Lukasz Poltorak

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

Alain Walcarius

Grégoire Herzog

In-situ Formation of Mesoporous Silica Films Controlled by Ion Transfer Voltammetry at the Polarized Liquid-Liquid Interface

Keywords: Electrochemically assisted self-assembly, ITIES, surfactant template, free-standing silica film, micro-interface, tetraethylammonium

published or not. The documents may come

In-situ formation of mesoporous silica films controlled by ion transfer voltammetry at the polarized liquid-liquid interface

Introduction

Mesoporous thin films are attractive for a number of application fields such as energy, material science and analytical chemistry [1,2]. These films, which can be functionalized to induce specific properties, are usually formed by deposition onto a solid support by sol-gel chemistry. They can be obtained either by evaporation-induced self-assembly or by electrochemically assisted self-assembly, giving rise to well-ordered and even oriented structures [3]. A silica precursor is hydrolysed in the presence of a template agent (e.g. surfactants). The material is then formed upon self-assembly polycondensation of the silica precursor and the surfactant, giving to the film its structure and orientation, which depends from the experimental conditions (sol composition, aging, etc.) [4,5]. We report here the generation of a surfactant-templated mesoporous silica film, in-situ at the liquid-liquid interface, based on the combination of sol-gel chemistry and electrochemistry at the interface between two immiscible electrolyte solutions (ITIES). The generation of mesoporous structures at non-polarised liquid-liquid interfaces is not straightforward, although cooperative assembly of silica and surfactants at the oil-water and air-water interfaces was reported [6][7][8], but these were based on microemulsions (i.e. not as thin films) [7] or on synthetic methods difficult to control due to very long processing (i.e. days) [8]. Previous studies have reported the ITIES modification by adsorption of phospholipid monolayers, proteins, by electropolymerisation of terthiophene, by electrogeneration of silica films and by the support of zeolite, polymer, or silicon-fabricated membranes [9][10][11][12]. We investigated here the influence of experimental conditions (pH, precursor and surfactant concentrations) on the film formation and structure. The films were characterized by small-angle X-ray scattering (SAXS) and transmission electron microscopy (TEM) analysis. In a similar fashion, µITIES were modified to demonstrate the possibility of using these modified liquid-liquid micro-interfaces as sensors.

Materials and Methods

The silica precursor (tetraethoxysilane, TEOS) was dissolved in an aqueous solution of 5 mM NaCl, pH 3 and stirred for 1 hour at room temperature for hydrolysis. The pH was then adjusted to the desired value (with HCl and NaOH solutions). The organic phase was made of bis-(triphenylphosphoranylidene) ammonium tetrakis(4chlorophenyl)borate (BTPPA + TPBCl -) as the organic solution electrolyte and of cetyltrimethylammonium tetrakis(4-chlorophenyl)borate (CTA + TPBCl -) as the template molecule. Electrochemistry experiments were run in a custom-made borosilicate glass cell for the macro-ITIES experiments (A = 2.83 cm 2 ) and at two sets of siliconfabricated array (30 pores of 10 µm radius and 110 pores of 5 µm radius) for the µITIES experiments [13]. The interface was polarised with a set of two reference Ag|AgCl electrodes and two Pt mesh as counter electrodes. The electrochemical cell was Ag | AgCl | Saturated BTPPA + Cl -in 10 mM LiCl | x mM CTA + TPBCl -+ 10 mM BTPPA + TPBCl -in 1,2-dichloroethane || y mM TEOS + 5 mM NaCl in H2O | AgCl | Ag. The pH of the aqueous phase was 9.5 unless stated otherwise; 1.5 mM < x < 14 mM; 50 mM < y < 300 mM. A potentiostat 302N from Autolab (Metrohm, Switzerland) was used for cyclic voltammetry experiments. All films formed were cured at 130 °C for 16 hours to ensure cross-linking. The film formed at the macro-ITIES was carefully collected using a glass slide. Samples were placed between two sheets of Kapton® before exposition to X-Ray beam for about 5 min.

Scattering of X-Ray beam was recorded according to the published procedure [START_REF] Sepulveda | [END_REF].

Results and Discussion

When the potential region is scanned from + 0.9 V down to + 0.05 V, a negative peak is observed around + 0.1 V if both CTA + and TEOS are present in the electrochemical cell (Figure 1). The adsorption of CTA + ions at the interface might destabilise the interface and cause these spikes seen in Figure 1 (dotted curve) [15]. The peak at + 0.1 V is attributed to the transfer of CTA + from the organic to the aqueous phase. As it is only visible if both CTA + and TEOS are present in the electrochemical cell, TEOS probably assists the transfer of CTA + to the aqueous phase.

