REF] ).

A final point to stress is related to the geometrical surface of µITIES that is likely to change upon modification with the hemispherical deposits oriented towards the aqueous phase, the surface of which being expected to be greater than the area of flat µITIES supported by array of unmodified pores, and hence the diffusion layer profile of single µITIES should increase upon modification.

Fig. 9 (b) shows the scan rate dependency for TBA + transfer across the µITIES supported with silica deposits. Since the positive current remains sigmoidal in shape and its intensity remains relatively constant among each scan rate, one can conclude that the TBA + transferring process from aqueous to organic phase remains controlled by radial diffusion, without overlap of diffusion layers, keeping the 𝑇𝐵𝐴 𝑎𝑞→𝑜𝑟𝑔 + reaction similar to that of an ensemble of ultramicroelectrodes, with the same advantages in terms of sensitivity. The shape of TBA + back transfer becomes peak-shaped as the scan increases. Indeed, the thickness of the diffusion layer in the organic phase becomes lower than the membrane thickness and hence TBA + ions are confined within the pores, leading to a linear diffusion.

3.4.Conclusion

Liquid-liquid micro-interfaces were modified by the electrochemically induced formation of mesoporous silica deposits. The modification process is based on the transfer of CTA + ions from the organic to an aqueous phase (controlled by the application of an interfacial potential difference), which catalyzes the condensation of hydrolyzed TEOS molecules in the aqueous phase. The silica deposition occurs on the aqueous side of the interface and cyclic voltammetric studies have shown that it is a process limited by the transfer of CTA + cations. Morphology (shape and dimension) of the deposit, determined by SEM and profilometry, is controlled by the amount of CTA + cations that is being transferred to the aqueous phase. Raman spectroscopy has confirmed that deposits are made of silica but it reveals the presence of both CTA + and ions from the electrolyte of the organic phase trapped in the silica matrix. After removal of these ions by calcination, the modified microinterfaces become porous to the transfer of model tetraalkylammonium ions (TMA + and TBA + ).

The modification in-situ of an array of micro-interfaces with mesoporous silica opens a wide range of possibilities in the design of new sensors. Indeed, it would offers the advantages of both microscopic and nanoscopic scale. Such sensors would benefit from improved mass transport linked to the microscopic dimension of the interface. Furthermore, functionalization of the silica matrix with chemical groups of interest would provide selectivity at the nanoscale.

Acknowledgements

LP

Figure 1 .

 1 Figure 1. Schematic illustration of the electrochemical cell used in this study. Designation: 1 -

Figure 2 .

 2 Figure 2. Typical cyclic voltammogram representing the formation of surfactant-templated silica

Figure 3 .

 3 Figure 3. Variations of current density as a function of the applied potential for two different

Fig. 4 ( 1 )

 41 Fig. 4 (part a) illustrates the influence of the scan rate on the current-potential curves

Figure 4 .

 4 Figure 4. (a) Cyclic voltammograms recorded at various scan rates during interfacial silica deposits

  by SEM and profilometry (based on shearing force measurement), and illustrative results are shown on Fig.5. They concern deposits generated using the same composition of solutions ([TEOS]aq = 50 mM and [CTA + ]org = 14 mM), but on the basis of different supporting silicon membranes and distinct deposition conditions (potential scan rates and number of cycles). The data are presented under the form of tilted views of single pore (row a) and pore arrays (row b), as well as 3D mapping profilometry while keeping constant the z axis scale in all cases (row c). The silica deposits are always formed on the aqueous side of the liquid/liquid interface (i.e. on the top of the macroporous silicon membrane). A second point is the good quality of the deposits, which have been formed

Figure 5 .

 5 Figure 5. SEM micrographs and 3D profilometry mapping based on shear force measurements

Figure 6 .

 6 Figure 6. Deposit height versus the difference between deposit and pore radii for materials prepared

Figure 7 .

 7 Figure 7. Typical RAMAN spectra recorded for an electrogenerated silica deposit in two distinct

Figure 8 .

 8 Figure 8. CV curves recorded in a blank electrolyte (A) or 70.9 µM TMA + (B), using µITIES

Figure 9 .

 9 Figure 9. a) Cyclic voltammograms showing the transfer of TBA + across the unmodified µITIES

Table 1 .

