Romain Viala

Vincent Placet

Sandie Le Conte

Stéphane Vaiedelich

Scott Cogan

L E Conte

Model-based decision support methods applied to the conservation of musical instruments: application to an antique cello

Keywords: Musical acoustics, virtual prototyping, dynamical modelling, cultural heritage conservation, finite element model

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

INTRODUCTION

Generally applied in industrial and research domains, model-based decision support has been used for decades as a powerful tool. Considering uncertainties and finely modeled geometries, it can be used for the virtual prototyping of parts and structures. In the musical instrument domain, these methods can be used as a support for the design of new instruments. Moreover, it can also be used for the conservation and restoration of antique and valuable instruments. Many models of musical instruments have been developed for decades, especially violins since the 1980's [START_REF] Knott | A Modal Analysis of the Violin Using MSC/NASTRAN and PATRAN[END_REF]. More recently, models have been developed for the reverse engineering of a violin [START_REF] Pyrkosz | Reverse Engineering the Structural and Acoustic Behavior of a Stradivari Violin[END_REF] or the study of dynamics and acoustics of violin body [START_REF] Gough | Vibrational Modes of the Violin Family[END_REF] and the way geometric properties interacts. Models have also been developed to screen material properties of the wooden constituents of the violin [START_REF] Viala | Model-based effects screening of stringed instruments[END_REF] and as a decision support tool for violins and guitars [START_REF] Viala | Towards a model-based decision support tool for stringed musical instrument making[END_REF].

It must be highlighted that curators deal with unique and irreplaceable instruments, which represents a main difference with usual industrial products, for which the critical aspects mostly deal with security and cost concerns. Musical instruments are made with different pieces of wood whose mechanical properties may be hard to evaluate and represent a considerable lack of knowledge. In the same manner, the fine knowledge of the geometry can be either impossible or costly, involving experiments and devices [START_REF] Van Den Bulcke | Nondestructive research on wooden musical instruments: From macro-to microscale imaging with lab-based X-ray CT systems[END_REF], [START_REF] Sirr | X-ray CT Measurements of the Internal Corpus Volume and a New Soundpost-Corpus Volume Relationship for Stringed Instruments of the Violin Family[END_REF].

So, a major concern must be pointed out about the geometrical and material uncertainties, and the way they can affect the results of a model. Moreover, the climatic conditions that an instrument can undergo have to be considered for such problematics, as musical instruments constantly undergo relative humidity and temperature changes when outside the showcase [START_REF] Le Conte | A Wood Viscoelasticity Measurement Technique and Applications to Musical Instruments: First Results[END_REF]. In this study, a cello is modeled using the finite element method, and a geometrical characteristic is changed, the height of the top and back plates, while the material properties are unchanged. Secondly, the impact of repairs and defaults on the dynamical response of the model is evaluated, as a potential starting point for a decision support tool for restoration of musical instruments. The object of study of this work is an antique cello which exhibits many previously repaired cracks, and galleries produced by xylophagous insects, whose activity can be experimentally detected by acoustic emission [START_REF] Conte | Acoustic Emission to Detect Xylophagous Insects in Wooden Musical Instrument[END_REF].

The cello is made of different species of wood: spruce, maple, ebony and sometimes rosewood that are assembled together. Different experimental studies in the dynamical fields have been focused on several parts of the cello, such as the tailpiece dynamics [START_REF] Fouilhé | String "After-Length" and the Cello Tailpiece: Acoustics and Perception[END_REF], [START_REF] Fouilhé | Vibration modes of the cello tailpiece[END_REF] and studied wolf notes [START_REF] Firth | The wolf in the cello[END_REF]. The body of the instrument is here the main object of concern and is made of carved maple back and bent sides, and a carved spruce soundboard. The aim of the study is to evaluate the impact of an incorrect geometrical modeling on the dynamical response. For this purpose, the geometry of the cello is finely modeled using computer aided design and the finite element method, described below.

ANALYSIS

The studied structure is a cello, made by Pietro Guarneri in the 18 th century and kept at the musée de la musique, Paris, under the label E.1555. The nominal model is made using the template of a model given in [START_REF] Wake | A 'Strad' Mode[END_REF] and the data collected by curators and instrument makers about some properties of the E1555 cello. The Computer aided design (CAD) of the cello model has been made using the software SOLIDWORKS and is shown in the figure 1. The repair wedges and galleries are schematized in the figure 2. The geometry has been meshed with tetrahedral elements with quadratic interpolation, the number of nodes is approximately equal to 350000, which leads to a value close to 1 million degrees of freedom. Most of the parts are made of wood (maple, spruce an ebony species). The wooden parts are modelled under the linear elastic hypothesis, with an orthotropic definition of the elastic parameters. The material properties identified are taken from [START_REF] Viala | Identification of the anisotropic elastic and damping properties of complex shape composite parts using an inverse method based on finite element model updating and 3D velocity fields measurements (FEMU-3DVF): Application to bio-based composite violin sounboard[END_REF] and [START_REF] Guitard | Modèles prévisionnels de comportement élastique tridimensionnel pour les bois feuillus et les bois résineux[END_REF] and given in the table 1. For the latter, three models will be considered: the first model made with nominal values obtained in [START_REF] Wake | A 'Strad' Mode[END_REF], labelled as V1 and the one with the correct arch height of both back and top, which corresponds to an increase of 10 mm and a corresponding global shape change, labelled as V2. The photogrammetry method has been used for the measurements of the arch dimensions. In addition, a model is created based on the model V2 without wedges and gallery, labelled as V2_2, to evaluate the impact of repairs or defaults on the behavior of the cello. 1: Orthotropic material properties for maple, spruce and ebony species, taken from [START_REF] Viala | Identification of the anisotropic elastic and damping properties of complex shape composite parts using an inverse method based on finite element model updating and 3D velocity fields measurements (FEMU-3DVF): Application to bio-based composite violin sounboard[END_REF] and [START_REF] Guitard | Modèles prévisionnels de comportement élastique tridimensionnel pour les bois feuillus et les bois résineux[END_REF]. The bent parts orientation has been considered by changing the orientation of the local coordinate frames of the corresponding elements. The modal basis computation of 100 modes lasts one hour. Once the modes are computed, the comparison between each case is made with a modal assurance criterion, proposed in [START_REF] Allemang | A correlation coefficient for modal vector analysis[END_REF]. To highlight the capability of the finite element models, the bridge admittances of each case are computed. The admittance is computed with the application of an input force and an observation of either the displacement velocity and acceleration at the same point in the same direction, as shown in the figure 2, right part. The bridge admittances are often considered as a signature of the soundboard musical instrument dynamics and has been widely measured [START_REF] Zhang | Motion of the cello bridge[END_REF] but never modeled previously on a numerical model of a cello. The synthesis of the admittance of a cello can unlock some issues that are common in experiments, such as the reproducibility of the measure [START_REF] Zhang | Reliability of the input admittance of bowed-string instruments measured by the hammer method[END_REF] and the interpretation of the results in the case of geometrical and material differences between instruments, since numerical models can change each parameter at once.

Maple

RESULTS

The computed modal bases are rich and only low frequency canonical modes will be considered. These modes, labeled as T1, C2, C3 and C4 according to usual nomenclature [START_REF] Askenfelt | Quarterly Progress and Status Report Eigenmodes and tone quality of the double bass[END_REF], are shown in the figure 3 and the evolution of the eigenfrequencies for the corresponding modes for each model V1, V2 and V2_2 are given in the table 2.

It is shown that changing the arch height can lead to a variation of the eigenfrequencies of up to 8 % for low frequencies mode. In addition, for higher frequencies, the computed and experimental modal bases are not correlated above the 20 th modes which states for a completely different behavior above 250 Hz. Thus, such geometrical parameters like arch heights and thickness are keys for the good correlation of a model and a real instrument and need to be characterized.

The admittances at the bridge for the cases V2 (without repairs) and V2_2 (without repairs) are given in the figure 4. First, the usual A0 acoustic mode is not displayed on the admittance since the fluid-structure interaction was not implemented in the model. The admittance shape below 230 Hz is like the one given in [START_REF] Askenfelt | Quarterly Progress and Status Report Eigenmodes and tone quality of the double bass[END_REF] (with lower frequencies) and, under this point, no significative differences are shown between the repaired and not repaired cases. Above this frequency, differences occur in the admittances' shapes, and increase with increasing frequencies, which highlights the facts that repairs affect the dynamical behavior of the cello, even in the low and mid-frequency domains.

CONCLUSION

The numerical models created have highlighted two main facts. Firstly, the geometry accuracy is a key for the predictive capability of a cello, and geometrical properties like arch shape and height are essential for the precision of the model. Nowadays, such geometrical features are evaluated effectively with photogrammetry and CT scans means. Secondly, the effects of repairs like wedges and defaults like insect galleries can modeled with such models. The effects of these elements are important on the dynamical behavior of the cello, and this can be considered as an effective tool for more refined and dedicated studies, like the design of repairs wedges that would affect as lower as possible the behavior of the musical instruments, when such repairs are inevitable for the structural integrity of the instrument. In conclusion, this study shows that the numerical models can simulate the effect of restorers and instrument maker's decisions on the dynamical behavior, which can be a starting point for a decision support tool in both static and dynamical domains. The perspective proposed is to evaluate the impact of geometric and material uncertainties on the static response of the cello, when undergoing prestresses due to assembly and strings.

Figure 1 :

 1 Figure 1: Computer aided design, (CAD) of the cello model; left: front view, right: back view

Figure 2 :

 2 Figure 2: Left: scheme of the wedges and cracks of the cello, based on radiographs provided by the laboratory of the Musée de la musique, Paris, right: bridge excitation and measurement point and direction on a Belgian cello.