The presence of CTA + favours the polycondensation of TEOS around CTA + ensembles to form the silica film (Figure 1). In the reverse scan, a sharp peak at + 0.4 V is attributed to surfactant molecules transferring back to the organic phase. A similar behaviour was observed for the tetraoctadecyl ammonium transfer in the presence of liquid glass [16]. The charge under the reverse peak, showed maximum values for pH 9-10 before decreasing for pH 11 (Figure 1B). CTA + transfer is the highest when polynuclear species ( ) are predominant in the aqueous phase [START_REF] Baes | Hydrolysis of Cations[END_REF]. At pH lower than 9 and higher than 11, mononuclear species are dominant, which may not facilitate the transfer of CTA + to the aqueous phase. The reverse peak charge increases upon repetitive cycling of the selected potential region until it reaches a plateau after 8 cycles approximately (Figure 1C), whereas the charge under the forward peak remains constant throughout the cycling. Silica is formed at the ITIES, induced by a voltammetrically controlled CTA + ion transfer. Indeed, no film formation was observed when an electrochemical cell was left for 16 hours at open-circuit potential at room temperature. These findings are consistent with previous observation of surfactant-templated silica films that were only obtained at the non-polarised oil-water interface after heating for 2 -10 hours at 150 °C [6]. The influence of CTA + and TEOS concentrations on the electrochemical process was investigated. The reverse peak charge, measured after 15 cycles, increased linearly with [CTA + ]org. within the 1.5 -14 mM range (not shown), regardless of [TEOS]aq.. However, the reverse peak charge remained constant although [TEOS]aq. increased (not shown).

These results indicate that the film formation is really limited by the CTA + transfer.

SAXS analysis of the films collected at the ITIES suggests the existence of a 'wormlike' structure as a broad signal was observed (especially visible on curves b and c in Figure 2). This level of order indicates that CTA + ions transferring to the aqueous phase are subject to cooperative self-assembly with TEOS molecules to form a surfactant-templated ordered film as shown by TEM analysis (Figure 2B&C). = 14 mM). The impact of [CTA + ]org. was expected as it influenced greatly the ion transfer charges measured during the cyclic voltammetry studies. The critical micelle concentration (cmc) in water for CTA + Cl -is 1.4 mM [START_REF] Sepulveda | [END_REF]. As [CTA + ]org. increases, the concentration of CTA + in aqueous diffusion layer is more likely to be above this cmc value leading to a structuration of the film formed. Note that decreasing the [TEOS]aq. has shown also an impact on the film structure, yet weaker than for [CTA + ]org, resulting in a small increase in the pore center-to-center distance (e.g. to 4.9 nm for [TEOS]aq. = 50 mM and [CTA + ]org. = 14 mM).

Miniaturization of the ITIES leads to higher sensitivities, lower limits of detection and smaller iR drop, analogously to phenomena observed for microelectrodes [11]. µITIES are generally obtained using a solid-state membrane pierced with holes of dimensions ranging from a few tens of nm up to a few tens of µm, bringing mechanical stability to the system [11]. A microfabricated membrane (r = 10 µm, N = 30 pores) is modified by ion transfer voltammetry with 1 cycle using the same electrochemical cell as for the macroscopic cell. The permeability of these modified membranes was then analysed by cyclic voltammetry of TEA + (Figure 3). The presence of the silica film at the interface renders the TEA + transfer more difficult as it is shifted to more positive potentials. However, at the modified µITIES, a limiting current ilim = (13.3±0.03) nA is obtained, which is higher than the current of (7.8 ± 0.02) nA obtained at a bare µITIES. This experimental value is reasonably close to the theoretical value of 8.1 nA obtained using the equation ilim = 4zFDCrN (z is the charge transferred, F is the Faraday constant, D the TEA + diffusion coefficient, C the TEA + concentration, r = 10 µm and N = 30 pores in the array). The ilim increase is probably linked to an increase of the interface surface area due to the film formation outside the pores. SEM images of a modified µITIES array (r = 5 µm, N =110 pores) show hemispherical films towards the aqueous side of the interface. The µITIES are uniformly covered by silica deposits, leaving the area between the µITIES unmodified (Figure 3B). This shape of films would correspond to the diffusion profile of the CTA + molecules when they transferred from the organic to the aqueous phase (Figure 3B&C) before condensation of TEOS occurred. Indeed, the diameter of the film formed is 21.27 ± 3.66 µm (twice as large as the actual pore diameter 10.41 ± 0.04 µm [13]). At the larger interfaces (r = 10 µm), films protruding inside the aqueous phase were also formed, higher at the side of the interface and lower at the centre of interface (SEM image not shown). These observations suggest that diffusion of CTA + ions might be responsible for the shape of film rather than the interface. These experiments show that µITIES can be modified and allow ion transfer once modified. Investigations for a more thorough understanding of the film formation mechanism is still under-going.

Conclusions

Mesoporous silica films were prepared in-situ at both macro-and µITIES by transfer of CTA + ions, catalysing the polycondensation of hydrolysed silica precursors. The films remain permeable, suggesting that the control of the film chemistry by either co-condensation or post-synthesis functionalization would confer selectivity to µITIES modified with organic-inorganic hybrids, en route to a new class of electrochemical sensors. Selectivity of the sensors could be provided by the charge, the chemical function and the size of the target analyte and its possible interferents.

Figure 1 .

 1 Figure 1. (A) Cyclic voltammograms (CV) in the presence of 50 mM TEOS, and in the presence of 1.5 mM CTA +

Figure 2 .

 2 Figure 2. (A) Variation of SAXS pattern for different CTA + concentrations in the organic phase. The films were obtained by CV with 300 mM TEOS in the aqueous phase and with (a) 5, (b) 10 and (c) 14 mM CTA + . images of films prepared by cyclic voltammetry with [TEOS]aq. = 300 mM and [CTA + ]org. = 10 mM (B) and [TEOS]aq. = 300 mM and [CTA + ]org. = 14 mM (C). Scale bars represent 20 nm.

