Charli Delayre

Patricia Patrier Mas

Paul Sardini

Philippe Cosenza
email: philippe.cosenza@univ-poitiers.fr

Anthony Thomas

Quantitative evolution of the petrophysical properties of andesites affected by argillic alteration in the hydrothermal system of Petite Anse-Diamant, Martinique

Keywords: argillic alteration, petrophysical properties, autoradiography, porosity, permeability, electrical conductivity, Petite Anse-Diamant hydrothermal system, Martinique

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

Introduction

Geothermal energy has been of increasing interest in recent decades, notably due to its environmentally friendly character with regard to the low carbon dioxide emissions per kWh produced by geothermal power plants [START_REF] Kagel | Promoting geothermal energy: air emissions comparison and externality analysis[END_REF]. In comparison with other energy sources, geothermal energy provides a base load power that is not subject to environmental variables such as sunshine or wind, as are photovoltaic and wind technologies.

On the other hand, the main limitations for the development of geothermal power plants are the availability and the capacity of efficiently targeting geological areas with a sufficient local temperature gradient and fluid circulation. These limiting factors could be overcome with the development of enhanced geothermal systems (EGS), seen as the future of geothermal energy by several authors [START_REF] Tester | The future of geothermal energy. Impact of Enhanced Geothermal Systems[END_REF][START_REF] Rybach | The future of geothermal energy and its challenges[END_REF][START_REF] Gerber | Environomic optimal configurations of geothermal energy conversion systems: application to the future construction of enhanced geothermal systems in Switzerland[END_REF]. Until this technology is fully operational, the success of finding new geothermal fields is reliant on exploration work intrinsically based on a cross-disciplinary approach with geological, geochemical and geophysical surveys.

Inherent to geothermal systems, hydrothermal alterations are the result of fluid/rock interactions inducing the dissolution of primary minerals of the host rocks and/or the crystallization of a wide range of minerals depending on several factors such as the fluid/rock ratio, time, temperature, pressure, fluid chemistry and permeability [START_REF] Browne | Hydrothermal alteration in active geothermal fields[END_REF][START_REF] Roberson | Smectite to illite conversion rates: effects of solution chemistry[END_REF][START_REF] Beaufort | Chemical variations in assemblages including epidote and/or chlorite in the fossil hydrothermal system of Saint Martin (Lesser Antilles)[END_REF][START_REF] Inoue | Formation of Clay Minerals in Hydrothermal Environments[END_REF][START_REF] Patrier | Role of permeability versus temperature in the distribution of the fine (< 0.2 Μm) clay fraction in the Chipilapa geothermal system (El Salvador, Central America)[END_REF][START_REF] Meunier | Clay minerals in hydrothermal systems[END_REF].

Such dissolution and crystallization processes lead to the modification of petrophysical properties such as porosity and permeability, which are essential for the understanding of the hydrogeological dynamics of hydrothermal systems, and in a more practical way, to predict the geothermal reservoir behaviour during exploitation [START_REF] Pruess | Modeling of geothermal reservoirs: fundamental processes, computer simulation and field applications[END_REF][START_REF] O'sullivan | State of the art of geothermal reservoir simulation[END_REF].