Figure 3 :

 3 Figure 3: Normalized eigenvectors of the eigenmodes modes of interest (blue=0, red=1; left side: top view, right side: back view. Eigenmode V1 V2 V2_2 T1 [Hz] 191.3 179.4 (-6.2%) 179.4 C2 [Hz] 179 164.2 (-8.2%) 164.5 C3 [Hz] 207 222.1 (+7.3%) 221.8 C4 [Hz] 213 212.2 (-0.4%) 212.1 Table 2: eigenfrequencies of the first three modes of interest of the cello and their evolution for three difference cases: nominal model V1, modified model V2, and modified model V2 without repairs. Relative changes between V1 and V2 are given in brackets.

Figure 4 :

 4 Figure 4: bridge admittance (acceleration) at the bridge with and without wedges and wormholes.


Anastasiia Kovalyk

Oksana Tananaiko

Anna Borets

Mathieu Etienne

Alain Walcarius

Voltammetric and microscopic characteristics of MnO2 and silica-MnO2 hybrid films electrodeposited on the surface of planar electrodes

Keywords: ITO electrodes, carbon nanostructured screen printed electrodes, MnO2 particles, silica films, electrochemical deposition, voltammetry, hydrogen peroxide

come

Introduction

The development of sensitive elements of disposable amperometric sensors for medical diagnostics or on-site analyses is important task of the modern analytical chemistry [START_REF]Biosensors: Fundamentals and applications[END_REF][START_REF] Evtugyn | Biosensors: Essentials[END_REF]. The planar printed electrodes are perspective for this purpose. Many commercial biosensors are based on electron mediator transport [START_REF] Thiyagarajan | Disposable electrochemical sensors: A mini review[END_REF][START_REF] Pumera | Electrochemical nanobiosensors[END_REF]. Metal and metal oxide nanoparticles immobilized on electrode surfaces can act as electro mediators in amperometric enzymatic sensors [START_REF] Evtugyn | Biosensors: Essentials[END_REF][START_REF] Monteiro | Electrochemical Enzyme Biosensors Revisited: Old Solutions for New Problems[END_REF], and they can be even used for the development of enzymatic free sensors [START_REF] Wang | Electrochemical glucose biosensors[END_REF][START_REF] Liu | Towards development of chemosensors and biosensors with metal-oxide-based nanowires or nanotubes[END_REF][START_REF] Chinnasamy | Synthesis and electrocatalytic properties of manganese dioxide for non-enzymatic hydrogen peroxide sensing[END_REF][START_REF] Cipolatti | Nanomaterials for biocatalyst immobilizationstate of the art and future trends[END_REF][START_REF] Beyene | Bio)sensors based on manganese dioxide-modified carbon substrates: retrospections, further improvements and applications[END_REF].

Because hydrogen peroxide is a common product of many enzymatic transformations, the development of the sensitive elements of amperometric sensors for H2O2 is a challenge for the construction of several enzymatic biosensors. The nanoparticles of noble metals (Au, Pt) are commonly used catalysts in amperometric sensors for hydrogen peroxide, thanks to their high activity and stability [START_REF]Biosensors: Fundamentals and applications[END_REF]. However, there are other more available and low cost mediators, such as transition metal oxide particles, i.e. MnO2.

MnO2 is known as mediator for hydrogen peroxide amperometric detection [START_REF] Liu | Towards development of chemosensors and biosensors with metal-oxide-based nanowires or nanotubes[END_REF][START_REF] Chinnasamy | Synthesis and electrocatalytic properties of manganese dioxide for non-enzymatic hydrogen peroxide sensing[END_REF][START_REF] Cipolatti | Nanomaterials for biocatalyst immobilizationstate of the art and future trends[END_REF][START_REF] Beyene | Bio)sensors based on manganese dioxide-modified carbon substrates: retrospections, further improvements and applications[END_REF][START_REF] Dontsova | Hydrogen Peroxide-Sensitive Amperometric Sensor Based on Manganese Dioxide Nanoparticles[END_REF]. In particular, MnO2 nanoparticles can be reduced by H2O2 on carbon, gold or ITO electrodes to Mn(II) or Mn(III) species and then re-oxidized to MnO2 electrochemically [START_REF] Beyene | Bio)sensors based on manganese dioxide-modified carbon substrates: retrospections, further improvements and applications[END_REF][START_REF] Dontsova | Hydrogen Peroxide-Sensitive Amperometric Sensor Based on Manganese Dioxide Nanoparticles[END_REF][START_REF] Dontsova | Screen-printed carbon electrode for choline based on MnO2 nanoparticles and choline oxidase/polyelectrolyte layers[END_REF]. The resulting anodic currents sampled at the electrode increased proportionally to H2O2 content in solution. The selectivity and sensitivity of H2O2 detection using an electrode modified with MnO2catalytistwere improved compared to the bare one [START_REF] Chinnasamy | Synthesis and electrocatalytic properties of manganese dioxide for non-enzymatic hydrogen peroxide sensing[END_REF][START_REF] Beyene | Bio)sensors based on manganese dioxide-modified carbon substrates: retrospections, further improvements and applications[END_REF]. The use of MnO2 as electron mediator is a good alternative to gold or platinum nanoparticles.

The presence of reducing agents such as ascorbic acid or urea can influence the results of hydrogen peroxide determination, which is a problem for amperometric enzymatic sensors. The oxidation currents of the mentioned compounds appear indeed at potential values overlapping with H2O2 signals, which caused an error in the results of determination of H2O2. In the goal to circumvent this limitation, MnO2 powder was used to reduce the influence of ascorbic acid on the results of choline amperometric determination with a help of the electrode modified with choline oxidase [START_REF] Xu | Application of MnO2 nanoparticles as an eliminator of ascorbate interference to amperometric glucose biosensors[END_REF][START_REF] Choi | Amperometric biosensors employing an insoluble oxidant as an interference-removing agent[END_REF][START_REF] Mazurenko | Amperometric Biosensor for Choline Based on Gold Screen-Printed Electrode Modified with Electrochemically Deposited Silica Biocomposite[END_REF]. Ascorbic acid was oxidized during the pretreatment of the solution with MnO2 particles. To avoid such pretreatment with addition of MnO2 particles into the solution, it would be interesting to use an electrode modified with MnO2 particles immobilized onto its surface for the development of the sensor element for amperometric determination of H2O2 in the presence of reducing agents such as ascorbic acid. The rate of ascorbic acid oxidation onto MnO2 should be lower than the catalytic oxidation of H2O2. Indium tin oxide (ITO) based electrodes can possess good affinity to MnO2 particles and thus ensure better holding of the particles on its surface, owing to favorable interactions between ITO and metal oxide particles [START_REF] Liu | A Review on the Synthesis of Manganese Oxide Nanomaterials and Their Applications on Lithium-Ion Batteries[END_REF][START_REF] Taleat | Screen-printed electrodes for biosensing: a review (2008-2013[END_REF][START_REF] Arduini | Electrochemical biosensors based on nanomodified screen-printed electrodes: Recent applications in clinical analysis[END_REF][START_REF] Chekin | Direct and mediated electrochemistry of peroxidase and its electrocatalysis on a variety of screen-printed carbon electrodes: amperometric hydrogen peroxide and phenols biosensor[END_REF][START_REF] Yamanaka | Printable Electrochemical Biosensors: A Focus on Screen-Printed Electrodes and Their Application[END_REF]. Carbon electrodes might be also of interest because they offer relatively easy surface modification platforms for mediators [START_REF] Xu | A third-generation biosensor for hydrogen peroxide based on the immobilization of horseradish peroxidase on a disposable carbon nanotubes modified screen-printed electrode[END_REF].

The use of screen printed electrodes is important for the development of portable disposable sensors. It was demonstrated earlier that carbon screen printed electrodes modified with enzymes were successfully applied for the construction of amperometric biosensors for determination of choline [START_REF] Mazurenko | Amperometric Biosensor for Choline Based on Gold Screen-Printed Electrode Modified with Electrochemically Deposited Silica Biocomposite[END_REF]22], H2O2 and phenols [START_REF] Chekin | Direct and mediated electrochemistry of peroxidase and its electrocatalysis on a variety of screen-printed carbon electrodes: amperometric hydrogen peroxide and phenols biosensor[END_REF][START_REF] Xu | A third-generation biosensor for hydrogen peroxide based on the immobilization of horseradish peroxidase on a disposable carbon nanotubes modified screen-printed electrode[END_REF], L-ascorbic acid [START_REF] Turkusic | Amperometric sensor for L-ascorbic acid determination based on MnO2 bulk modified screen printed electrode[END_REF], glutamate [START_REF] Beyene | A Stable Glutamate Biosensor Based on MnO2 Bulk-modified Screen-printed Carbon Electrode and Nafion Film-immobilized Glutamate Oxidase[END_REF], biogenic amines [START_REF] Telsnig | Design of an Amperometric Biosensor for the Determination of Biogenic Amines Using Screen Printed Carbon Working Electrodes[END_REF] and glucose [START_REF] Abellán-Llobregat | A stretchable and screen-printed electrochemical sensor for glucose determination in human perspiration[END_REF]. The immobilization of nanostructured materials together with enzymes onto the surface of the screen printed electrodes is also a perspective for the development of third generation biosensors [START_REF] Arduini | Electrochemical biosensors based on nanomodified screen-printed electrodes: Recent applications in clinical analysis[END_REF][START_REF] Chekin | Direct and mediated electrochemistry of peroxidase and its electrocatalysis on a variety of screen-printed carbon electrodes: amperometric hydrogen peroxide and phenols biosensor[END_REF][START_REF] Yamanaka | Printable Electrochemical Biosensors: A Focus on Screen-Printed Electrodes and Their Application[END_REF].

In the present work we studied nanostructured screen printed carbon electrodes (nanoSPCE), which possess higher surface area compared to carbon screen printed electrodes described earlier.