Figure 3 .

 3 Figure 3. (A) CVs of 70 µM of TEA + at a bare µITIES array (solid line), and at an array of µITIES modified with a

Acknowledgments 8

The project was partly supported by the Région Lorraine. The authors are grateful to J. Ghanbaja for the TEM imaging, to V. Jacque for preliminary studies, to A. Pasc, M.-J. Stébé and M. Emo for the SAXS measurements and to the Tyndall National Institute for provision of the microfabricated membranes.


Patricia Vazquez
email: patricia.vazquez@tyndall.ie

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

Conor O'mahony

Joseph O'brien

James Scully

Alan Blake

Cian O'mathuna

Paul Galvin

Microscopic gelliquid interfaces supported by hollow microneedle array for voltammetric drug detection

Keywords: Microneedles, micro-interfaces, ITIES, -blocker drug, minimally invasive, stripping voltammetry

This report describes a method for integration of a gel-liquid interface in hollow microneedles compatible with minimally invasive, electrochemical detection of drugs in vivo. The electrochemical sensor was characterised using cyclic voltammetry of tetraethyl ammonium. The experimental work demonstrated the detection of propranolol as a representative drug in physiological buffer with the microneedle system. A calibration curve for propranolol was built from measurements with differential pulse stripping voltammetry, indicating a sensitivity of 43 nA µM -1 , a limit of detection of 50 nM and a linear range between 50 -200 nM.

Introduction

The past 20 years have seen the development of microneedles as an alternative to traditional subcutaneous needles for transdermal delivery of substances that are of medical interest. Microneedles have been used for the delivery of pharmacological compounds [START_REF] Wermeling | Microneedles permit transdermal delivery of a skin-impermeant medication to humans[END_REF] and for vaccination [START_REF] Van Damme | Safety and efficacy of a novel microneedle device for dose sparing intradermal influenza vaccination in healthy adults[END_REF][START_REF] Chabri | Microfabricated silicon microneedles for nonviral cutaneous gene delivery[END_REF]. Research efforts have also been devoted to extend the use of microneedles beyond their traditional purpose of drug delivery, as it was the case in the use of metalized microneedles for electroporation therapy [START_REF] Wilke | Silicon microneedle electrode array with temperature monitoring for electroporation[END_REF].

Hollow microneedles applied onto the skin are able to extract interstitial fluid [START_REF] Mukerjee | Microneedle array for transdermal biological fluid extraction and in situ analysis[END_REF][START_REF] Wang | Minimally invasive extraction of dermal interstitial fluid for glucose monitoring using microneedles[END_REF], offering a solution for a minimally invasive sensor of biomarkers in bodily fluids.

However, detection of biomarkers of interest is done off-line [7], which requires the extraction of interstitial fluid from the body. Regardless of the method of extraction, the concept is hindered by the need of high vacuum pressures (200-500 mm Hg) for the extraction and collection of fluid [START_REF] Wang | Minimally invasive extraction of dermal interstitial fluid for glucose monitoring using microneedles[END_REF]. Additionally, the volumes of fluid collected are variable [START_REF] Wang | Minimally invasive extraction of dermal interstitial fluid for glucose monitoring using microneedles[END_REF][START_REF] Stout | A novel approach to mitigating the physiological lag between blood and interstitial fluid glucose measurements[END_REF] and little is known of the reliability of extraction in a continuous basis. In conclusion, the complexity of a sensor able to provide continuous information of biochemical analytes in a physiological fluid would be drastically reduced with a design that did not require the uptake of biological fluids.

The use of electrochemical sensors can be a solution for an on-line detection without the need of sample extraction. Furthermore, miniaturised electrochemical sensors are more sensitive than macroscopic sensors [START_REF] Wang | Portable electrochemical systems[END_REF] and electrochemical instrumentation is relatively easily miniaturised [START_REF] Miller | Multiplexed microneedle-based biosensor array for characterization of metabolic acidosis[END_REF]. Recently, hollow polymeric microneedle arrays were filled with microelectrodes and used for the amperometric detection of hydrogen peroxide, lactate, glutamate and glucose [START_REF] Windmiller | Microneedle array-based carbon paste amperometric sensors and biosensors[END_REF][START_REF] Malzahn | Wearable electrochemical sensors for in situ analysis in marine environments[END_REF].

We report here a novel sensor based on an array of hollow microneedles filled with an organogel able to sense ionic species in a physiological fluid. This platform takes advantage of the possibility to polarise the interface formed between the organogel, located inside the hollow microneedle, and the physiological fluid. Electrochemical sensors based on micro-interfaces between two immiscible electrolyte solutions (µITIES) have been developed in recent years [START_REF] Herzog | Stripping voltammetry at micro-interface arrays: A review[END_REF][START_REF] Arrigan | Bioanalytical detection based on electrochemistry at interfaces between immiscible liquids[END_REF]. This electrochemical technique is based on ions transferring across a polarized liquid-gel interface upon application of a potential difference. Ion transfer from one phase to another gives rise to a current that can be monitored. Silicon-based micro and nano ITIES have been developed for the detection of analytes that are not redox active molecules [START_REF] Zazpe | Ion-transfer voltammetry at silicon membrane-based arrays of micro-liquidliquid interfaces[END_REF][START_REF] Scanlon | Ion-transfer electrochemistry at arrays of nanointerfaces between immiscible electrolyte solutions confined within silicon nitride nanopore membranes[END_REF].