Prospecting tools such as electrical and electromagnetic (EM) methods are commonly used for the characterization of geothermal settings as well as to target high-conductivity anomalies of the subsurface [START_REF] Komori | Vertical mapping of hydrothermal fluids and alteration from bulk conductivity: simple interpretation on the USDP-1 Site, Unzen Volcano, SW Japan[END_REF][START_REF] Gonzales | Asymmetrical structure, hydrothermal system and edifice stability: the case of Ubinas volcano, Peru, revealed by geophysical surveys[END_REF][START_REF] Muñoz | Exploring for geothermal resources with electromagnetic methods[END_REF]Revil et al., 2018a;2018b;[START_REF] Ahmed | 3D electrical conductivity tomography of volcanoes[END_REF][START_REF] Lévy | The role of smectites in the electrical conductivity of active hydrothermal systems: electrical properties of core samples from Krafla volcano, Iceland[END_REF]Gailler et al., 2019). These anomalies are generally related to the presence of clay minerals resulting from fluid/rock interactions and constitute the so-called caprock formation of hydrothermal systems. The associated high conductivity results from the high cation exchange capacity (CEC) of these minerals, particularly of smectite minerals exhibiting a large amount of cations adsorbed onto their platelet surfaces (e.g., [START_REF] Lévy | The role of smectites in the electrical conductivity of active hydrothermal systems: electrical properties of core samples from Krafla volcano, Iceland[END_REF]. However, as [START_REF] Muñoz | Exploring for geothermal resources with electromagnetic methods[END_REF] reported, the highconductivity anomalies observed in the field do not always indicate the presence of a caprock and can lead to the incorrect targeting of geothermal wells. Thus, we understand that upfront costs as well as the risk related to the drilling of unproductive wells have a determinant impact on potential investments. Aimed at reducing the risk of unsuccessful wells, laboratory studies investigating hydrothermal alterations and their effects on petrophysical rock properties are essential to better understand the operation of hydrothermal systems and to interpret the data arising from the exploration phase. In the literature, a significant proportion of available papers focuses entirely or partially on the evolution of petrophysical properties depending on parameters such as temperature, pressure and lithology, without taking into account hydrothermal alterations [START_REF] Björbsson | A survey of geothermal reservoir properties[END_REF][START_REF] Boitnott | Permeability, electrical impedance, and acoustic velocities on reservoir rocks from the Geysers geothermal field[END_REF][START_REF] Nielson | Fracture permeability in the Matalibong-25 corehole, Tiwi geothermal field, Philippines[END_REF][START_REF] Dobson | Porosity, permeability, and fluid flow in the Yellowstone geothermal system, Wyoming[END_REF][START_REF] Stimac | Porosity and permeability of the Tiwi geothermal field, Philippines, based on continuous and spot core measurements[END_REF]. It is only recently that hydrothermal alterations are considered qualitatively in parallel with the above-cited parameters for the study of the petrophysical property evolution of volcanic rocks in hydrothermal contexts. Notably, and through several papers, [START_REF] Frolova | Geothermal reservoir study through petrophysical data[END_REF][START_REF] Frolova | Petrophysical properties of argillitization zone in geothermal fields[END_REF][START_REF] Frolova | Petrophysical alteration of volcanic Rocks in hydrothermal systems of the Kuril-Kamchatka Island Arc[END_REF][START_REF] Frolova | Effects of hydrothermal alterations on physical and mechanical properties of rocks in the Geysers Valley (Kamchatka Peninsula) in connection with landslide development[END_REF] studied the influence of hydrothermal alterations on the petrophysical properties of rocks from the Kuril-Kamchatka island arc by dividing the hydrothermal alterations encountered into low-temperature alterations and high-temperature alterations. [START_REF] Mordensky | Increasing the permeability of hydrothermally altered andesite by transitory heating[END_REF] on their side focused their study on high temperature hydrothermal alterations developed in Rotokawa geothermal field. Nevertheless, hydrothermal alterations are sorted from weak to high depending on the mineral paragenesis identified usually through optical microscopy observation [START_REF] Kristinsdóttir | Electrical conductivity and P-Wave velocity in rock samples from high-temperature icelandic geothermal fields[END_REF][START_REF] Wyering | Mechanical and physical properties of hydrothermally altered rocks, Taupo volcanic zone, New Zealand[END_REF][START_REF] Mielke | Effect of hydrothermal alteration on rock properties in the Tauhara Geothermal Field, New Zealand[END_REF][START_REF] Nehler | Monitoring fluid-rock interactions at In-Situ conditions using computed tomography[END_REF][START_REF] Pola | Influence of alteration on physical properties of volcanic rocks[END_REF][START_REF] Muñoz | Exploring for geothermal resources with electromagnetic methods[END_REF], 2016, Navelot et al. 2019, among others). Only a few papers quantified clay phases in low temperature environments that constitute upper parts of geothermal systems [START_REF] Heap | A multidisciplinary approach to quantify the permeability of the Whakaari/White Island volcanic hydrothermal system (Taupo Volcanic Zone, New Zealand)[END_REF][START_REF] Heap | Quantifying the role of hydrothermal alteration in creating geothermal and epithermal mineral resources: the Ohakuri ignimbrite (Taupō Volcanic Zone, New Zealand)[END_REF]. To date, only papers addressing the electrical conductivities of rocks in geothermal systems have quantitatively considered the impact of clay minerals through empirical relationships, as reported by [START_REF] Ussher | Understanding the resistivities observed in geothermal systems[END_REF] and [START_REF] Caldwell | Resistivity of rocks in geothermal systems: a laboratory study[END_REF]. This is precisely the aim of this paper, where we investigate the effect of clay mineral abundances on the porosity, permeability and electrical conductivity of a series of andesitic samples collected from the caprock formation of the hydrothermal system of Petite Anse-Diamant (Martinique). Moreover, due to the mutual dependence between the porous network and petrophysical properties, autoradiographs of rock sections obtained from 14 C-PMMA method will be analysed in parallel with mineralogical maps obtained using Quantitative Evaluation of Minerals by Scanning electron microscopy (QEMSCAN®) to identify the development of the porous network with regard to the mineral phases.