Such electrodes can be perspective sensitive elements of amperometric sensors. The modification of nanoSPCE with MnO2 particles and their application for H2O2 determination was not yet investigated to our knowledge.

MnO2 particles can be synthesized in solution and then immobilized onto an electrode surface in the form of thin films using organic or inorganic binders [START_REF] Beyene | Bio)sensors based on manganese dioxide-modified carbon substrates: retrospections, further improvements and applications[END_REF][START_REF] Dontsova | Screen-printed carbon electrode for choline based on MnO2 nanoparticles and choline oxidase/polyelectrolyte layers[END_REF][START_REF] Liu | A Review on the Synthesis of Manganese Oxide Nanomaterials and Their Applications on Lithium-Ion Batteries[END_REF]. Beside this, MnO2 films can be directly electrodeposited onto the surface of the electrode from a solution containing Mn(II) species by applying a positive potential to the working electrode [START_REF] Guo | 3D Porous CNT/MnO2 Composite electrode for High-Performance Enzymeless Glucose Detection and Supercapacitor Application[END_REF][START_REF] Thiagarajan | Electrochemical Fabrication of Nano Manganese Oxide Modified Electrode for the Detection of H2O2[END_REF][START_REF] Yu | A facile and practical biosensor for choline based on manganese dioxide nanoparticles synthesized in-situ at the surface of electrode by one-step electrodeposition[END_REF]. Good control of the amount and size of the metal oxide particles on the electrode surface can be achieved by controlling the electrodeposition parameters (applied potential and deposition time).

On the other hand, sol-gel-derived silica films can be generated on solid electrodes by the electrochemically-assisted deposition (EAD) method, which is also a convenient procedure for the immobilization and holding of nanoparticles and enzymes onto electrode surfaces [START_REF] Chaudhuri | A Review on Porous Silicon Based Electrochemical Biosensors: Beyond Surface Area Enhancement Factor[END_REF][START_REF] Walcarius | Template-directed porous electrodes in electroanalysis[END_REF][START_REF] Walcarius | Exciting new directions in the intersection of functionalized sol-gel materials with electrochemistry[END_REF][START_REF] Owens | Knowles Sol-gel based materials for biomedical applications[END_REF][START_REF] Collinson | Electrodeposition of porous silicate films from ludox colloidal silica[END_REF][START_REF] Shacham | Electrodeposition of Methylated Sol-Gel Films on Conducting Surfaces[END_REF][START_REF] Liu | Electrochemical Deposition of Sol-Gel Films[END_REF][START_REF] Nadzhafova | Direct electrochemistry of hemoglobin and glucose oxidase in electrodeposited sol-gel silica thin films on glassy carbon[END_REF]. The method is easy to use, it is quick and it allows to control the thickness and surface characteristics of SiO2 on the electrode by adjusting the time and value of applied potential [START_REF] Nadzhafova | Direct electrochemistry of hemoglobin and glucose oxidase in electrodeposited sol-gel silica thin films on glassy carbon[END_REF][START_REF] Walcarius | Electrochemically assisted selfassembly of mesoporous silica thin films[END_REF][START_REF] Sibottier | Factors Affecting the Preparation and Properties of Electrodeposited Silica Thin Films Functionalized with Amine or Thiol Groups[END_REF][START_REF] Levy | The Sol-Gel Handbook -Synthesis, Characterization, and Applications, Synthesis and Processing[END_REF]. The EAD method allows precise modification of the target electrode because the catalyst is being generated only in close vicinity of negatively-polarized (working) electrode. Recently this procedure was successfully applied for the modification of screen printed planar electrode with biocomposite film silica-choline oxidase [22]. It was also used to generate extremely porous coatings by adding surfactants in the synthesis medium [START_REF] Walcarius | Template-directed porous electrodes in electroanalysis[END_REF][START_REF] Walcarius | Electrochemically assisted selfassembly of mesoporous silica thin films[END_REF][START_REF] Goux | Oriented mesoporous silica films obtained by electro-assisted self-assembly (EASA)[END_REF][START_REF] Etienne | Electrochemical approaches for the fabrication and/or characterization of pure and hybrid templated mesoporous oxide thin films: a review[END_REF][START_REF] Vila | Electrochemically Assisted Generation of Highly Ordered Azide-Functionalized Mesoporous Silica for Oriented Hybrid Films[END_REF], which can be otherwise applied as a useful matrix to encapsulate functional microparticles in a durable way (e.g., clays [START_REF] Maghear | Clay-mesoporous silica composite films generated by electro-assisted self-assembly[END_REF]).

The objective of the work is modification of the nanoSPCE and ITO electrodes with MnO2 particles using direct electrodeposition technique followed by their coating/embedding with a silica film generated by EAD method. The comparison of the electrocatalytic characteristics of obtained modified electrodes using H2O2 as a model substrate is then made in respect to their further application as sensitive elements of amperometric sensors.

Experimental section

Materials, Reagents and Apparatus

Tetraethoxysilane (TEOS, 98%) was purchased from Fluka (Switzerland).

Cetyltrimethylammonium bromide (CTAB) was provided by Sigma (USA). Phosphate buffer solutions (PBS) were prepared from Na2HPO4•2H2O and KH2PO4 (99.5%, Merck, Germany). All the other reagents were of analytical grade. All solutions were prepared using doubly distilled water. The nanoSPCE ensembles were made of a nanostructured carbon screen-printed working electrode, a carbon counter-electrode and an AgCl reference electrode, which were purchased from Orion Technology (OHT-000). The working electrode (WE) was obtained using carbon nanoparticles-based inks. The size of carbon nanoparticles constituting the electrode was in the 20-60 nm range according to the manufacturer. The diameter of the WE was 4 mm. For comparison purposes, a classical carbon screen printed electrode (SPCE, purchased from Ital Sens) was tested. In this case, the WE (3 mm in diameter) was obtained from carbon inks without particles size control.

Glass slides covered with an ITO film (1.1 mm thickness) were used as ITO electrodes (Sigma-Aldrich); a Pt wire and Ag/AgCl were used as counter and reference electrodes, respectively. All electrochemical experiments were conducted using either a model µStat400 bipotentiostat/galvanostat (DropSens, Spain) or a EmStat2 potentiostat (Palm-Sens, Netherlands). SEM measurements of MnO2 and ITO modified with MnO2 were carried out using the model JCM-6000 scanning electron microscope (JEOL).

Electrochemical deposition of MnO2 onto electrode surfaces

Synthesis of manganese dioxide particles was carried out by electrochemical deposition in accordance with the method described in [START_REF] Liu | A Review on the Synthesis of Manganese Oxide Nanomaterials and Their Applications on Lithium-Ion Batteries[END_REF]. For this purpose, ITO and nanoSPCE electrodes were immersed in a solution of 0.05M MnSO4 and 0.1M CH3COOK and a constant potential (typically between +0.6 and +1.0 V) was applied to the working electrode: the time of such potentiostatic electrodeposition was varied in the range of 10-240 s (see optimization detail at section 3.2). Obtained modified electrodes, respectively ITO-MnO2 and nanoSPCE-MnO2, were dried in an oven at 100 0 C for 5 hours and then kept at room temperature for 12 hours prior to use.

Modification of the electrodes with SiO2 film

The ITO-MnO2 and nanoSPCE-MnO2 electrodes were then modified with silica film using EAD method [START_REF] Nadzhafova | Direct electrochemistry of hemoglobin and glucose oxidase in electrodeposited sol-gel silica thin films on glassy carbon[END_REF][START_REF] Maghear | Clay-mesoporous silica composite films generated by electro-assisted self-assembly[END_REF]. The composition of the sol for deposition was adapted from the literature [22,[START_REF] Nadzhafova | Direct electrochemistry of hemoglobin and glucose oxidase in electrodeposited sol-gel silica thin films on glassy carbon[END_REF]: the acidic hydrolysis of tetraethoxysilane was performed by mixing TEOS, H2O and 0.01M HCl with the volume ratio 1.4 : 1:1.73 for 8 hours. Then a calculated amount of CTAB was added to obtain 1mM solution of surfactant in the final sol, 0.3 mL of 67 mM PBS (pH 7.0). The mixture was carefully mixed and 50 µL were dropped on the sensitive part of screen printed chip covering all three electrodes. The negative potential (E= -1.1 V) was applied to the working electrode for 15 s. In the case of modification of ITO electrodes they were dipped into the mixture of silica sol then the same potential of -1.1 V was applied for 15 s. After film formation, the electrodes were left for 1 min covered with sol, then carefully washed with water and dried at room temperature for 30 min. The obtained modified electrodes (ITO-MnO2-SiO2 and nanoSCPE-MnO2-SiO2) were kept at room temperature for 8 hours and washed with phosphate buffer pH-8.0 for 30 min prior to use. They were stored in desiccator if not in use.

Results and discussion

Electrochemical response of MnO2 modified electrodes toward H2O2.

Before investigating the effect of MnO2 on the cyclic voltammetric (CV) response of H2O2 (i.e., using nanoSPCE and ITO electrodes modified with MnO2 particles), some preliminary experiments were performed on bare screen-printed carbon electrodes (i.e., inks containing carbon nanoparticles or not) in order to select the most promising one for further experiments. The CV curves recorded in the absence and presence of hydrogen peroxide at the bare carbon screen printed electrode (CSPE) and at the nanostructured carbon screen printed electrode (nanoSPCE) were compared (Fig. 1a). Both background and oxidation currents of H2O2 are much higher at the nanostructured carbon electrode (curves 3&4), and the presence of H2O2 was even not noticeable on classical (i.e., not nanostructured) screen-printed electrode (curves 1&2 superimposed). The overpotential for H2O2 oxidation was much less at the nanoSPCE, indicating its interest for further modification with MnO2.