Examples like detection of dopamine [START_REF] Beni | Cyclic and pulse voltammetric study of dopamine at the interface between two immiscible electrolyte solutions[END_REF] and peptides [START_REF] Yuan | Facilitated Protamine Transfer at Polarized Water/1,2-Dichloroethane Interfaces Studied by Cyclic Voltammetry and Chronoamperometry at Micropipet Electrodes[END_REF][START_REF] Herzog | Ion-Transfer Voltammetric Behavior of Protein Digests at Liquid|Liquid Interfaces[END_REF] show the potential of this technique as an alternative to traditional electrochemical techniques.

We propose the use of a novel diagnostic platform that combines a hollow microneedle array with electrochemical detection at µITIES. The presence of the µITIES in the inner bore of the microneedles offers several advantages: it avoids the need of fluid extraction, as the ions cross the gel-liquid interface due to the potential difference applied at the interface; additionally, the reduced dimensions of the interface enhance rapid diffusion of species, which improves detection limits of the analytes. This effect is illustrated in different works on µITIES [START_REF] Yuan | Facilitated Protamine Transfer at Polarized Water/1,2-Dichloroethane Interfaces Studied by Cyclic Voltammetry and Chronoamperometry at Micropipet Electrodes[END_REF][START_REF] Herzog | Ion-Transfer Voltammetric Behavior of Protein Digests at Liquid|Liquid Interfaces[END_REF][START_REF] Scanlon | Electrochemical Detection of Oligopeptides at Silicon-Fabricated Micro-Liquid|Liquid Interfaces[END_REF][START_REF] Herzog | Sensing via Voltammetric Ion-Transfer at an Aqueous-Organogel Micro-Interface Array[END_REF] and even used for the development of bioanalytical sensors [START_REF] Pereira | Amperometric glucose biosensor based on assisted ion transfer through gel-supported microinterfaces[END_REF].

We have selected propranolol as a model analyte. This drug belongs to the βadrenergic blocking group [START_REF] Baker | Evolution of β-blockers: from anti-anginal drugs to ligand-directed signalling[END_REF]; it reduces heart rate and blood pressure, which makes it useful for treatment of hypertension and chest pain related to heart failure (angina) [START_REF] Baker | Evolution of β-blockers: from anti-anginal drugs to ligand-directed signalling[END_REF]. Blood pressure and heart rate correlate with propranolol levels in plasma [START_REF] Coltart | Plasma propranolol levels in the quaniitative assessment of beta-adrenergic blockade in man[END_REF]. Therefore, its direct measure in a physiological matrix could be useful for correct drug dosage and the monitoring of patient response to treatment.

Propranolol has already been detected by ion transfer voltammetry at µITIES at concentration levels that are physiologically relevant [START_REF] Collins | Ion-Transfer Voltammetric Determination of the β-Blocker Propranolol in a Physiological Matrix at Silicon Membrane-Based Liquid|Liquid Microinterface Arrays[END_REF][START_REF] De Eulate | Detection of haemoglobin using an adsorption approach at a liquid-liquid microinterface array[END_REF][START_REF] Sisk | Assessment of ion transfer amperometry at liquid-liquid interfaces for detection in CE[END_REF], thus allowing useful comparison with the results reported here.

We present also the characterisation of the silicon microneedle system by scanning electron microscopy (SEM) and cyclic voltammetry (CV) of tetraethyl ammonium (TEA + ). This model analyte has been used extensively in the characterization of ITIES, as it presents rapid and mechanistically simple transfers in these systems [START_REF] Girault | Charge Transfer across Liquid-Liquid Interfaces[END_REF][START_REF] Samec | Charge Transfer Kinetics at Water-Organic Solvent Phase Boundaries[END_REF][START_REF] Benjamin | Molecular structure and dynamics at liquid-liquid interfaces[END_REF][START_REF] Wang | Kinetic study of rapid transfer of tetraethylammonium at the 1, 2dichloroethane/water interface by nanopipet voltammetry of common ions[END_REF]. The array is then used for the detection of propranolol in physiological medium by differential pulse stripping voltammetry (DPSV).

Material and methods

Reagents

All the reagents were purchased from Sigma-Aldrich Ireland Ltd. and used as received except for 1,6-dichlorohexane (1,6-DCH) (Merck), that was purified according to the published procedure [START_REF] Katano | Ion-transfer voltammetry at 1, 6-dichlorohexane| water and 1, 4-dichlorobutane| water interfaces[END_REF]. The organic electrolyte salt was prepared by metathesis of bis-(triphenylphosphoranylidene) ammonium chloride (BTPPA + Cl -) and of potassium tetrakis(4-chlorophenyl)-borate (K + TPBCl -) to obtain BTPPATPBCl, following the published experimental procedure [START_REF] Lee | Amperometric Ion Detector for Ion Chromatography[END_REF]. The organogel phase was prepared using purified 1,6-DCH and low molecular weight polyvinylchloride (PVC) 10:1 (v:w) [START_REF] Arrigan | Bioanalytical detection based on electrochemistry at interfaces between immiscible liquids[END_REF]. For the experiments of propranolol detection at the µITIES array, the aqueous phase was composed of magnesium pyrophosphate (1.6 mg L -1 ), carboxymethyl cellulose (4 g L -1 ), urea (4 g L -1 ), disodium hydrogen phosphate (0.6 g L -1 ), anhydrous calcium chloride (0.6 g L -1 ), potassium chloride (0.4 g L -1 ), and sodium chloride (0.4 g L -1 ).