Geological settings

Located in the eastern Caribbean Sea, Martinique Island belongs to the Lesser Antilles Island arc, which has experienced volcanic activity from the late Oligocene to the present, as evidenced by the recent and deadly eruptions of Mount Pelée from 1902-1905 and from 1929-1932. The volcanic activity of the entire island arc results from the subduction of the Atlantic plate under the Caribbean plate at a rate of 2-4 cm y -1 [START_REF] Macdonald | The Lesser Antilles volcanic chain: a study in arc magmatism[END_REF]. Due to its unique position, Martinique Island has recorded the most complete history of the island arc leading to the formation of the following volcanic units, from east to west: the Basal Complex and the Sainte Anne series; the Vauclin Pitault Chain and the South Western Volcanism unit; and the Morne Jacob, Trois-Ilets, the Carbet Complex, Mount Conil and Mount Pelée, belonging respectively to the old, intermediate and recent arcs (Figure 1) [START_REF] Germa | The volcanic evolution of Martinique Island: insights from K-Ar dating into the Lesser Antilles Arc migration since the Oligocene[END_REF]. The distribution from east to west of the erupted rocks is thought to be related to the penetration of an aseismic ridge under the Caribbean plateau, which led to the flattening of the subducting slab and therefore shifted the volcanic front towards the west [START_REF] Bouysse | Subduction of atlantic aseismic ridges and late Cenozoic evolution of the Lesser Antilles Island Arc[END_REF]. Thus, the western part of the island should be the most suitable region for geothermal exploration due to the potential remnant heat sources allowing the development of geothermal systems.

The first investigations on the geothermal potential of Martinique Island were initiated in the late 1960s in the Lamentin area where thermal sources have been recognized [START_REF] Cormy | Prospection géothermique aux Antilles Françaises, Guadeloupe et Martinique[END_REF]. Several areas were investigated afterwards during exploration campaigns in the 1980s and 2000s, where the two regions of Mount Pelée and Trois-Ilets showed a strong geothermal potential, particularly the latter, in the area of Petite Anse located in the southwest of the island (Figure 1) [START_REF] Gadalia | Compléments d'exploration géothermique en Martinique: conclusions et recommandations pour les zones de La[END_REF][START_REF] Gadalia | The Petite Anse-Diamant geothermal system (Martinique Island, Lesser Antilles): Results of the 2012-2013 exploration[END_REF].