The typical CV response of the nanoSPCE after electrodeposition of MnO2 (nanoSPCE-MnO2) is presented in Fig. 1b, curve 1. The small signals observed in the buffer solution can be attributed to the Mn(IV) / Mn(III), Mn(II) redox systems in their oxide states, as described previously [START_REF] Beyene | Bio)sensors based on manganese dioxide-modified carbon substrates: retrospections, further improvements and applications[END_REF][START_REF] Zbilji | Determination of H2O2 by MnO2 modified screen printed carbon electrode during Fenton and visible light-assisted photo-Fenton based removal of acetamiprid from water[END_REF]. As no peak current was observed at unmodified nanoSPCE, these results confirm the presence of Manganese species on the surface of the nanoSPCE. Anodic current of nanoSPCE-MnO2 at E = 0.6 V noticeably increases in the presence of 1 . 10 -3 M H2O2 (Fig 1b ,curve 2). This is due to the electrocatalytic oxidation of H2O2 at nanoSPCE-MnO2 which was described earlier for the other types of the electrodes modified with MnO2 [START_REF] Beyene | Bio)sensors based on manganese dioxide-modified carbon substrates: retrospections, further improvements and applications[END_REF][START_REF] Dontsova | Hydrogen Peroxide-Sensitive Amperometric Sensor Based on Manganese Dioxide Nanoparticles[END_REF].

Please insert here Figs. 1a, 1b &2a, 2b A similar set of CV experiments has been performed with ITO electrodes, respectively before and after electrodeposition of MnO2, in the absence and presence of H2O2, as presented in Fig. 2. Two slightly visible oxidation-reduction peaks at E 0.4 and 0.3 V can be noticed in CV recorded in the background electrolyte at bare ITO (Fig 2a ,curve 1), possibly due to some impurities in solution. A well-defined oxidation peak at E = 0.7 V appeared in the presence of H2O2 in solution (Fig. 2a, curve 2). Much larger peak currents were observed when using the MnO2-modified ITO electrode either with or without H2O2 (Fig. 2b). The two well defined oxidation-reduction peaks observed at ITO-MnO2 (at +0.65 and +0.4 V, respectively) in phosphate buffer at pH 8.0 (Fig. 2b, curve 2) confirm the presence of MnO2 on the ITO surface and testify that MnO2 is electrochemically active. The oxidation current of ITO-MnO2 at E = 0.65 V noticeably increased in the presence of hydrogen peroxide while the reduction current was not changed (Fig. 2b, curve 2). As for nanoSPCE-MnO2, this is the result of the catalytic oxidation of H2O2 at ITO-MnO2, but the difference between the CV curves (peak current ratio) recorded in the background electrolyte and in the presence of 1 . 10 -3 M H2O2 is more clear at nanoSPCE-MnO2.

Optimization studies of MnO2 electrodepositon.

It was investigated the influence of the deposition potential, time and concentration of Mn(II) solution on the electrocatalytic properties of the studied electrodes. The oxidation current of H2O2 at nanoSPCE-MnO2 and ITO-MnO2 was used as analytical signal.

Effect of electrodeposition potential. The deposition potential of MnO2 at nanoSPCE and ITO electrodes was varied in the range of +0.6 to +1.0 V. The range of potentials is limited by the value of the formal redox potential of the (Mn (IV) / Mn (II) system (E = 0.72 V at pH 8.0) and the beginning of water decomposition at E = 1.0 V. The optimal electrodeposition potential of MnO2 on the both nanoSPCE and ITO electrodes was +0.8 V, leading to the highest analytical signal observed for H2O2 detection (please see supplementary material, Fig. 1S).

Effect of the modifier precursor concentration. The concentration of the MnSO4 solution used for electrodeposition process was varied in the range of 0.05 -0.5 M. The oxidation potential of H2O2 depended of the concentration of MnSO4, and increased from 0.65 to 0.8 V when passing from more diluted to more concentrated solutions. 0.05 M solution of MnSO4 was thus used further for electrode modification as it gave rise to the best electrocatalytic behavior (the oxidation potential of H2O2 was the lowest in this case, please see supplementary material, Fig. 2S).

Effect of electrodeposition time. The time of electrodeposition of MnO2 onto ITO electrodes was checked in the range of 10 -240 s. In agreement with previous observations [START_REF] Beyene | Bio)sensors based on manganese dioxide-modified carbon substrates: retrospections, further improvements and applications[END_REF], varying the the deposition time causes the formation of either MnO2 particles or films on the surface of the electrodes. Increasing the deposition time from 10 to 240 s resulted in an anodic shift in the oxidation potential of hydrogen peroxide, from 0.65 to 0.85 V (please see supplementary material, Fig. 3S).

In conclusion, the highest electrocatalytic current for H2O2 oxidation and the lowest potential values was observed for the electrodes modified with MnO2 particles generated from 0.05 M solution of MnSO4 (in 0.1M CH3COOK) at a deposition potential of E = +0.8 V and deposition times of 10 -60 s.

The SEM micrographs of ITO electrode modified with MnO2 from 0.05 M MnSO4 at potential E = +0.8 V for various deposition times are presented in Fig. 3. Uniformly distributed disaggregated particles are observed on the surface of ITO-MnO2 at a deposition time up to 60 s.

The diameter of the metal oxide particles was 200 ± 50 nm. For longer deposition times (i.e., 120 or 240 s) a MnO2 film is formed onto the surface instead of particles. It can be concluded that particles caused better electrocatalytic behavior compared to the film, indicating a significant role in the morphology of MnO2 deposits on their catalytic effect.

Please insert here Fig. 3a,3b, 3c, 3d

3.3.Effect of pH

The effect of pH on the oxidation peak potential of H2O2 at ITO-MnO2 and nanoSPCE-MnO2 electrodes was investigated (Fig. 4).

Please insert here Fig. 4 A decrease in the anodic peak potential values from 1.0 to 0.6 V with increasing pH of the solution (in the presence of H2O2) was observed at both electrodes. The sharp potential shift corresponding to lower overpotentials for hydrogen peroxide oxidation was observed in the pH ranges 5-8 and 6-7, respectively at ITO-MnO2 and nanoSPCE-MnO2 electrodes. This indicates that hydroxyl ions play an important role in the electrocatalytic detection of H2O2 at electrodes modified with MnO2. As the concentration of hydroxyl ions increases, the oxidation of manganese ions is going on easier. According to the literature [START_REF] Beyene | Bio)sensors based on manganese dioxide-modified carbon substrates: retrospections, further improvements and applications[END_REF], the scheme of the catalytic cycle of oxidation and reduction of hydrogen peroxide, can be assumed as follows:

(y + 2z)MnO2+ 2H2O2 = 2H2O + O2 + yMnO2 + zMn2O3 chemical reaction [START_REF]Biosensors: Fundamentals and applications[END_REF] 2MnO + Mn2O3 + 6OH -= 4MnO2 + 3H2O + 6e - electrochemical reaction (2)

Initially, a chemical reaction occurs with the oxidation of hydrogen peroxide and the reduction of manganese dioxide to the lower oxidation levels. Then electrochemical oxidation of Mn(II,III)

to Mn(IV) occurs at the electrode surface. Thus an alkaline medium is required to make the process easier. The optimum pH for H2O2detection starts at 7 for nanoSCPE-MnO2 while pH=8 is necessary to reach similar electrocatalytic effectiveness at ITO-MnO2 electrode. It can be assumed that the optimal pH value slightly depends on the properties of the working electrode. Phosphate buffer of pH 8.0 was used for the determination of H2O2 in the further studies. The effect of scan rate on the oxidation peak of 1 . 10 -3 M K3Fe(CN)6 was studied by cyclic voltammetry. The plot of peak current vs. square root of scan rate (ν 1/2 ) was linear over the whole studied range of scan the rate from 10 to 250 mV . s -1 for both nanoSPCE-MnO2 and ITO MnO2 electrodes (see supplementary material, Figs. 4S&5S). This indicates a typical diffusion-controlled behavior of the studied systems. The electron transfer rate constant (ks) and the transfer coefficient () were calculated using the Laviron equation [START_REF] Bard | Electrochemical Methods, Fundamentals and Applications[END_REF]. The results are presented in Table 1. The value is almost similar for all studied electrodes. The value of ks increased twice on nanoSPCE-MnO2 compared to ITO electrodes. This contributes to explain why MnO2 on the SPCE significantly promoted the electrocatalytic detectionof H2O2, as discussed above.

The effective surface area (A, сm 2 ) of the modified electrodes was determined using the Randles-Sevcik equation [START_REF] Bard | Electrochemical Methods, Fundamentals and Applications[END_REF]. Slight increase in the effective surface area of nanoSPCE-MnO2 compare to bare SPCE is observed. This feature was even more pronounced for ITO-MnO2 (i.e., almost 100 times increase compared to bare ITO), suggesting that MnO2 particles substantially contribute to the large surface area of ITO-MnO2 while the nanostructured carbon particles constituting the nanoSPCE electrode already possesses large surface area. Overall, the value of effective surface area of the SPCE-MnO2 was 10 times larger than of ITO-MnO2.

Please insert here Table 1.

Effect of SiO2 film

The ITO-MnO2 and nanoSPCE-MnO2 electrodes were covered with silica film using EAD technique. SEM image of the ITO-MnO2-SiO2 is presented in Fig. 5. The film is highlighted in light on the micrograph. The black part on the right is an edge of the film. The dark circles in the center can be ascribed to MnO2 particles.

Please insert here Fig. 5.

The stability of the analytical signal of H2O2 at the electrodes covered with SiO2 film was improved. The Both operational and long term stability of nanoSPCE-MnO2-SiO2 (i.e., the analytical signal of H2O2 at the electrodes covered with SiO2 film) was improved compared to nanoSPCE-MnO2. The intensity decrease of the analytical signal for 30 successive detection scans in 0.5 mM H2O2 was respectively 30% and 15% at nanoSPCE-MnO2 and at nanoSPCE-MnO2-SiO2 (see supplementary material, Fig. 6S). The analytical signal of H2O2 diminished by four times at nanoSPCE-MnO2 but only 20% at nanoSPCE-MnO2-SiO2 after one month of testing.