 1 Characteristics of silicon membranes used to support µITIES.

	Design

number Radius / µm Spacing / µm Quantity of pores Interfacial surface area / cm 2

	The electrochemical set-ups, depending on experimental condition, can be written as:
	Cell 1:
	(𝑎𝑞)𝐴𝑔 | 𝐴𝑔𝐶𝑙 | 5 𝑚𝑀 𝑁𝑎 + 𝐶𝑙 -𝑥 𝑚𝑀 𝑇𝐸𝑂𝑆	||	10 𝑚𝑀 𝐵𝑇𝑃𝑃𝐴 + 𝑇𝑃𝐵𝐶𝑙 -𝑦 𝑚𝑀 𝐶𝑇𝐴 + 𝑇𝑃𝐵𝐶𝑙 -𝑖𝑛 𝐷𝐶𝐸 | 𝐴𝑔(𝑜𝑟𝑔)
	Cell 2:
	(𝑎𝑞)𝐴𝑔 | 𝐴𝑔𝐶𝑙 |	10 𝑚𝑀 𝐿𝑖 + 𝐶𝑙 -𝑧 µ𝑀 𝑖𝑛𝑡𝑒𝑟𝑓𝑎𝑐𝑖𝑎𝑙 𝑎𝑐𝑡𝑖𝑣𝑒 𝑖𝑜𝑛	|| 10 𝑚𝑀 𝐵𝑇𝑃𝑃𝐴 + 𝑇𝑃𝐵𝐶𝑙 -𝑖𝑛 𝐷𝐶𝐸 | 𝐴𝑔(𝑜𝑟𝑔)
	1	5		20	2900	2.28×10 -3
	2	5		50	460	3.61×10 -4
	3	5		100	110	8.64×10 -5
	4	5		200	30	2.36×10 -5

  is grateful to Ecole Doctorale SESAMES (ED 412, Université de Lorraine) for his PhD grant and to Région Lorraine for financial support to the project. The authors are grateful to L. Salsi for SEM imaging, to Dr M. Dossot for his huge engagement and help with RAMAN experiments and data treatment, and to Dr Mathieu Etienne for his help with profilometry based on shear force measurements. Tyndall National Institute (Cork, Ireland) is acknowledged for supplying the silicon membranes.


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

Recent developments in electrochemistry at the interface between two immiscible electrolyte solutions for ion sensing

HAL is

I. Introduction

The control and understanding of phenomena occurring at the interface between two immiscible electrolyte solutions (ITIES) have triggered the interest of scientists for over a century. Indeed, in 1902, Nernst and Riesenfeld observed visually the transfer of coloured ions from water to phenol upon electrolysis. 1 From the initial experiments until the mid-1980s, research efforts were focused mainly on the understanding of fundamental phenomena. Since the mid-1990s, the number of research groups interested in electrochemistry at the ITIES has grown and they have contributed to

develop applications to answer specific needs. Analytical chemistry is one of the applications of choice when it comes to electrochemistry in general. [START_REF] Wang | Analytical Electrochemistry[END_REF] Electrochemical sensors are part of a very dynamic field in terms of both academic research [START_REF] Kimmel | [END_REF] and commercial products (e.g. glucose sensors and ion-selective electrodes). In some electroanalytical applications, the ITIES can replace the working electrode, which is the centre of the transduction mechanism in classical electrochemistry. It offers the opportunity for electrochemical detection of ionic species that are generally not detected by a redox process, with the advantages often associated with electrochemistry (e.g. relatively lowcost analysis and equipment with a short response time). This review will focus on the developments in electroanalytical chemistry at the ITIES reported in the literature during the period 2010-2015. The general principles of electrochemistry at the ITIES will be briefly presented (Part II). Electrochemical events at the ITIES can be associated with direct detection of target analytes (part III). Finally, future perspectives will be given (Part IV).

II. General principles of electrochemistry at the ITIES

In traditional electrochemistry, electron transfer reactions take place at the interface between a working electrode and an electrolyte solution. Such reactions require the presence of redox active species in the solution, which can exchange electron with the electrode. The originality of electrochemistry at the ITIES resides in the replacement of the working electrode by an interface between two immiscible liquid phases, allowing the charge transfer study of both redox-active species (by electron transfer) and ionic species (by ion transfer). Electron transfer reactions at the ITIES require the redox reaction between an oxidised form of a redox couple in one phase and the reduced species of a second redox couple in the other phase. Electron transfer has scarcely been used for sensing and it won't be reviewed here. More interesting from the analytical point of view, the ITIES allows the electrochemical study of ionic species that are not necessarily detected by a redox process. The electrochemical signal recorded is generated by the transfer of ionic species through the interface. In the case of ionic species, their transfer can be induced by the application of a Galvani potential difference,  w o  , across the interface. Equation (1) presents a form of the Nernst equation that applies to the ITIES:

) ( ) ( ln 0 w a o a F z RT i i i i w o w o +  =    Eq. (1)
zi is the charge of i and ai stands for the activities in each phase. transfer is observed gives an indication of the nature of the ionic species transferring, whereas the charge recorded is proportional to the amount of ionic species transferred. As a convention, cations transferring from the aqueous phase to the organic phase and anions transferring from the organic phase to the aqueous phase give rise to a positive current. Inversely, anions transferring from the aqueous phase to the organic and cations transferring from the organic phase to the aqueous phase are responsible for negative currents. Typically, the organic solvent used is 1,2-dichloroethane, 1,6dichlorohexane or nitrobenzene in which highly hydrophobic electrolyte salts are dissolved. Recent studies have explored new organic solvents (trifluorotoluene 4 and 5-nonanone 5 ) or mixture of solvents 6 for electrochemistry at the ITIES. Hydrophobic room temperature ionic liquids can also be used as a replacement to the organic phase. 7 The aqueous phases are usually prepared from common electrolyte salts (LiCl, KCl, NaCl, MgCl2, Li2SO4…). The full range of techniques available at solid electrodes can be used at the ITIES, making possible the electrochemical studies of ions that cannot be detected by redox processes. For further details on electrochemistry at the ITIES, readers are invited to consult text books, [START_REF] Bard | Eletrochemical Methods: Fundamentals and Applications[END_REF][START_REF] Oldham | Electrochemical Science and Technology: Fundamentals and Applications[END_REF] detailed reviews and monographs [START_REF] Samec | [END_REF][11][12][13][14][START_REF] Dryfe | Advances in Chemical Physics[END_REF][START_REF] Girault | Electroanalytical Chemistry[END_REF][START_REF] Méndez | [END_REF][18][19][START_REF] Arrigan | Electroanalytical Chemistry[END_REF][START_REF] Amemiya | [END_REF][22][23][24][25] already published on the subject. For example, the principles of electrochemistry at the ITIES were thoroughly described [START_REF] Samec | [END_REF]12,[START_REF] Dryfe | Advances in Chemical Physics[END_REF][START_REF] Girault | Electroanalytical Chemistry[END_REF]19,25 or with an emphasis on interface modification, 13 electrocatalysis 17 or electroanalysis. 11,14,18,[START_REF] Arrigan | Electroanalytical Chemistry[END_REF][START_REF] Amemiya | [END_REF][22][23][24]

III. Sensing

Ion sensing is a major challenge in both environmental monitoring and clinical diagnosis. A wide range of ions have been detected by ion transfer controlled by electrochemistry. However, simple electrochemical cells with a macroscopic interface do not favour a sensitive detection method. A number of strategies (i.e. microscopic ITIES and hydrodynamic conditions) are employed to improve mass transport and, hence, the sensitivity. This improvement is often accompanied with an optimisation of the electrochemical method employed.