In the latter area, geochemical analysis of the local thermal spring Eaux-Ferrée revealed a reservoir temperature of 190-210°C, according to chemical geothermometers: Na-Li, Ca-K, Sr-K, Fe-K, 18O(H2O-SO4)) even if the flow rate was low (0.03 l s -1 ), presumably due to the clayey caprock preventing substantial recharge of the hydrothermal system [START_REF] Sanjuan | Geothermal exploration in the Mount Pelée volcano-Morne Rouge and Diamant areas (Martinique, West French Indies): geochemical data[END_REF][START_REF] Gadalia | Compléments d'exploration géothermique en Martinique: conclusions et recommandations pour les zones de La[END_REF][START_REF] Coppo | Exploration géophysique (magnétotellurique et gravimétrie) du potentiel géothermique de La Martinique (Montagne Pelée, Anses d'Arlet, Pitons Du Carbet et Lamentin)[END_REF]). The measured salinity of the latter thermal spring was 20 g l -1 (i.e., 2 wt %). This salinity will be used in this article as a reference during the examination of the electrical conductivities exhibited by the sample series. The dating work performed by [START_REF] Germa | The volcanic evolution of Martinique Island: insights from K-Ar dating into the Lesser Antilles Arc migration since the Oligocene[END_REF] allowed to constrain the timeline of the island's volcanic activity and to estimate that the activity in the area of Trois-Ilets occurred between 2.36 Ma and 346 ka ago. At the foot of Morne Jaqueline, where the thermal source of Eaux-Ferrée is located, evidence of the potential existence of a caprock formation has been revealed by a flank collapse, outcropping andesitic/dacitic rocks affected by argillic alteration with a montmorillonite-pyrite mineral paragenesis associated with zeolite, sulphate, calcite and iron oxides [START_REF] Genter | Réévaluation du potentiel géothermique dans les régions de Morne Rouge -Montagne Pelée et du Diamant (Martinique): Étude Géologique[END_REF][START_REF] Traineau | Compléments d'exploration géothermique en Martinique: volet géologie, Rapport Intermédiaire[END_REF]. Other types of fossil hydrothermal alteration have been recognized in the vicinity of Morne Jacqueline, with notably, a more acidic alteration represented by kaolinite-alunite mineral associations expressing an advanced argillic alteration, characteristic of fumarolic activity [START_REF] White | Epithermal environments and styles of mineralization: variations and their causes, and guidelines for exploration[END_REF]Hedenquist 1990, Gadalia et al. 2015). Silica deposits as well as scarce smectite/illite interstratified species have also been identified that should indicate the circulation of past or present fluid flows originating from higher reservoir temperatures. Materials and methods

Sampling

On the outcrop located at the foot on the side of Morne Jacqueline, a series of 11 samples with an andesitic lithology were collected (Figure 1). To obtain an increasing clay abundance through the whole sample's series, rock samples were collected depending on their apparent consolidation (poorly consolidated = clay rich) and colour, where a grey facies corresponds to low-alteration andesite and lighter/yellowish facies corresponds to a higher argillic alteration progression (Table 1). The detailed petrographic features of the whole sampling is given below section 4.1.

Quantitative Evaluation of Minerals by Scanning Electron Microscopy (QEMSCAN® analysis)

QEMSCAN® is an automatic quantitative analysis system that allows spatially resolved mineralogical data to be obtained. The system consists of the sequential acquisition of energy dispersive X-ray (EDX) spectra and back-scattered electron images (BSI) of a polished rock section. The raw chemical data obtained are then interpreted using a mineralogical database to associate each measurement point (or pixel) to a mineral. Spatial reconstruction of the interpreted data leads to a mineralogical image of the rock section investigated. In this work, the mineralogical images as well as the quantitative results acquired from the QEMSCAN® technique will be used to spatially correlate the mineral phases analysed with the connected porosity observed from 14 C-PMMA autoradiographs and to study the evolution of petrophysical properties depending on the mineral phases resulting from hydrothermal alteration.

QEMSCAN® analysis was carried out by the ERAMET Research group on 7 thin sections (from samples PA 3, PA 4, PA 7.2, PA 10.1, PA 11, PA 12 and PA 13). Chemical analysis was performed using an FEI Quanta 650 FEG scanning electron microscope mounted with two energy dispersive spectrometers (EDSs) (Bruker; Synergie 4) and a back-scattered electron detector (BSE), operating at an acceleration voltage and current intensity of 15 kV and 5 nA, respectively. The QEMSCAN® software ensured the automatic displacement of the microscope stage with a step size of 10 µm. The acquisition of the X-ray spectrum for each measurement point was considered complete for a total count of 2000 photons. The acquisition time of each thin section was approximately 10 hours. For each area analysed, the recorded EDS spectrum was attributed to one mineral phase by using the Species Identification Protocol (SIP) database [START_REF] Pirrie | Rapid quantitative mineral and phase analysis using automated scanning electron microscopy (QemSCAN); potential applications in forensic geoscience[END_REF][START_REF] Ayling | QEMSCAN (Quantitative Evaluation of Minerals by Scanning Electron Microscopy): Capability and application to fracture characterization in geothermal systems[END_REF]. To supplement the SIP database results, complementary chemical analysis of the mineral phases was performed in our laboratory using an SEM JEOL 5600 LV operating with a probe current of 1 nA and an acceleration voltage of 15 kV (detector: SiLi EDS AXS Brucker).