The characteristics of the calibration graphs for the determination of H2O2 by CV method using bare and MnO2-modified nanoSPCE and ITO electrodes, with and without silica film, are presented in the Table 2. The oxidation potentials of H2O2 are similar for the electrodes modified with MnO2 and MnO2-SiO2. The relative standard deviation (sr values) of the analytical signal of 5 . 10 -4 M H2O2 were found to be respectively 0.06 and 0.02 at nanoSPCE-MnO2 and nanoSPCE-MnO2-SiO2 electrodes (n=10, P=0.95).

Please insert here Table 2.

The lowest oxidation potential, widest linear range of calibration graph and lowest LOD for the determination of H2O2 is observed for the nanoSPCE-MnO2-SiO2. This indicates not only that silica does not prevent the electrocatalytic properties of the MnO2-modified electrodes, but also contributes to improve somewhat their performance, which could be related to the fast mass transport properties featured by such mesoporous silica films [START_REF] Goux | Oriented mesoporous silica films obtained by electro-assisted self-assembly (EASA)[END_REF][START_REF] Etienne | Electrochemical approaches for the fabrication and/or characterization of pure and hybrid templated mesoporous oxide thin films: a review[END_REF].

Interference studies

The possible interference effect of ascorbic acid on the analytical signal of H2O2 at the electrodes modified with MnO2 was investigated. This compound is indeed present in biological fluids, together with the target analyte, and can influence the results of its amperometric determination as it is expected to be oxidized in similar potential region. Equimolar amounts of ascorbic acid interfere on H2O2 detection at the bare electrodes, especially at ITO (Fig. 6a). Such deleterious effect of ascorbic acid was substantially less at the electrodes modified with MnO2 (Fig. 6c). It is interesting to note that the oxidation peak of ascorbic acid decreased with increasing scan rate from 25 to 100 mV . s -1 at the ITO-MnO2 while the oxidation current of H2O2

was not noticeably changed (Fig. 6, compare a&b and c&d).

Please insert here Fig. 6 a,b,c,d It can be a result of the differences in the kinetics of the oxidation of the studied compounds at metal oxide particles. The difference between oxidation current of H2O2 and ascorbic acid was more important at a scan rate of 100 mV . s -1 . The hydrogen peroxide can be determined in the presence of equimolar amounts of ascorbic acid at both nanoSPCE-MnO2-SiO2 and ITO-MnO2-SiO2 electrodes. (2) at bare ITO electrode. Other conditions as in Fig. 1a.

Conclusions

3. 4 .

 4 Electrochemical characteristics of nanoSPCЕ, nanoSPCE-MnO2, ITO and ITO-MnO2 electrodes The cyclic voltammetric response of another redox probe (1 . 10 -3 M K3Fe(CN)6) was used to evaluate the electrochemical characteristics of the nano SPCЕ, nanoSPCE-MnO2, ITO and ITO-MnO2 electrodes. It can help to compare the effect of MnO2 particles on the electrochemical characteristics of the studied electrodes.

  Nanostructured screen printed carbon (nanoSPCE) and ITO electrodes modified with MnO2 by an electrodeposition method have demonstrated good electrocatalytic properties in the presence of H2O2. The uniformly distributed particles of MnO2 obtained under optimal conditions provided high electrocatalytic currents for H2O2 at nanoSPCE-MnO2 and ITO-MnO2 electrodes. Larger effective surface areas and faster charge transfer rates were observed at the nanoSPCE-MnO2 electrode. This latter was characterized by lower LOD and wider linear range of the calibration curve for the determination of H2O2 compared to ITO-MnO2 electrode. The modification of MnO2-functionalized electrodes with a mesoporous silica film contributed to provide better stability of the analytical response of the studied electrodes. NanoSPCE-MnO2-SiO2 is perspective element of amperometric sensor for the detection of hydrogen peroxide in the presence of equimolar amounts of ascorbic acid.

  Figure caption

Fig. 1b .

 1b Fig.1b. CV curves recorded in background electrolyte[START_REF]Biosensors: Fundamentals and applications[END_REF] and in the presence of 1 . 10 -3 М H2O2[START_REF] Evtugyn | Biosensors: Essentials[END_REF]

Fig. 2a .

 2a Fig.2a. CV curves recorded in the background electrolyte[START_REF]Biosensors: Fundamentals and applications[END_REF] and in the presence of 1 . 10 -3 М H2O2

Fig. 2b .

 2b Fig. 2b. CV curves recorded in the background electrolyte (1) and in the presence of 1 . 10 -3 М

Fig. 3b .

 3b Fig. 3b. SEM image of ITO-MnO2 electrode surface; electrodeposition time 60 s,

Fig. 3c .

 3c Fig. 3c. SEM image of ITO-MnO2 electrode surface; electrodeposition time 120 s,

Fig. 3d .Fig. 4 .

 3d4 Fig. 3d. SEM image of ITO-MnO2 electrode surface; electrodeposition time 240 s,

Fig. 5 .

 5 Fig. 5. SEM image of ITO-MnO2-SiO2 electrode surface.

Fig. 6 a

 6 Fig. 6 a,b. CV curves recorded in the background electrolyte (1) and in the presence of 1 . 10 -3 M

Fig. 6 c

 6 Fig. 6 c,d. CV curves recorded in the background electrolyte (1) and in the presence of 1 . 10 -3 M

Table 1 .

 1 Electrochemical characteristics of non modified and modified with MnO2 ITO and nanoSPCE electrodes, n=3.

	Electrode	α	ks, s -1 ± 0.05	A, сm 2
	ITO	0.89	1.15	0.03
	ITO-MnO2	0.89	1.16	2.70
	nanoSPCE	0.86	2.50	15.30
	nanoSPCE-MnO2	0.85	2.20	20.50
	ks -charge transfer rate constant, α -charge transfer coefficient, A -effective surface area.

Table 2 .

 2 The characteristics of the calibration graphs for the determination of H2O2 using different

	types of the electrodes
	Electrode type	Eox (V)*	Linear range,	LOD (3s criteria),
			mM	mM
	ІТО	0.65	2-11	1.8
	ІТО-MnO2
	ІТО-MnO2-SiO2

Acknowledgement

One of the authors (O.T.) acknowledges a guest professorship fellow from the Université de Lorraine (France).


Mamta Yadav

Vellaichamy Ganesan

Biswajit Maiti
email: bmaiti@bhu.ac.in

Rupali Gupta

Piyush Kumar Sonkar

Dharmendra Kumar

Alain Walcarius
email: alain.walcarius@univ-lorraine.fr

Sensitive determination of acetaminophen in the presence of dopamine and pyridoxine facilitated by their extent of interaction with single walled carbon nanotubes

Keywords: Acetaminophen oxidation, Dopamine oxidation, Pyridoxine oxidation, Electrocatalysis, Single walled carbon nanotubes, DFT calculations

Single walled carbon nanotubes (SWCNTs) were immobilized on glassy carbon (GC) electrode by drop casting The resulting modified electrode (represented as GC/SWCNTs) efficiently oxidizes acetaminophen (AC), dopamine (DA) and pyridoxine (PY) by decreasing the respective oxidation potentials and increasing peak currents in comparison to bare GC electrode. The extent of lowering overpotentials is in the order of AC > PY > DA, in agreement with the order of decrease in the HOMO-LUMO energy gap (E) of these analytes, as determined from Density Functional Theory (DFT) calculations. DFT calculations further reveal that due to the adsorption of the analytes on the SWCNT(10,10) there is a negative charge density transfer (higher probability of electron transfer, lower E value) to the frontier molecular orbitals of the analytes, which eases their oxidation. Since AC, DA and PY oxidize distinctly at distinct potential values, the present SWCNTs modified electrodes could be used to simultaneously determine them. Cyclic voltammetry, differential pulse voltammetry and amperometry techniques are utilized to understand the electrochemical characteristics of the analytes (AC, DA and PY) and subsequent sensing of them at the GC/SWCNTs electrode. The electrode is then applied to the determination of Ac as a case study. Sensitivity, detection limit and linear calibration range for the AC are found to be 7.9 µA µM -1 cm -2 , 1.1 µM and 2.0-100.0 µM, respectively.

Introduction

Acetaminophen (AC) is a common antipyretic and analgesic drug which is mainly employed to decrease fever and to relieve pains associated with muscular, arthritis, post-operation, toothache, headache, backache, etc. [1][2][3][4]. In case of contraindication to aspirin, for instance, AC is considered as the best alternative for patients. Small dosages of AC generally do not cause any harmful side-effects; however over-dosage may lead to kidney and liver damages [3]. The long term use of AC may have fatal neurotoxic and hepatotoxic effects [2]. Thus, it is essential to get an accurate, easy to handle, fast and sensitive analytical technique for the frequent determination of AC [2]. Dopamine (DA) is a neurotransmitter which exists in central and peripheral nervous systems of mammals [1,5,6]. Low concentration levels of DA in human body generally lead to Parkinson's disease [1]. DA is supplied as medication to such patients, however excess dosage may cause increased heart beat and blood pressure because of its effect on sympathetic nervous system [1]. Therefore, regular monitoring of DA levels in human body is necessary [7]. Pyridoxine (PY), otherwise known as vitamin B6 is a water soluble vitamin which plays significant role in body cells maintenance and in amino acids metabolism [8][9][10]. Three forms of PY are available which are known as, pyridoxal, pyridoxol and pyridoxamine [8][9][10]. PY is also necessary for physical and mental growth of the individual [11]. It is reported that vitamin B6 imposes major effects on gene modulation, sexual behaviour, and immune modulation in HIV-1 infection [9]. Insufficient concentration level of PY in body leads to illness and various sicknesses such as hypertension, cardiovascular problems, hyperlipidemia, obesity and anaemia [8,10,11]. Hence, accurate PY determination is essential in human health diagnostics. Due to their complementary features/properties (nutrient, biologically important molecule, or drug), these compounds have been used as representative analytes in the context of the present study.