Microneedle array-µITIES

The silicon microneedle arrays were fabricated using a combination of potassium hydroxide (KOH) wet-etching and Bosch dry-etching techniques [START_REF] O'mahony | A Microneedle-based Miniature Syringe for Transdermal Drug Delivery[END_REF][START_REF] O'brien | Hollow Microneedles for Pain-Free Drug Delivery[END_REF]; the final microneedle array is shown in the scanning electron micrograph of figure 1A. Briefly, square oxide/nitride masks were patterned using standard photolithography tools on a <100>, boron doped, 100 mm diameter monocrystalline silicon wafer. These masks are aligned with the <110> direction and the patterned silicon wafer is then etched in a 29 % w/v aqueous KOH solution. Convex corner mask undercut takes place, leading to the formation of two angled planes at each corner, which eventually intersect to form an octagonal microneedle. This needle is comprised of eight {263} planes, a base of {212} planes and has a height to base aspect ratio of 3:2.

To form hollow bores through the microneedles, a 3 µm Plasma-Enhanced Chemical Vapour Deposition (PECVD) oxide layer is deposited and patterned on the rear of the wafer to act as a hard mask; a 2 µm layer of aluminium is sputtered on the front side of the wafer to act as an etch stop layer. 50 µm diameter capillary conduits are then micromachined from the back side of the wafer using a Surface Technology System (STS) ASE ICP dry etching tool incorporating Bosch dry etching technology; the etch gases are SF6 and C4F8. Afterwards, the aluminium layer and thick oxide layers are stripped from the front and back sides of the wafer, respectively.

In the case of the array used, the 300 µm high microneedles were separated by 1 mm. The bore is of a cylindrical shape with a diameter comprised between 55 and 62 µm as it is shown on the scanning electron microscope (SEM) image of figure 1A. The creation of a micro ITIES at the microneedles is done by filling their bores with the organogel. A glass cylinder of 9 mm of inner diameter was sealed to the back of the microneedle array with silicone rubber. This configuration encloses a total of 52 hollow microneedles available for the experiments. The fabricated needles feature hydrophobic walls, which allowed an easy filling of the microneedle holes with the organogel; the latter had been heated gently at 50 °C in order to reduce its viscosity.

The filling was done by pipetting the organogel onto the back of the microneedle array. Once the holes were filled (see figure 1B), the array was left to rest for at least 1 h before proceeding with the experiments.

Electrochemical set-up

In order to perform the electrochemical measurements, the system was configured as shown in figure 2. For all experiments, the electrodes were connected to a CHI660B electrochemical analyser (CH Instruments, Texas, purchased from IJ Cambria, Burry Port, Wales, United Kingdom). Cyclic voltammetry was used as characterisation method, and differential pulse stripping voltammetry (DPSV) was chosen as the analytical detection method. The stripping method consisted of three steps: (i) preconditioning, (ii) preconcentration and (iii) detection. First, the ITIES went through an optimised preconditioning step during which a potential of 0.45 V was maintained for 120 s to clean the gel from any residuals of the analyte that may remain from a previous experiment. A preconcentration step was then used to transfer the analyte from the aqueous phase to the organic phase; the potential was held at 0.86 V for a period between 15 and 240 s to transfer the analytes to the organogel. These two steps allow concentrating the propranolol in the organogel. Finally, the detection step consisted of the back transfer of the preconcentrated propranolol to the aqueous phase by the scanning of a predefined potential window. The parameters for DPSV were: amplitude = 80 mV; pulse width = 0.1 s; sample width = 0.05 s; pulse period = 0.2 s. This DPSV protocol has been described previously [START_REF] Collins | Ion-Transfer Voltammetric Determination of the β-Blocker Propranolol in a Physiological Matrix at Silicon Membrane-Based Liquid|Liquid Microinterface Arrays[END_REF].

Results and discussion

System characterization by cyclic voltammetry analysis

The electrochemical response of the hollow microneedle system was characterized by ion transfer voltammetry of the model analyte tetraethyl ammonium cation (TEA + ) at the ITIES formed in the bore of the needles. Before proceeding with any experiments, we determined the optimal scan rate values for the geometry of the needle array. The diffusion layer thickness (δ) should ideally be larger than the µITIES formed in the microneedles bores but not as large as to cause adjacent diffusion layers to overlap. This condition is highly desired as it results in the maximum faradaic current to background current ratio. To ensure non-overlapping diffusion areas between the holes of the array, it is sufficient to have 𝑑 > 2𝛿 [START_REF] Davies | The cyclic and linear sweep voltammetry of regular and random arrays of microdisc electrodes: Theory[END_REF], d being the distance of the holes from centre to centre.