Crystal lattice water abundance (H2O + )

Clay minerals are known to embed a significant amount of water in the form of hydroxyls (OH) in their crystal structure. For this reason, the amount of structural water (H2O + ), generally obtained from bulk-rock chemical analysis, has been used by several authors as an indicator of the weathering grade of rocks (Jayawardena andIzawa 1994a, 1994b;[START_REF] Udagedara | Evaluation of geomechanical and geochemical properties in weathered metamorphic rocks in tropical environment: a case study from Samanalawewa hydropower project, Sri Lanka[END_REF]). In the present study, bulk measurement of the H2O + content is used to support the clay mineral abundances determined from the mineralogical maps of the QEMSCAN® system, and furthermore, we use the H2O + values as proxies for the clay mineral abundances of the whole sample series. The bulk-rock chemical analysis was conducted at the SARM laboratory of Nancy (CRPG, France) on 1 g of each sample, previously ground to reach an average size between 80 µm and 1 mm. The powder samples were heated at 105°C to remove the free and adsorbed water and then placed in an oven at 1000°C under a nitrogen atmosphere for determination of the H2O + abundance, following the Karl Fisher titration protocol.

Methylene blue

Cost-effective and easy to implement, the methylene blue (MB) method can be used during the exploration step of a geothermal project to rapidly evaluate the abundance of smectite minerals on a semi-quantitative basis. The MB method is commonly applied in civil engineering, to estimate the clay content of soils, or to determine the cation exchange capacity (CEC) of clay minerals [START_REF] Hang | Methylene blue absorption by clay minerals. Determination of surface areas and cation exchange capacities (clay-organic studies XVIII)[END_REF][START_REF] Kahr | Determination of the cation exchange capacity and the surface area of bentonite, illite and kaolinite by methylene blue adsorption[END_REF][START_REF] Lorenz | Determination of the cation exchange capacity (CEC) of clay minerals using the complexes of copper (II) ion with triethylenetetramine and tetraethylenepentamine[END_REF][START_REF] Yukselen | Suitability of the methylene blue test for surface area, cation exchange capacity and swell potential determination of clayey soils[END_REF]. MB is an organic dye with the corresponding chemical formula of C16H18ClN3S, which is positively charged when dissolved in water (C16H18N3S + ). Therefore, when the organic dye is added to a suspension containing clay minerals, the cationic dye can be adsorbed onto the negatively charged surfaces as well as onto the interlayer surfaces of the clay minerals. The MB test was conducted by preparing a suspension of 30 cm 3 using 2 g of powder with a grain size of <50 µm. The suspensions were stirred overnight to ensure disintegration of any remaining aggregates. The spot test was performed following the methodology of [START_REF] Santamarina | Specific surface: determination and relevance[END_REF], which consists of adding a methylene blue solution to the sample suspension in increments of 0.5 mL and waiting for 1 min between each MB addition. After 1 min, a drop of the suspension is placed on paper filter to evaluate if all MB molecules have been adsorbed. When MB molecules are in excess (i.e., all adsorption sites are occupied), a blue halo is formed around the aggregate. The MB value is then calculated from the volume of the MB solution used, VMB sol, and the mass of the sample (Equation ( 1)):

=

(1)

Bulk Connected Porosity

The bulk connected porosity of the samples was evaluated following the triple weight method (TW) [START_REF] Franklin | Suggested methods for the determining water content, porosity, density, absorption and related properties and swelling and slake-durability index properties[END_REF]. The method consists of the measurement of three different weights, depending on the state of the sample: dry, water-saturated and watersaturated suspended in water. Rock pieces of ~10 cm 3 were partially immersed in purified water under vacuum for imbibition. This volume was considered as sufficient in consideration of the representative elementary volume needed for the grain or mineral size distribution of the samples. Any potential fluid excesses present on the sample's surface were removed using a wet paper before measurement of the sample's weight in the saturated state. The saturated samples were dried for 48 h in an oven at 105°C to determine the dried sample weight. The sample bulk volume Vs is calculated using Archimedes' principle by Equation (2):

= (2)
ww is weight of water-saturated sample measured at ambient air. wa is weight of watersaturated sample plunged in water. ρw is the mass unit weight of water.