Variety of methods which include HPLC, flow injection analysis, liquid chromatography, etc.

were reported for the sensing of the above analytes [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18]. However, they can suffer from low selectivity, poor detection limit, tedious sample preparation procedure, high cost, etc.

Electrochemical methods can offer several advantages like low detection limit, high high sensitivity and good selectivity if the electrode surface is modified with suitable catalytic materials. The electrode modification requires only small amount of additional materials and the resulting electrodes can decrease the electrode fouling effects and detect the target analytes at low overpotentials [19]. Recently, carbon based nanomaterials enticed the electrochemists extensively due to efficient promotion of electron transfer events on their surfaces [20][21][22][23][24][25][26][27][28]. Carbon nanotubes (CNTs) are attractive nanomaterials which possess several interesting properties from the electroanalytical point of view, including high conductivity, durable mechanical stability and high surface area [20][21][22][23][24]. These unique properties of CNTs are exploited in electrochemical science and technology to immobilize electrocatalysts and to increase the electrical communication within the immobilized molecules [20][21][22][23][24]. For example, increased oxidation currents are reported for cysteine, glucose, DA, epinephrine and ascorbic acid detection thanks to presence of CNTs on electrodes [22,24,29]. These huge improvements facilitated by the CNTs may be attributed to specific interactions of CNTs with the target analytes through - interaction and/or Van der Waals forces, or the presence of edge-plane-like defect sites [30][31][32][33][34]. In addition to these interactions, depending upon the functional groups of the analytes (phenolic, hydroxyl, amine and -NHCOCH3 groups) they may have specific interactions to CNTs. Though numerous CNTs based composite materials are described to be beneficial in electrochemical sensing [20][21][22][23][24][30][31][32][33], it should be noted here that the influence of CNTs alone on the electrochemical sensing process is not so studied in detail so far [1,17,29,[35][36][37][38]. Hence, in this study a simple, sensitive and cost effective electrochemical route for the efficient quantification of AC, DA and PY based on single walled CNTs immobilized glassy carbon electrodes is reported. The originality not only relies on the simultaneous analysis of these three analytes, but also on the use of additional theoretical calculations carried out to confirm the results obtained from electrochemical studies, which remains underexplored to date [39][40][41]. This work is expected to strengthen the existing knowledge on the adaptability of CNTs for the electroanalysis of biologically-relevant molecules.

Experimental

Reagents and chemicals

Single walled carbon nanotubes (SWCNTs), AC and DA were obtained from Sigma-Aldrich.

Phosphate buffer of pH 7.0 (0.1 M) was prepared by mixing appropriate amounts of K2HPO4 and KH2PO4 (Qualigens, Mumbai, India) stock solutions. N, N-dimethyl formamide (DMF) and PY were purchased from S.D. Fine Chemicals, India. All chemicals were used as received and all solutions were prepared from triple distilled water.

Instrumental details

All electrochemical experiments have been performed using the CH Instruments' electrochemical workstation (model: CHI-660C), along with a three electrodes-single compartment cell system. Bare glassy carbon (GC) electrode or modified GC was used as working electrodes. A Pt wire and KCl saturated calomel electrode were employed as counter and reference electrodes, respectively. Electrochemical studies were carried out at room temperature in 0.1 M phosphate buffer (pH 7.0) unless otherwise mentioned. Prior to each electrochemical analysis, nitrogen gas was purged into the supporting electrolyte solution for 20 min to remove the dissolved oxygen.

Modification of working electrode

GC electrode was cleaned by rubbing it over the Buehler-felt pad with alumina slurry followed by ultra-sonication in triple distilled water. Colloid of SWCNTs was prepared in DMF (1.0%) by ultra-sonicating the mixture for 20 min. A 10 µL of the colloid was dropped over the clean GC electrode and dried for 12 h (represented as GC/SWCNTs).

Theoretical calculations

To understand the interaction between the SWCNTs and AC, DA and PY at the molecular level and hence to find out the change in oxidation properties of these molecules after adsorption onto SWCNTs, quantum chemical calculations were done based on the density functional theory (DFT, M06-2X/6-31G(d,p)). Gaussian 09 suits of program were used for the calculations [42]. For the SWCNT model, zigzag (10,10) nanotube was used (represented as SWCNT (10,10)). It contains five aromatic-ring chains and length of this SWCNT model can hold the target analytes (AC, DA and PY) well within the SWCNT structure.

3 Results and discussion 3.1 Cyclic voltammetry characterization of AC, DA, and PY at GC and GC/SWCNTs electrodes Fig. 1 shows typical cyclic voltammograms (CV) recorded at a scan rate of 25 mV s -1 using GC and GC/SWCNTs electrodes for the oxidation of AC, DA, and PY in 0.1 M pH 7.0 phosphate buffer. CV responses clearly manifest the fact that both GC and GC/SWCNTs provide a potential window wide enough to examine the electrochemical behaviour of these target analytes. The CV responses of 0.2 mM AC at bare GC and GC/SWCNTs electrodes are shown in Fig. 1a, corresponding to a well-established redox process (Eq. 1) [43].

Compared to the quasi-reversible signal for AC on bare GC electrode (with an anodic peak occurring at 510 mV), the use of GC/SWCNTs electrode results in lower overpotential for AC oxidation (anodic peak potential at 386 mV) and currents larger by more than one order of magnitude. The decrease in oxidation potential (124 mV) and huge increase in peak current (34 times) at GC/SWCNTs electrode indicate the efficient electrocatalytic effect exhibited by SWCNTs towards the oxidation of AC. During the reverse scan, it exhibits a reduction peak at 330 mV which may be ascribed as per the Eq. 1.

DA exhibits an oxidation peak around 245 mV at bare GC, while it shows a decreased oxidation peak potential (190 mV) with higher current at GC/SWCNTs (Fig. 1b), due to the oxidation of DA according to the process reminded at Eq. 2 [44]. The noticeable shift in the oxidation peak potential (55 mV) towards the less positive values with higher current (17 times) at GC/SWCNTs indicate confirm the electrocatalytic properties of SWCNTs towards DA oxidation [35]. The oxidized DA is reduced back in the reverse scan at 160 mV. The anodic to cathodic peak-to-peak separation of 30 mV is in good agreement with a fast electron transfer process. Fig. 3 shows CV response for the oxidation of AC, DA, and PY at various potential scan rates, as recorded using the GC/SWCNTs electrode. On increasing the scan rate, an increase in the currents is observed (Fig. 3a-3c), with anodic peak currents being directly proportional to the square root of the scan rate (see insets in Fig. 3a-3c), indicating redox processes controlled by diffusion.

Fig. 4 shows the simultaneous oxidation of AC, DA, and PY at bare GC and GC/SWCNTs electrodes. Three distinct peaks for the oxidation of AC, DA, and PY are obtained at the GC/SWCNTs electrode at the oxidation peak potentials 374, 195 and 705 mV, respectively.

These oxidation peak potentials are matching well with the oxidation peak potentials of individual oxidation of the respective analytes. These observations specify that the efficiency of individual analyte oxidation process is not affected by the presence of other components in the system. Since the peak potential difference between the individual anodic peaks are distinctly high (c.a., 180 mV between DA and AC and 330 mV between AC and PY), it is expected to carry out the simultaneous determination of these analytes in mixture. At bare GC electrode, for a mixture of AC, DA and PY, only two oxidation peaks are observed with quite low currents and overlapping considerably (see inset on Fig. 4). Thus, bare GC electrode is not appropriate for the quantification of the analytes in presence of each other. 5a), DA (5-750 µM range, see Fig. 5b), and PY (50-1000 µM range, see Fig. 5c). In all cases, when the analyte concentration is incrementally increased there is a regular enhancement in the oxidation peak currents. One can also see from the corresponding calibration plots (insets on Fig. 5) that variations are linear for all the analytes (with an r values of more than 0.99). These plots enable estimating sensitivity values of 7.86 µA µM -1 cm -2 , 3.66 µA µM -1 cm -2 and 0.31 µA µM -1 cm -2 , respectively for AC, DA and PY. DPV is also employed for the quantification of AC in the presence of DA and PY (Fig. 6).

The concentration of DA and PY is kept constant (0.1 mM each) and the concentration of AC is increased incrementally. As the concentration of AC is increased in the 10-500 µM range, the DPV current response corresponding to its oxidation also increases regularly and no change in the current due to the oxidation of DA and PY is observed (Fig. 6). The inset in the Fig. 6 shows a linear calibration plot with an r value of 0.98. Comparison of electroanalytical data for the present method along with previously reported methods for the electrochemical oxidation and determination of AC.

[c] Cu-poly-TTCA = copper terthiophene carboxylic acid polymer; GCE/Nafion/RuO = Nafion@/ruthenium oxide pyrochlore modified glassy carbon electrode; Nano-TiO2/poly(acid yellow 9) = poly(Acid Yellow 9) coated nano-TiO2 modified GC electrodes; FeTPyPz/GCE = glassy carbon electrode surface with a Nafion membrane doped with iron tetrapyridinoporphyrazine; GCE/NiHCF-Bt = Nickel hexacyanoferrate incorporated bentonite modified glassy carbon electrode.