The diffusion area can be approximated by Einstein's equation for the root meansquare displacement of diffusing particles with Brownian motions:

𝛿 = √2𝐷 ∆𝐸 𝜈 ⁄ (1)
In eq. ( 1), D is the diffusion coefficient (m 2 s -1 ); ΔE is the difference of potential between the onset of electrolysis and the peak of limiting current (V);  is the scan rate (V s -1 ). The substitution of this expression in the aforementioned condition for independent diffusion layers leads to the following equation for the scan rate:

𝝂 > 𝟖𝑫 𝜟𝑬 𝒅 𝟐 ⁄ (2)
In the particular case of our experiments, eq. ( 2) gives  = 2.35 mV s -1 as the lower limit that present radial diffusion with no overlapping areas (taking D = 9.8•10 -6 cm 2 s - 1 for TEA + and ΔE = 0.3 V). All the experimental work was carried out with  values higher than this limit. Therefore, the distance between adjacent microneedles (20 times the radius of the inner needle bore) is sufficiently large to avoid overlapping of the diffusion layer.

Figure 3A shows the cyclic voltammogram in the absence and in the presence of TEA + in the aqueous phase. In the absence of TEA + , the potential window is limited by the transfer of the background electrolytes, noticeable in the steep variations of current at potentials greater than + 0.9 V and lower than + 0.2 V. The capacitance at + 0.6 V was estimated to be of (16.46 ± 0.40) µF cm -2 .

With the experiments conditioned as by the above calculations, asymmetry in the CVs was expected, as it has already been observed in a number of µITIES systems [START_REF] Liu | Electrochemistry at micro-and nanoscopic liquid/liquid interfaces[END_REF]. In these reported cases, a sigmoidal wave is observed in the forward scan, which results from the radial diffusion of species in the aqueous phase. On the other hand, the reverse scan showed a peak that corresponded to the linear diffusion of species inside the pores.

According to this, the microneedle system should present a steady state response during the forward sweep in the form of a sigmoidal curve for the TEA + transfer.

However, as shown in figure 3A, it is hard to distinguish the TEA + transfer from the background electrolyte transfer. This could be explained by the hindered diffusion towards the interface. The bore of the microneedle is not centred but slightly shifted to the side as it is shown in figure 1A and schematically represented in figure 3B. As a consequence, diffusion of species towards the micro-interface is not hemispherical as it can be observed at regular disk micro-interfaces. It presents a diffusion profile mixed between radial and linear as represented by the arrows in figure 3B. By contrast, the reverse sweep presents a peak-shaped voltammogram indicating a diffusion-controlled return of the analyte from the organic to the aqueous phase (see again figure 3). This was further corroborated by subsequent experiments where the scan rate was varied between 5 and 17 mV s -1 (see figure 4). The measured reverse peak current is directly proportional (with R 2 =0.997) to the square root of the scan rate, which confirms the presence of the diffusion-controlled process when TEA + transfers back to the aqueous phase. The gel phase forms a recessed interface at the aqueous side of the ITIES (as it can be seen in figure1B). Computational simulations have shown that this configuration is optimal for stripping voltammetry, as transport of analyte species to the gel microinterface improves with a hemispherical diffusion regime during the preconcentration step [START_REF] Strutwolf | Optimisation of the conditions for stripping voltammetric analysis at liquid-liquid interfaces supported at micropore arrays: a computational simulation[END_REF] . Once they are transferred to the organic phase, they remain in the vicinity of the interface because of the reduced mass transport caused by both the linear diffusion and the lower diffusion coefficient in the PVC gel. Simulation studies have shown that the diffusion coefficient of TEA + was estimated to be nine times lower in the gel phase than in the aqueous solution [START_REF] Strutwolf | Electrochemical ion transfer across liquid/liquid interfaces confined within solid-state micropore arrays-simulations and experiments[END_REF].

Voltammetric detection of propranolol at the microneedle array

Figure 5 shows the CV of propranolol at the liquid-gel interface supported by the microneedle array. A sigmoidal wave is observed at + 0.7 V for the transfer of propranolol from the aqueous to the organic phase, whereas a peak is observed at + 0.25 V for the back transfer. The CV from propranolol shows the same features than with the experiments with TEA + : hemispherical diffusion during the forward scan, and linear diffusion on the reverse sweep. The CV behaviour allows us to use eq. ( 3) to calculate the radius of the microneedle bore based on the propranolol limiting current, ilim, measured on the forward scan.

𝑖 𝑙𝑖𝑚 = 𝑛4𝑧𝐹𝐷𝐶𝑟 (3)
This equation defines the limiting current that is expected for an array of disc interfaces, where n is the number of microneedle interfaces (n = 52); z is the number of charge transferred (z = 1); F is the Faraday constant; D is the diffusion coefficient (D = 1.15 × 10 -5 cm 2 s -1 , [START_REF] Fantini | Influence of the presence of a gel in the water phase on the electrochemical transfer of ionic forms of beta-blockers across a large water 1,2-dichloroethane interface[END_REF]); C is the propranolol concentration (C = 100 × 10 -9 mol cm -3 ) and r is the radius in cm. The theoretical limiting current of 57 nA (from equation 3) agrees well with the experimental value of 55 ± 3 nA (measured in CVs modified with background subtraction, not shown) corresponds to a microneedle interface radius of 25 µm, which agrees with the radius suggested by the SEM images (figure 1).