Interface types. Different methods exist to build a liquid-liquid interface for electrochemical measurements (Figure 1). Classical macroscopic glass cells generally offer an interface with a surface area in the cm 2 range (Figure 1A) and have been used to investigate electroanalytical behaviour of myoglobin 26 and poly-Lysine dendrigrafts. 27 Although widely used for other applications, their use for analytical purposes is rather limited due to linear diffusion of species and unstable mechanical properties. The other types of interfaces were developed to overcome the limitations of the classical approach. The mechanical stability can be improved by the use of a porous membrane that supports the ITIES (Figure 1B). Creatinine, 28 heparin, 29 albumin 30 and inorganic cations [31][32][33][34] and anions 35 have been detected at such membrane-supported ITIES. Bakker and colleagues used a tubular liquid-liquid cell for ion transfer coulometry. 31 The cell was made of a silver -silver chloride wire inserted in a porous polypropylene tube impregnated with the organic phase. A thin aqueous layer (approximately 50 µm thick) is formed between the polypropylene tube and the wire. Ions are transferred from the aqueous layer to the organic phase supported by the porous polypropylene tube (Figure 2A). The thin layer of aqueous solution and its known volume allows total transfer of the ions monitored by coulometry, resulting in a calibration-free determination of ion concentration. A liquid-liquid interface can also be formed using a solid electrode impregnated with a thin organic layer and then immersed in an aqueous electrolyte solution (Figure 1C). This method offers the advantage of using low volumes of organic phase. The organic solution contains a redox species, which can exchange electron with the electrode. Ions then transfer from the aqueous phase to the organic phase to maintain electroneutrality. The transfer potential depends on the hydrophobicity / hydrophilicity of the ions constituting the aqueous electrolyte. This approach has been used for the detection of inorganic cations [36][37][38][39][40][41] and perfluoroalakanesulfonates. 42 Nevertheless, the sensitivity of macroscopic ITIES can be restricted by a linear diffusion of species. Amemiya and colleagues circumvented this drawback by forming the organic gel containing a conducting polymer on a rotating disc electrode made of either gold or carbon. 42,39,41 The improved mass transport due to the convection resulted in limits of detection in the sub-nanomolar concentration range. Mass transport was also be improved by the miniaturisation of ITIES and resulted in better sensitivities and limits of detection. Different fabrication strategies to prepare these µITIES exist and have been reviewed elsewhere. [START_REF] Arrigan | Electroanalytical Chemistry[END_REF]22 ITIES of microscopic dimensions are now routinely used under the form of a single µITIES [43][44][45][46][47][48]37,[49][50][51] (Figure 1D) or of an array of µITIES [52][53][54][55][56][57][58][59][60][61][62][63][64][65][66][67][68][69][70] (Figure 1E) allowing to reach limits of detection in the range of tens of nM with an analysis time of a couple of minutes. Diffusion of ionic species at µITIES has shown an asymmetric profile. 18 In the case where the micropipette or the pores are filled with the organic phase (i.e. analogous to inlaid microelectrodes), radial diffusion of species is observed and a sigmoidal signal is recorded for the ion transfer. If the ionic species remain confined within the capillary or within the pore of the membrane, linear diffusion will predominate and a peak shaped voltammogram will result. The electrochemical behaviour of reversible ion transfer has been theoretically modelled for a variety of electrochemical techniques (cyclic, linear sweep, square wave, normal pulse and differential pulse voltammetries). [71][72][73] The influence of adsorption of the ionic species on the pore walls after the transfer was also investigated by finite element simulations. 74 The critical dimension of the ITIES was further reduced down to the nanometer scale using single nanoITIES 75 or nanoITIES arrays fabricated by e-beam lithography 76 or focused ion beam. 77 NanoITIES arrays were characterised by cyclic voltammetry [76][77][78][79] and chronoamperometry 80 of model ions such as tetraethylammonium or tetrapropylammonium. Scanlon and Arrigan demonstrated that nanoITIES arrays were 50 times more sensitive than µITIES arrays and 1000 times more sensitive than a single macroITIES. 79 However, the sensitivity obtained at such nanoITIES was only half of the theoretical one. The authors attributed this discrepancy to the non-equivalence of the nanoITIES in the array and to an overlap of the diffusion zones, 78 a claim later supported by finite element simulations. 81 Detection of propranolol by cyclic voltammetry at an array of nanoITIES resulted in a limit of detection of 0.8 µM, which is 5-10 times lower than the limit of detection achieved at larger interfaces. 81 All the examples above described the variety of the physical nature (membrane-supported ITIES, thin organic layer and miniaturised ITIES) of interfaces that was used to improve the analytical performances. The type of interface can also be changed by modifying the nature of the hydrophobic phase. The properties of hydrophobic ionic liquids of low volatility and of high conductivity have established them as a good alternative for organic solvent in electroanalytical applications 82 . Ding

and colleagues investigated the transfer potential of inorganic cations (Sr 2+ , 48 Rb + , 49 Cs +50 and uranyl species 83 ) assisted by octyl(pheny)-N,N-diisobutylcarbamoylmethylphosphine oxide dissolved in a hydrophobic room-temperature ionic liquid. Their studies have estimated the stoichiometry of complexes formed between the metal ions and the ligand and their complexation constant, . The stoichiometry of the complexes determined by electrochemistry was correlated with the one obtained by mass spectrometry, 49,50 establishing electrochemistry at the liquid-liquid interface as a simple analytical method for the studies of metal on-ligand complexation.