Modified electrode [c] The above data show that the GC/SWCNTs electrode looks promising for the sensitive and selective determination of AC and this is confirmed by Table 1 giving a comparison of the analytical parameters for the determination AC at the GC/SWCNTs together with other reported electrochemical methods [12][13][14][15][16][17]46]. As compared to the reported methods [12][13][14][15][16][17]46], the present method shows low LOD, high sensitivity and low oxidation potential for the AC determination. Careful comparison of the parameters based on the data in Table 1 infers that the present method is superior over many other methods and therefore this route can be utilized for the selective quantitative recognition of AC even in the presence of DA and PY.

The superior performance of the present electrode is mainly due to the different degree of interaction of SWCNTs, with the target analytes. Among the three analytes, AC interacts more with the SWCNTs probably due to the -NHCOCH3 group of AC [29,33,47].

In addition to DPV, the determination of the above analytes can be also performed by amperometry, as exemplified hereafter in the case of AC electroanalysis. Amperometry is an analytical technique in which a constant potential is applied to the working electrode and the resulting current is continuously monitored with addition of target analyte to the stirred supporting electrolyte at certain intervals of time. This approach is well suited to interference studies as it enables the alternative addition of either the analyte or the interference, for instance.

Selectivity of GC/SWCNTs on the determination of AC is evaluated by interference analysis.

Supporting information, Figure S1 shows the amperometric determination of AC together with other potential interferent species. Accordingly in the present study, a potential of 0.25

V is applied to the GC/SWCNTs electrode and after 300 s, calculated amount of AC or possible interferents are injected to the supporting electrolyte (0.1 M pH 7.0 phosphate buffer). Whenever AC is injected, an immediate increase in current is observed (Supporting information, Figure S1) due to the oxidation of AC. Conversely, whenever the possibly interfering species, i.e., alanine, glycine, glucose, uric acid and tyrosine (0.1 mM each) are injected, no change in the amperometric response is observed. Thus, the decreased AC oxidation potential avoids the interference from several oxidizable co-existing molecules, which can be useful in the determination of AC in real samples.

The reproducibility of the AC determination at the GC/SWCNTs electrode is tested by recording the DPV response (ten times at the interval of one minute, see Supporting information, Figure S2) of the oxidation of 0.1 mM AC. The oxidation peak current for AC shows a relative standard deviation (RSD) of 3.8%, signifying the high reproducibility of the determination. The stability (Supporting information, Figure S3) of GC/SWCNTs is established by keeping the electrode under room conditions and recording the CV response for 0.1 mM AC determination on the 3 rd and 5 th days. The CV response retains 96.5% and 87.6% of the current respectively, as that of the initial response observed on the 1 st day indicating satisfactory storage stability.

To understand the practicability of the method for real sample analysis the response of GC/SWCNTs electrode towards the determination of AC present in a pharmaceutical tablet, DOLO (a product of Micro Labs Ltd., South Sikkim, India) is estimated. The tablet is carefully crushed into powder and solubilized in a mixture of water and ethanol (70:30). The DPV is recorded and the amount of AC is determined with the previously constructed calibration graph. In addition, the tablet solution is added with known amounts of standard AC and the recovery percentage is calculated. The results of the recovery analyses are shown in supporting information, Table S1. The average recovery was calculated to be 105.8%

which is typical for such analysis. The RSD value for three measurements varies from 2.4 to 8.9% which indicate the acceptable reliability of this method.

Conclusions

This work demonstrates the electrochemical quantification of AC, DA and PY using GC/SWCNTs modified electrode. It oxidizes AC, DA and PY at distinctly different potentials with decreased overpotentials due to the different degree of interaction with the SWCNTs. It also shows higher currents for the oxidation which is expected to increase the sensitivity in contrast to bare GC electrode. DFT calculations predict the order of interaction of the target analytes with the SWCNTs as AC > PY > DA which is in accordance with the electrochemical observations. This efficient electrocatalytic oxidation property of SWCNTs leads to the sensitive determination of AC, DA and PY. Interference studies demonstrate that this electrode is appropriate for the analysis of AC in samples of simple matrixes. The sensitivity (7.9 µA µM -1 cm -2 ), LOD (1.1 µM) and linear calibration range (2.0-100.0 µM) obtained in this study are superior to many other methods reported earlier for the determination of AC.

Fig. 1 .

 1 Fig. 1. CV curves recorded at GC/SWCNTs electrode in absence (dashed line) and presence (solid line) of 0.2 mM AC (a), 0.2 mM DA (b) and

Fig. 2 .Fig. 3 .

 23 Fig. 2. Isosurfaces for molecular electrostatic potential of individual analytes and their adsorption complexes with SWCNT(10,10). The negative

Fig. 4 .

 4 Fig. 4. CV responses recorded at GC/SWCNTs electrode in absence (a) and presence (b) of a

15 Fig. 5 .Fig. 6 .

 1556 Fig. 5. DPV curves at GC/SWCNTs for various concentrations of AC (a, 2.0-100.0 µM), DA (b, 5.0-750.0 µM) and PY (c, 50.0-1000.0 µM) in

Table 1

 1

Acknowledgements

Generous financial support from DST-ASEAN (IMRC/AISTDF/R&D/P-16/2018) program is gratefully acknowledged. We thank Prof. S. A. John, Gandhigram Rural Institute, India for useful suggestions.


Christophe Marcel Trouillefou

Yann-Seing Law-Kam Cio

Mario Jolicoeur

Bilel Said

Anne Galarneau

Sofiane Achiche

Giovanni Beltrame

An autonomous plant growing miniaturized incubator for a Cubesat

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


Tauqir Nasir

Natalya A Vodolazkaya

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

Alain Walcarius
email: alain.walcarius@univ-lorraine.fr

Critical Effect of Film Thickness on Preconcentration Electroanalysis with Oriented Mesoporous Silica Modified Electrodes

Keywords: Mesoporous silica, Thin film, Voltammetric detection, Paraquat, Sensitivity

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Thin films made of nanostructured and highly porous metal oxides exhibiting well-ordered mesochannels of monodisperse size are very attractive for applications in many fields, such as dyesensitized solar cells [1], energy storage [2], electrochromism [3], photocatalysis [START_REF] Matsuoka | Anpo in Photocatalysis and Water Purification[END_REF], gas sensors [START_REF] Hyodo | [END_REF], or electrochemistry [6]. Ordered mesoporous materials are also of outstanding interest for sensing and biosensing [7]. Periodically organized nanoporous thin films can be obtained via surfactant templated growth of inorganic or hybrid polymers [8], but an important point governing their performance relies on nanoscale control of compositions and mesochannel orientation [9], to ensure the accessibility of mesoporous thin films from surfaces [10]. An effective way to reach this goal is via the vertical orientation of mesopore channels [11]. Nanostructured thin films with vertically aligned mesochannels are very attractive for electroanalysis [12] because this pore configuration enables fast mass transfer processes from the solution to the electrode surface (as easily characterized by electrochemical methods [13]). Such films can be prepared by electroassisted self-assembly (EASA) [11a,14] or by the Stöber solution growth process [11b], which are the two most versatile methods available to date to prepare vertically oriented mesoporous silica films on electrodes giving rise to well-defined electrochemical characteristics [15]. Good adhesion to the support is ensured for films generated as such onto electrode surfaces bearing surface hydroxyl groups, whereas using "molecular glue" is mandatory for other electrode substrates (such as electrografting of 3-aminopropyltriethoxysilane on glassy carbon [16]) prior to film deposition.

Electroanalytical interests have been reported for surfactant-containing, template-extracted, organically-functionalized, and free-standing oriented mesoporous silica thin films [12]. Surfactantcontaining films have been used as permselective sensors for lipophilic analytes (e.g., pharmaceutical drugs [17] or trace nitroaromatic explosives and pesticides [18]) based on their extraction/dissolving in the surfactant phase and subsequent detection onto the electrode surface while rejecting ionic interferences [19]. Applications of surfactant-extracted films include preconcentration electroanalysis of redox cations (via electrostatic binding to the negatively charged silica surface) [20,21], permselective coatings (based on charge selectivity [22], hydrophobic/ hydrophilic balance [23] or gated molecular transport [24]), or molecular sieving membranes [25] which are also likely to be exploited as anti-fouling surfaces [26]. Oriented mesoporous silica films bearing organo-functional groups can be also manufactured [27]. They have been exploited in electroanalysis based on preferential accumulation/detection of target species via complexation [28] or electrostatic binding [29], as electrochemical bioassays [30], or as electrocatalytic sensors [31].

Electrochemiluminescence sensors have been also described [32,33]. Finally, free-standing mesoporous silica membranes can be used for molecular filtration [34]. In a previous work [21], we have shown that perpendicularly oriented mesoporous silica thin films electrogenerated by EASA on glassy carbon electrode can be successfully applied to the preconcentration electroanalysis of paraquat, a cationic 4,4'-bipyridinium-based pesticide likely to bind quickly to the negatively charged silica surface. Here we discuss the critical effect of film thickness of the sensor's sensitivity, on the basis of deposits obtained after distinct synthesis durations.