The use of differential pulse stripping voltammetry (DPSV) as the analytical technique offers the possibility to detect lower concentrations of the analyte as capacitive/background current is eliminated.

Figure 6 shows the DPSVs for increasing preconcentration times ranging from 15 to 240 s. Stripping peak currents increase slowly with the preconcentration time.

Noticeably, the stripping peak current after 240 s of preconcentration is approximately 5 times higher than the one measured for 15 s. Based on these results, a preconcentration time t = 240 s was selected for the rest of experiments, as it provided the maximum current peak that was recorded. A sensitivity of 43 nA µM -1 was calculated as the slope of the calibration curve (R 2 = 0.99423). The stripping peak for propranolol shifts towards less positive potential values as the preconcentration time or the concentration increase. This could be explained by two different phenomena (or a combination of both): (i) the kinetic limitation due to the increasing amounts of propranolol back-transferring or (ii) the uncompensated resistance due to an increasing ohmic drop. At concentrations higher than 200 nM, the stripping peak currents present a saturation effect. This could be justified by the fact that diffusion of propranolol is reduced due to the viscosity of the gel. The lowest concentration that the system was able to detect was 50 nM, which is within the detection limits published for propranolol [START_REF] Collins | Ion-Transfer Voltammetric Determination of the β-Blocker Propranolol in a Physiological Matrix at Silicon Membrane-Based Liquid|Liquid Microinterface Arrays[END_REF][START_REF] Álvarez | Reusable phosphorescent probes based on molecularly imprinted polymers for the determination of propranolol in urine[END_REF][START_REF] Radi | Adsorptive behaviour and voltammetric analysis of propranolol at carbon paste electrode[END_REF][START_REF] El-Tohamy | Propranolol hydrochloride-selective membrane electrode and their application to pharmaceutical preparations and biological fluids[END_REF][START_REF] Ghoneim | Indirect determination of propranolol by cathodic adsorptive stripping voltammetry[END_REF].

Conclusions

This report presents for the first time the development of a microneedle array with an embedded µITIES platform that is electrochemically able to detect propranolol.

Although there is a wealth of literature reporting the use of µITIES for electrochemical detection, previous works were based in the use of micropipettes or arrays of micro and nano-holes as support structures for the miniaturized ITIES.

The array of sensors was capable of detecting propranolol in artificial saliva with good sensitivity and a limit of detection that is of pharmacological relevance. The use of miniaturized interfaces and DPSV as the analytical method of detection deliver a low limit of detection, 50 nM, which compares well with previously published results

for propranolol. This includes 0.2 μM with adsorptive stripping differential pulse voltammetry [START_REF] Sartori | Square-wave voltammetric determination of propranolol and atenolol in pharmaceuticals using a boron-doped diamond electrode[END_REF]; 5 μM for potentiometric membrane electrode [START_REF] El-Tohamy | Propranolol hydrochloride-selective membrane electrode and their application to pharmaceutical preparations and biological fluids[END_REF]; 0.02 μM with cathodic adsorptive stripping voltammetry [START_REF] Ghoneim | Indirect determination of propranolol by cathodic adsorptive stripping voltammetry[END_REF]; 0.1 μM for capillary electrophoresis [START_REF] Li | Simultaneous analysis of six cardiovascular drugs by capillary electrophoresis coupled with electrochemical and electrochemiluminescence detection, using a chemometrical optimization approach[END_REF]. The platform presents a linear range between 50 and 200 nM.

Our future line of work will address the application of the microneedle-µITIES system to the detection of biomarkers for in vivo applications. The characteristics of the microneedle-ITIES could be beneficial in a scenario for transdermal analysis, as the system can overcome the skin barrier with minimal pain (thanks to the microneedles) and without physiological fluid extraction (due to the µITIES embedded).

Nevertheless, the chemicals used for the organogel are toxic and will not be used as such for in-vivo applications. Future developments will need the investigation of new materials for the organic phase. We will look into compounds that present both biocompatibility and stability in the long term (the organogel presently used is volatile at air atmosphere and degrades slowly with time).

Additionally, even though the presence of the organogel in the bore of the needles should avoid potential clogging in transdermal applications, we will study this issue during future experimental work.

on CMOS microsystems that provide intelligent platforms of non-standard functions such as sensors, actuators, power and cooling.

In January 2013, he was named an IEEE Fellow in the field of power electronics "for leadership in the development of power supply using micro-magnetics on silicon". from light green to black, respectively). The experiments correspond to an array of needles with 56 micropores.

Dr

(Inset) Reverse current peaks against square root of . The peaks present a linear slope with the square root of , which is a typical response of diffusion-prevailing systems (R 2 =0.997).

Figure 1 :

 1 Figure 1: (A) SEM image of an array of hollow silicon microneedles. (B) Optical micrograph of the cross-section of a microneedle filled with the organogel. The microneedle system was encased in epoxy glue in order to facilitate subsequent slicing of the silicon material.

Figure 2 :

 2 Figure 2: Experimental set up for electrochemical measurements. The aqueous phase consists of an Ag/AgCl wire and a meshed Pt wire as reference and counter electrodes respectively. Both are immersed in a solution with the analyte to be detected. As for the organic phase, a Ag/AgCl electrode behaves as both the reference and counter electrode.