Detection methods. Stripping voltammetry at the ITIES has been widely used since the early 1990s, 22 and it remains used as it is one of the most sensitive techniques for ion sensing. Since 2010, it has been used for the detection of proteins, 54,55,58,59 ionic drug molecules, 61,63,64 pollutants, 44,45,27,42 and inorganic species (both cations 47,39,41 and anions [67][68][69] ). Stripping voltammetric studies improved the limits of detection and the sensitivities by at least one order of magnitude compared to cyclic voltammetry. Computational simulations have shown that the asymmetric diffusion behaviour, discussed in the following section, is beneficial for the analysis by stripping voltammetry. 84,85 Indeed, the radial diffusion in the aqueous phase ensures a high mass transport rate for the preconcentration step, whereas the linear diffusion in the organic phase limits the diffusion of the target analyte away from the interface. The preconcentration can be further improved by using a poly-(vinyl chloride) gel as the organic phase, which restraints diffusion of species. More recently, Arrigan's group harnessed the specificity of protein electrochemistry at the ITIES (described in a previous section) to devise an adsorptive stripping voltammetry protocol. The target protein is accumulated at the ITIES via potential-dependent adsorption. 55 They demonstrated that several layers of proteins accumulated at the interface and that the saturation of the analytical signal was the result of protein reorganisation at the interface rather than diffusion-limited. First demonstrated with lysozyme, this approach was then extended to insulin 58 and haemoglobin. 59 Stripping voltammetry determination of ions has used the current as the analytical signal, whereas

Bond and colleagues have developed voltammetric ion-selective electrodes for the detection Na + , K + and Ca 2+ , 36,40 based on ITIES formed at electrodes modified with thin-organic films (Figure 1C). The transfer peak potential varied linearly with the logarithm of the target ion activity and was used as the analytical signal for the detection of Na + , K + and Ca 2+ in a variety of real samples.

In most analytical applications, calibration of the sensors remain a critical issue. Bakker's group has developed a coulometric method using the thin layer cell described in the previous section and in Figure 2. 31,32,86 This experimental set-up could be used for the calibration-free determination of ion concentration. A series of potentiostatic and galvanostatic pulses was used to compensate the contribution of interfering ions to the coulometric response. First, a potential equal to the open circuit potential was applied to verify the absence of ion transfer (i.e. current is 'zero'). The transfer potential was then applied and the current was measured as a function of time. During this chronoamperometric step, both the target analyte and interfering ions are transferred, and hence both contributing to the current response. After a resting period with zero current applied, the second potentiostatic step was applied, during which only the interfering ions transferred. Indeed, since a thin layer cell was used, the aqueous phase is completely depleted from target ions and only interfering ions, which are at a much higher concentration, remain. The current of the second potentiostatic pulse is subtracted from the first one to determine the contribution of the sole target ions. This method was successfully used for the selective detection of Ca 2+ (10 µM), K + (0.1 µM) and NO3 -(1.1 µM). The same type of ion selective membrane was then adapted for the indirect determination of heparin in undiluted blood samples, using a thin layer coulometric cell. 29 Heparin is an anticoagulant used during surgical procedures, known to interact with protamine. Protamine is transferred across the interface and the charge transfer recorded increased linearly with the concentration in both laboratory and undiluted blood samples. In the presence of heparin, protamine was bound to it and thus, was not available for transfer anymore, leading to a drop in the charge transferred. Quantitation of the unbound protamine remaining has led to the heparin concentration in the analysed sample.

Target analytes. The sensing of molecules of biological interest (small organic molecules, 52,53,28,87 carbohydrates, 88,29 and proteins 26,[54][55][56]30,[57][58][59][60] ), synthetic organic molecules (drug molecules, 61,43,62- 64,81,89 pollutants 44,45,27,42 ) and inorganic species (both cations 36,46,31,32,47,48,33,37,38,49,65,34,39,40,51,50,66,41 and anions 67,35,68 ) has been extensively studied in the 2010-2015 period covered in this mini-review (see Table 1 of the Supplementary Information section for more details). Although the detection of some analytes was already targeted before 2010 (e.g. dopamine, 14,[START_REF] Arrigan | Electroanalytical Chemistry[END_REF] propranolol, 90 and heparin 91 ), other types of ionic species have recently been detected at the ITIES. Proteins (albumin, 30 amylin, 57 haemoglobin, 59 insulin, 58 lysozyme, 55 myoglobin 56 ) and peptides issued from the enzymatic proteolysis 54 have been detected by ion transfer voltammetry. This analytical development resulted from fundamental studies on the electrochemical behaviour of proteins. 92 The detection of proteins at the ITIES is generally based on two different types of facilitated ion transfers (Figure 3A-B). In the first case, the protein is dissolved in the aqueous phase. Upon the application of an interfacial potential, proteins adsorbed at the interface and facilitates the transfer of anions of the supporting electrolyte salt of the organic phase (as schematised on Figure 3A). The current arising from this ion transfer is used as the analytical signal for protein detection. (Figure 3C). Earlier works have shown that proteins can only be detected at the ITIES when they were positively charged (i.e. pH < pI) as their detection resulted from an interaction with the anion of the organic electrolyte. 92 This approach was used successfully for the detection of myoglobin 56 by cyclic voltammetry and for the detection of lysozyme, 55 insulin 58 haemoglobin 59 using adsorptive stripping voltammetry with detection limits in the range of tens of nanomolar. The sensitivity of the protein detection depends on its charge and on the possibility of the organic anion to interact with hydrophobic pockets within the protein structure.