Figure 1 shows typical cyclic voltammograms recorded in 0.5 mM Ru(NH3)6 3+ using glassy carbon electrodes (GCE) covered with mesoporous silica films prepared by EASA at increasing synthesis times, respectively before (Fig. 1A) and after surfactant extraction (Fig. 1B). This cationic redox probe has been selected as this is the only one likely to distinguish the coating uniformity (neutral probes can dissolve in the surfactant phase and negative probes suffer from restricted diffusion through the film [35]). Part C of the figure represents the relative variations of peak current differences between bare and modified electrodes as a function of film deposition time. Prior to template removal, and as far as thin films are produced as uniform deposits over the whole electrode surface area, no signal for the redox probe is expected to be seen [11a,12a,25]. Fig. 1A shows that it is indeed the case for the films obtained after sufficient synthesis time (> 16 s) whereas significant defects or incomplete electrode coverage was observed for deposition times shorter than 12 s (see black curve on Fig. 1C). After template removal, all modified electrodes gave rise to significant response of the probe (Fig. 1B), even for the films covering uniformly the electrode surface thanks to their open mesoporosity, yet with some decrease in intensity for the much thicker deposits (see red curve on Fig. 1C) which can be explained by increased resistance to mass transport through the film. The presence of particles/aggregates over thicker films, i.e., those formed at longer deposition times, as previously reported [11a,14] and confirmed by scanning electron microscopy (Fig. SI 1), might also contribute to explain slower transport rates. Figure 1 Profilometry measurements were then carried out to estimate film thicknesses (Table 1). Due to the important roughness of the GCE surface, thinner films were not analysable by the technique (wellknown limitation [36]), but other data gave rise to a variation expected from previous studies, with thickness values in the sub-micrometric range, increasing with the deposition time, and characterized by high dispersion for the thicker ones as a result of the presence of particles/aggregates [14]. To confirm the observed trend, similar series of experiments have been performed with films deposited on ITO (flatter than GCE) and comparable variations were observed in both cases (Table 1). Note that the absence of exploitable profilometry signal for deposits generated at short synthesis time can also be due to incomplete coverage of the electrode surface, in agreement with permeability data obtained by cyclic voltammetry, as discussed above (Fig. 1). [a] for comparison purposes, as films have been prepared differently on GCE and ITO (respectively by galvanostatic and potentiostatic EASA), and deposition charges (Dep. charge) are given as charge densities (i.e., divided by the electrode surface area). [b] non measurable.

The film modified electrodes were then used to discuss the influence of deposition time on paraquat detection by square wave voltammetry (SWV). Typical results are depicted on Figure 2 for various paraquat concentrations in the µM range. No noticeable difference can be seen when passing from bare GCE to a film generated for 5 s (compare parts A1 & A2 of Fig. 2), suggesting that no (or very little) silica has been deposited. Then, the films prepared at longer deposition times led to proportional increases in sensitivity (see illustrative raw data in parts A3 & A4 and calibration curves in part B of Fig. 2). This confirms the accumulation ability of the mesoporous silica film towards cationic paraquat species and indicates most effective accumulation behavior when increasing the film thickness (due to more available binding sites, i.e., silanolate groups, onto the electrode surface). In parallel, a negative shift in peak potentials (by ca. 100 mV with respect to the bare GCE) was observed, as a consequence of the interactions between the analyte and the film. The optimum sensitivity (2.1 A cm -2 M -1 ) was obtained for the film deposited for 16 s (corresponding to ca. 120 nm thickness), as determined from calibration curve in the 0.5 to 10 µM concentration range (Fig. 2B). In this case, a preconcentration factor of almost 30 can be achieved with respect to bare ITO. For thicker films, however, a gap to the ideal linear variation was observed, the calibration curves becoming S-shaped (Fig. 2B). A possible explanation for lower response than expected at low paraquat concentration when using thicker films is that the few quantity of accumulated paraquat is mostly located in the upper part of the film (i.e., close to the solution side where preconcentration in the film started to occur) and thus less available to be detected at the electrode surface. An additional observation supporting this hypothesis is the fact that this limiting effect was more pronounced with the thickest film (compare the responses of films 20 & 30 s in Fig. 2B). At larger analyte concentrations, the electrode response became again more or less linear but rapidly tended to level off. Such a behavior can be explained by stronger resistance to mass transport through thicker films (slower diffusion rates), as also supported by permeability measurements discussed above (see red curve in Fig. 1C). Note that thick films contain indeed a large amount of binding sites (as ascertained by peak current increase when performing multiple cyclic voltammetric scans, see Fig. 3), but they cannot be exploited for single SWV measurements.

From these cyclic voltammetry data, one can also confirm the strong interactions between paraquat and the mesoporous silica film (progressive shift of peak potentials towards more negative values)

and the real accumulation of the probe into the film (decrease in peak potential difference from 60 mV for the first scan to ca. 40 mV for the 40 th scan, due to progressive shift of diffusion-controlled processes to thin layer behavior). These results indicates that when performing preconcentration electroanalysis with porous film electrodes, a compromise has to be found for getting the optimal sensitivity: the film should be thick enough to offer a high number of accumulation sites, but not too thick to avoid limitations for trace analysis (analyte binding far away from the sensing electrode surface) and a levelling off in the modified electrode response (due to restricted mass transport through thick films). From the sensor point of view, since the recognition/preconcentration is based just on the binding of cationic species to the negatively charged silica surface, the selectivity of this approach in view of practical applicability in samples where other cations may coexist can be questionable but we have shown in a previous work that analysis of real water samples (i.e., river water) by the standard addition method is indeed possible [21]. The electrodes can be used for more than 20 successive measurements on the same day without cleaning but suffer from poor long-term stability beyond that period due to the well-known degradation of silica gel materials over prolonged use in aqueous media. Good reproducibility values were found for both intra-electrode (99.1 %, n=9) and inter-electrode (95.3%, n=3) measurements. In order to further confirm this behavior, we have performed additional experiments using the optimal 16 s film, but in the form of monolayer, bilayer and trilayer configurations (it is indeed possible to get multi-layered mesoporous silica films by EASA [37]). The results (Fig. SI 2A)

indicate that almost no improvement can be obtained when passing from the mono-to the bilayer film, showing the same type of limitations as discussed above, and the situation was even worse with the trilayer film, suffering from stronger diffusional restrictions. Again, the limitation in the SWV was of kinetic kind, as multisweep cyclic voltammetry revealed that the number of binding sites was indeed increased when passing from the mono-to the bi-and trilayer films (see the 100 th cycles obtained for these three films in Fig. SI 2B).

In summary, the above results indicate clearly that increasing the thickness of mesoporous silica films resulted in significant increase in the number of binding sites present at the electrode surface, which can indeed contribute to dramatically increase its sensitivity when applied to preconcentration electroanalysis, but too thick films have to be avoided because they induce mass transport restrictions (accumulation too far from the sensing electrode surface in case of trace analysis, or signal levelling off effects).

Experimental

The origin and purity of chemicals used here, as well as the apparatus characteristics, have been detailed in Supporting Information. Glassy carbon plates (SIGRADUR G, 20 mm × 10 mm × 1 mm) were purchased from HTW Germany. These plates were polished and cleaned using (3, 1 and 0.05 μm) alumina particles at polishing cloth followed by 5 minutes sonication with ultrapure water.

The mesoporous silica films were prepared by electrochemically assisted self-assembly on APTESelectrografted glassy carbon electrodes (GCE), by adapting a previously described method [16].

(1 mM in 0.1 M Bu4NBF4 in acetonitrile) was grafted on cleaned GCE (A = 3.4 cm 2 ) by single CV scan (between +0.7 and +2.0 V at a rate of 100 mV s -1 ), which was then rinsed abundantly with acetone. Typical sol for silica films deposition was prepared by using ethanol and 0.1 M aqueous NaNO3 in 1/1 v/v ratio to which TEOS (100 mM) and CTAB (32 mM) were added and pH was adjusted to 3 with HCl. The sol was stirred for 2.5 hours for proper hydrolysis of TEOS. A portion of the electrode area (A = 0.5 cm 2 ) was contacted to the sol and film formation was achieved under galvanostatic conditions by applying a current density, j, of -0.74 mA cm -2 (optimized value [14]). Silica films were grown for various deposition times: 5, 8, 10, 12, 16, 20, and 30 s for monolayer films. Multilayered films were also deposited by EASA process (typically for 16 s repeated two or three times to get either bilayer or trilayer films), as previously described [37]. All mesoporous silica modified electrodes were treated overnight at 130 °C, and the surfactant template was extracted using 0.1 M HCl in ethanol under moderate stirring for 15 min to 1 h depending upon film deposition time (IR spectroscopy was used to check the time required for complete extraction).

Figure 3

 3 Figure 3

Figure 1 .

 1 Figure 1. (A,B) Cyclic voltammograms recorded in 0.5 mM Ru(NH3)6Cl3 (in 0.1 M KCl) using GCE electrodes modified with mesoporous silica films generated by EASA for various times, respectively before (A) and after surfactant removal (B). (C) Relative ratios of peak current intensities obtained at film electrodes with respect to bare GCE, as expressed as a function of film deposition time. Potential scan rate: 100 mV s -1 .

Figure 2 .

 2 Figure 2. (A) Square wave voltammograms (SWV) recorded in 0.5, 2, 3.5, 5, 6.5, 8 and 10 µM paraquat (in 0.07 M NaNO3, pH 6) using GCE electrodes modified with mesoporous silica films generated by EASA for 0 s (bare GCE, A1), 5 s (A2), 10 s (A3) and 16 s (A4). (B) Corresponding calibration curves, including also additional data obtained with other film electrodes.

Figure 3 .

 3 Figure 3. Multisweep cyclic voltammograms (40 first scans) recorded in 5 µM paraquat (in 0.07 M NaNO3, pH 6) using a GCE electrode modified with a mesoporous silica film generated by EASA for 30 s. Potential scan rate: 20 mV s -1 .

Table 1 .

 1 Film thicknesses measured by stylus profilometry for samples deposited galvanostatically (current density = 3.7110 -4 A cm -2 ) on glassy carbon electrode (GCE) and (for comparison) potentiostatically (potential = -1.3 V) on indium-tin oxide (ITO) electrode, by EASA for increasing

	deposition times.
	GCE

Acknowledgements

This work was supported in part by the French Programme Investissement d'Avenir (PIA) "Lorraine Université d'Excellence" (Reference No. ANR-15-IDEX-04-LUE). T. N. is grateful to the Higher Education Commission of Pakistan for funding his Ph.D. N. A. V. thanks the French Embassy in Ukraine for a mobility financial help through the "Campus France" program. The authors are also thankful to Dr. Liang Liu for help in profilometry measurements.