Figure 3 :

 3 Figure 3: (A) Cyclic voltammetry curves in the presence (black) and in the absence (grey) of 160 µM TEA + . The scan rate was 3.5 mV s -1 . (B) Schematic representation of the top view and side views of a microneedle filled with an organogel. The arrows represents the diffusion of species in the aqueous phase towards the organogel.

Figure 4 :

 4 Figure 4: CVs of 160 µM TEA + in 10 mM LiCl, at increasing values of  (5, 6, 7, 8, 9, 10, 11, 12, 15 and 17 mV/s; from light green to black, respectively). (Inset) Reverse current peaks against square root of . The peaks present a linear slope with the square root of , which is a typical response of diffusion-prevailing systems (R 2 =0.997).

Figure 5 :

 5 Figure 5: CV of 100 µM propranolol (black) and background electrolyte (grey). scan rate was 35 mV s -1 )

Figure 6 :

 6 Figure 6:(A) DPSVs of 100 µM propranolol for increasing preconcentration times: 15, 30. 45, 60, 90, 120, 180, 210, 240 s. (B) Stripping peak current as a function of the preconcentration time Once the preconcentration step was defined, DPSVs for increasing concentrations of propranolol in aqueous solution were recorded (figure 7A). The stripping peak (figure 7B) increased linearly with propranolol concentrations within the range 50 -200 nM.

Figure 7 :

 7 Figure 7: (A) DPSVs of different concentrations of propranolol in artificial saliva (propranolol concentrations, from lightest grey to black: 0.03, 0.05, 0.07, 0.09, 0.11, 0.13, 0.15, 0.17, 0.2, 0.4, 0.6,0.8 and 1 µM). (B) Current calibration curve for DPSVs of propranolol. Lower concentrations show a marked behaviour typical of spherical diffusion (a steep sloped line), whereas higher concentrations (typically, from 200 nM onwards) present a linear diffusion progress (flat line). R 2 = 0.994.

Figure 1 :

 1 Figure 1: (A) SEM image of an array of hollow silicon microneedles. (B) Optical micrograph of the cross-section of a microneedle filled with the organogel. The microneedle system was encased in epoxy glue in order to facilitate subsequent slicing of the silicon material.

Figure 2 :

 2 Figure 2: Experimental set up for electrochemical measurements. The aqueous phase consists of an Ag/AgCl wire and a meshed Pt wire as reference and counter electrodes respectively. Both are immersed in a solutionwith the analyte to be detected. As for the organic phase, a Ag/AgCl electrode behaves as both the reference and counter electrode.

Figure 3 :

 3 Figure 3: (A) Cyclic voltammetry curves in the presence (black) and absence (grey) of 160 µM TEA+, for an array of 56 micropores/needles. The scan rate was 35 mV s -1 . (B) Schematic representation of the top view and sideviews of a microneedle filled with an organogel. The arrows represent the diffusion of species in the aqueous phase towards the organogel. The addition of 160 µM TEA + to the aqueous solution leads to an increase of the current starting from + 0.6 V, indicating the TEA + transfer. On the reverse scan, a peak is visible at + 0.4 V, corresponding to the transfer of the TEA + from the organic phase back to the aqueous phase.

Figure 4 :

 4 Figure 4: CVs of 160 µM TEA+ in 10 mM LiCl, at increasing values of (5, 6, 7, 8, 9, 10, 11, 12, 15 and 17 mV s -1 ;

Figure 5 :

 5 Figure 5: CV of 100 µM propranolol (black) and background electrolyte (grey). Scan rate was 8 mV s -1 ).

Figure 6 :

 6 Figure 6: (A), DPSVs of 100 µM propranolol for increasing preconcentration times: 15, 30. 45, 60, 90, 120, 180, 210, 240 s. (B), Stripping peak current as a function of the preconcentration time.

Figure 7 :

 7 Figure 7: (A) DPSVs of different concentrations of propranolol in artificial saliva. (B) Corresponding currentcalibration curve for DPSVs of propranolol. Measurements were performed for the following concentrations of propranolol: 0.03, 0.05, 0.07, 0.09, 0.11, 0.13, 0.15, 0.17, 0.2, 0.4, 0.6, 0.8 and 1 µM (figure A shows a selection of plots for visual clarity, corresponding to concentrations of propranolol of 0.07, 0.09, 0.11, 0.15, 0.17, 0.2, 0.8 and 1 µM). Lower concentrations show a marked behaviour typical of spherical diffusion (a steep sloped line), whereas higher concentrations (typically, from 200 nM onwards) present a linear diffusion progress (flat line). R 2 = 0.994.

  . Paul Galvin was awarded his PhD in 1995 and joined the Tyndall National Institute (then NMRC) in 2000, where he now leads the Life Sciences Interface Group. Research within his group is focussed on exploiting the extensive design, fabrication and characterisation tools available in Tyndall, for applications related to health and environment. Dr Galvin is a PI in the Science Foundation Ireland INFANT centre, collaborates in the SFI BDI and INSIGHT centres, and has led two international multidisciplinary research projects. He has over 60 international publications, and has presented his research at numerous international conferences.

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Acknowledgements

The authors would like to acknowledge support by Science Foundation Ireland (SFI) under grant 07/CE/I1147. P. Galvin would also like to acknowledge support from SFI through the INSIGHT, Biomedical Diagnostics and INFANT centres.