Electroanalytical studies have shown that the technique is more sensitive for proteins than for dendrimers deprived of these hydrophobic patches. 27 In the second case, proteins are present in the aqueous phase and anionic surfactants are dissolved in the organic phase (Figure 3B). Anionic surfactants allow the transfer of the protein across the interface, giving rise to a current, which can be used as the analytical signal (Figure 3D). 26,60 Osakai et al. have investigated the use of a variety of surfactants for the transfer of cytochrome c, ribonuclease A, lysozyme, albumin, myoglobin and lactalbumine. 26 They suggested that the anionic surfactants interacted with the positively charged amino acids (lysine, arginine and histidine) of the proteins. Cyclic voltammograms for cytochrome c, haemoglobin and myoglobin in the absence (Figure 3C) and in the presence (Figure 3D) of anionic surfactant in the organic phase showed that both approaches allowed the detection of protein.

However, the use of anionic surfactant as an ionophore assisting protein transfer through the interface, improved the peak current by a factor 6 in comparison to the signal obtained in the absence of surfactants. 60 The majority of proteins investigated at the ITIES did not transferred across the interface unless a surfactant was used to facilitate the transfer. The mechanism for the detection of amylin at the ITIES was different as it transferred unassisted across the interface. The polypeptide could then be detected at physiological pH from a protein mixture. 57 Small organic molecules such as ionic drugs (propranolol, 61,43,63,81 daunorubicin 62 and ractopamine 64 ) were also detected by ion stripping voltammetry at micro-interface arrays. Limits of detection of 50 nM were reached for propranolol, 63 800 nM for daunorubicin 62 and 100 nM for ractopamine. 64 These concentrations are suitable for detection in real samples, however the interactions between drugs and plasma proteins required the deproteinisation of the serum before detection. 61,62,64 A single µITIES was also used to investigate the chiral interactions of (S)-and (R)-propranolol with a serum protein: 1-glycoprotein. 43 Lopes and Kataky have demonstrated that the transfer signal for propranolol decreases in the presence of the protein as a consequence of the protein-drug complexation and that the interaction of (S)-propranolol with 1-glycoprotein is greater than in the case of (R)-propranolol. Amemiya's group has focused its attention on the detection of perfluoroalkanesulfonates and perfluoroalkanecarboxylates by ion transfer voltammetry. 42 A limit of detection of 50 pM was reached, which is below the maximum authorised concentration set in the US. The detection of perfluoroalkanesulfonates ion transfer voltammetry is superior to its detection by electroanalysis at solid electrodes as the oxidation of perfluoroalkanesulfonates is more difficult than their nonfluorinated analogues. On the contrary, perfluoroalkanesulfonates are more hydrophobic and are hence more easily detected at the liquid-liquid interface.

The variety of interface types and of detection methods has led to the improvement of sensitivities and limits of detection for an increasing number of target analytes. However, most of the studies described above were done in clean laboratory solutions. The number of target analytes detected in real samples remained limited to a few biological samples (urine 28,30 whole blood, 29,40 and plasma 40 ) fresh water, 40,35,68 and drinks. 40 Target analytes such as heparin, Na + , K + and Ca 2+ , NO3 -and ClO4 -were detected in those complexes matrices despite the presence of possible interferents.

IV. New horizons

Recent developments in the field of electrochemistry at the ITIES have emerged over the past few years. Although they have not necessarily resulted in analytical applications yet, they offer promising perspectives for the future. They include the electrochemistry of two interfaces at supported liquid membranes, the coupling of ITIES with other analytical techniques and the modification of the ITIES.

Electrochemistry at supported liquid membranes systems. In such experiments, an organic phase is supported by a porous membrane and is sandwiched between two aqueous electrolyte solutions (Figure 4A). This method has found analytical applications for extraction and sample preparation. 93,94 When combined with electrochemistry at the ITIES, a hydrophobic electrolyte is dissolved in the organic phase, leading to two polarisable liquid-liquid interfaces. A potential difference is applied across the supported liquid membrane to favour the transfer of ionic species from one aqueous solution to the other across the supported organic electrolyte solution. 95 Dryfe and co-workers investigated the ion transfer mechanism at such supported liquid membranes systems. 96,97 Their experimental setup consisted of two aqueous phases separated by a supported liquid membrane. In both aqueous phases, the same ion was dissolved. 97 As a consequence, the potential window was the double of the one observed for a single ITIES setup, resulting in the appearance of two couples of ion transfer peaks, each couple corresponding to the reversible transfer from one of the aqueous phases to the membrane (Figure 4B). Transfer potentials of these ions are dependent on their hydrophilicity.

As seen on Figure 4B, the most hydrophilic ions require more energy and the couples of transfer peaks are more separated. As the hydrophobicity increases, the separation between the couple of transfer peaks decreases, although their transfer potentials remain distributed symmetrically about the zero potential difference. The understanding of the double transfer of ions across the supported liquid membrane has an impact on the development of applications in the field of ion separation, purification and detection.

Coupling with other analytical techniques. Since the origin of electrochemical studies at the ITIES, there has been an interest to correlate electrochemical information with other analytical techniques (e.g. spectroscopy techniques being among the most popular ones). 12,[START_REF] Fermin | Diffraction and Spectroscopic Methods in Electrochemistry[END_REF] Recently, electrochemical cells for ITIES studies were adapted for the interfacing spectroscopy methods such as Raman spectroscopy, [START_REF] Booth | [END_REF][100][101] X-Ray absorption spectroscopy 102 and spatial scanning spectroscopy. 103 Girault's group has harnessed electrochemistry at the ITIES with electrospray ionisation mass spectrometry. 104,105 A number of experimental arrangements was devised to follow interfacial electrochemical processes by mass spectrometry. Hence, a biphasic electrospray ionisation combined with the ITIES allowed the elucidation of the interactions between lysozyme and organic electrolyte anions as they are described in Figure 3A. 104 Although electrospray ionisation is a powerful technique for the mass spectrometry study of proteins, it suffers from the occurrence of electrochemical reactions at the electrospray electrode. This drawback was circumvented by the introduction of the electrostatic spray ionisation (ESTASI) method, in which the electrospray electrode is separated from the sample by an insulating substrate. This method was then used for fingerprinting the tertiary structure of lysozyme, previously adsorbed by electrochemistry at the ITIES on a gel phase (Figure 5). 105 It consisted of three steps: (i) the electroadsorption of proteins at the gel; (ii) the transfer of the gel onto the ESTASI substrate and (iii) the running of the ESTASI-MS experiments. The mass spectra confirmed the hypothesis based on electrochemical results, 55 that multiple layers of proteins were adsorbed at the ITIES during the preconcentration step of adsorptive stripping voltammetry.

Modification of the liquid-liquid interface. Although the lack of sensitivity of electrochemistry at the ITIES has been overcome, the issue of selectivity remains. Ionophores, used for the elaboration of ion-selective electrodes, have been extensively used at the ITIES. Unfortunately, they do not solve all the issues related to selectivity. The modification of the liquid-liquid interface may contribute further to improve the selectivity, analogously to the modification of solid electrodes. Among the various strategies to modify an electrode surface, silica mesoporous has appeared over the years as a simple and efficient way of introducing selectivity and functionality to an electrode. 106 In a similar fashion, mesoporous silica materials have been deposited at the liquid-liquid interface by the means of evaporation 107,108 or electrochemistry. [109][110][111] Mesoporous silica is generated at the liquid-liquid interface by ion transfer of voltammetry of a surfactant, cetyltrimethylammonium, which is transferred from an organic phase to an aqueous phase containing hydrolysed silica precursor. The presence of surfactant in the aqueous phase catalyses the condensation of silica. 109,110 Ion transfer voltammetry of six model ions (3 different tetraalkylammonium ions, 4-octylbenzenesulfonate, and two poly dendrimers) was used to characterise the electrodeposited mesoporous silica. Experiments have shown that the transfer of these ions differs depending on their size and charge. 111 Chen and co-workers have used micro-interfaces modified with mesoporous silica for the detection of folic acid by differential pulse stripping voltammetry. 108 Similarly, the behaviour of nanoparticles at the liquidliquid interface has been investigated for potential applications in the field of sensors or catalysis. 112 Although the liquid-liquid interface was not polarised in most cases, there has been a substantial number of studies at the ITIES on the in-situ electrodeposition of metallic nanoparticles 113 or on their self-assembly at the ITIES after ex-situ synthesis. 114 These studies are essentially fundamental but they may pave the way for new (spectro)electrochemical sensing methods at the ITIES. These recent efforts for the modification of the interface have confirmed the need to investigate interfacial processes in-situ. A range of spectroscopic techniques was coupled to electrochemistry at the ITIES to monitor interfacial reactions such as metallic nanoparticles nucleation 102,103 and adsorption 99 and silica condensation. 100

Conclusions

The frontier in ion sensing at the ITIES is constantly pushed forward as new target analytes are being detected, new methods are being developed and more and more analytical techniques are coupled to the electrochemistry at the ITIES. The possibility of detecting ions in real samples as complex as whole blood or environmental matrices also testifies of potential applications and eventually commercial products. The increasing interest for electrochemistry at the ITIES as an analytical Ref. 29 Copyright 2014 American Chemical Society. (TEA + ) tetrabutylammonium (TBA + ) and Crystal Violet (CrV + ). Adapted with permission from Ref. 97 Copyright 2014 American Chemical Society.

  ion i , which is related to the standard Gibbs energy of ion transfer

  .e. they will transfer at a largely positive  Addition of an ionophore to the electrochemical cell can facilitate the ion transfer, by lowering the Gibbs energy of transfer required. The potential difference at which a

  technique over the years and the continued research efforts in interface modification with nanoobjects (nanoparticles, mesoporous silica or graphene) and in the coupling with other analytical techniques will contribute to the dynamism of the field over the next few years. Grégoire Herzog received his PhD in Chemistry from University College Cork (Ireland) in 2004. He then moved to Grenoble for a post-doc. In 2005, he joined the Tyndall National Institute in Cork, where he worked on the development of miniaturised electroanalytical tools based on ion transfer voltammetry at the liquid-liquid interface. Since 2011, he is a researcher at the Laboratoire de Chimie Physique et Microbiologie pour l'Environnement (CNRS-Université de Lorraine, Nancy, France) where he is working on the electrochemical modification of the liquid-liquid interface with mesoporous silica films.

Figure 1 :

 1 Figure 1: Most common experimental set-ups for electrochemistry at the ITIES: (A) classical macroscopic ITIES, (B) membrane-supported ITIES, (C) thin organic film, (D) single µITIES and (E) µITIES array. The aqueous phase is represented in blue and the organic phase in orange. REorg: reference electrode for the organic phase, REaq: reference electrode for the aqueous phase, CEorg: counter electrode for the organic phase, CEaq: counter electrode for the aqueous phase, M: Membrane; : ionic species of charge Zi in the aqueous phase (w) or in the organic phase (o); Ared: reduced form of an electroactive species; AOx: oxidised form of an electroactive species.

Figure 2 :

 2 Figure 2: (A) Schematic representation of the thin layer coulometric cell used for (B) the indirect determination of heparin concentration in undiluted blood samples. Adapted with permission from

Figure 3 :

 3 Figure 3: Origin of the analytical signal for protein detection at the ITIES in (A) the absence and (B) the presence of surfactants in the organic phase. The arrows indicate which ionic species are transferring. Cyclic voltammograms for haemoglobin (Hb), cytochrome c (Cyt c) and myoglobin (Mb) in (C) the absence and (D) the presence of surfactant in the organic phase. Adapted with permission from Ref.60 Copyright 2013 American Chemical Society.

Figure 4 :

 4 Figure 4: (A) Schematic representation of the supported liquid membrane, M, separating two aqueous phases A and B. REA ad REB are the reference electrodes and CEA and CEB are the counter electrodes. (B) Cyclic voltammograms for tetramethylammonium (TMA + ), tetraethylammonium

Figure 5 :

 5 Figure 5: (A) Experimental procedure for the electrostatic spray ionisation mass spectrometry involving (1) electrochemical preconcentration, (2) gel removal and (3) mass spectrometry analysis. (B) Mass spectrum obtained for the electroadsorption of 10 µM lysozyme for 30 min at an interfacial potential of 1.0 V. Adapted from Ref. 105

Figure 2

 2 Figure 2

Acknowledgments

GH is grateful to the Agence Nationale pour la Recherche for the funding of the HYPERION project (grant number: ANR-14-CE14-0002-01).

Biographical sketch