The pore volume Vp is calculated with Equation (3):

= ( 3
)
where wd is the weight of the dried sample.

Finally, the connected porosity ϕtw is calculated from Equations ( 2) and (3):

= = (4)
Here, it was assumed that the swelling of expandable clay minerals resulting from the complete water saturation of samples did not significantly influence porosity measurement.

Connected porosity from the 14 C-PMMA method

Bulk measurements of properties such as the electrical conductivity and permeability are intimately linked to the characteristics exhibited by the porous network of rocks (e.g., connectivity, pore type, pore size distribution, anisotropy and tortuosity) [START_REF] Avellaneda | Rigorous link between fluid permeability, electrical conductivity, and relaxation times for transport in porous media[END_REF][START_REF] Milsch | The relationship between hydraulic and electrical transport properties in sandstones: An experimental evaluation of several scaling models[END_REF][START_REF] Wright | Pore structure of volcanic clasts: measurements of permeability and electrical conductivity[END_REF]. In this section, connected porosity maps of the samples obtained by the 14 C-PMMA technique are analysed: (1) to characterize the type of porosity involved in the samples and (2) to analyse the porosity distribution with regard to the mineral phases identified in the QEMSCAN® analysis.

Quantification and imaging of the connected porosity of the samples were performed using the 14 C-PMMA methodology developed in the 1990s [START_REF] Hellmuth | Investigation of the porosity of rocks: impregnation with 14 C-Polymethylmethacrylate (PMMA), a new technique[END_REF][START_REF] Hellmuth | Study of porosity and migration pathways in crystalline rock by impregnation with 14C-Polymethylmethacrylate[END_REF][START_REF] Siitari-Kauppi | Development of 14 C-Polymethylmethacrylate method for the characterisation of low porosity media. Application to rocks in geological barriers of nuclear waste storage[END_REF]. The method consists of measuring the radiation arising from samples saturated beforehand with a 14 C-labelled tracer. Polished rock sections of impregnated samples are put into contact with an autoradiographic film (FA)

composed of an emulsion of silver bromide crystals (AgBr) of which the Ag + cations can be reduced to metallic silver by absorption of the electrons arising from the 14 C-labelled tracer.

The latter process results in a blackening of the autoradiographic film corresponding to the distribution of the radionuclide tracer and therefore the connected porosity. After exposure, the autoradiographic film is developed and digitized for image analysis. In this study, core samples with dimensions of 4 cm in diameter and 3 cm in height were dried in an oven at 40°C for one month and placed under vacuum for three hours to remove any residual water.

Impregnation of the samples with a 14 C-MMA solution of an activity of 178 kBq mL -1 was performed under vacuum for one month at the Laboratory of Radiochemistry (Helsinki, Finland). Polymerization of the 14 C-MMA solution in the porous network was ensured by heating the sample in a bath at 55°C for 16 hours. Finally, the impregnated samples were sawed and polished to ensure the optimal contact between the autoradiographic film and the rock sections. The autoradiographic films used in this work were KODAK BioMax MR films (resolution: 20 µm) for a total exposure time of ~4 days. Digitization of the autoradiographic film into grey-level 16-bit depth images was performed using a Microtek Artix Scan F1

scanner with a resolution of 2400 dpi (pixel size: 10.6 µm). Due to the non-linear response of the autoradiographic film to irradiation, 14 standards of pure 14 C-PMMA with known activities were exposed at the same time to provide an empirical relationship between the blackening of the film and the connected porosity. However, the maximal range of electrons emitted by the 14 C in PMMA has been estimated by [START_REF] Kanaya | Penetration and energy-loss theory of electrons in solid targets[END_REF] at ~224 µm, which is significantly higher than the thickness of the thin sections used for the acquisition of the autoradiographs (~30 µm). Following this condition and since the thickness of the 14 C-PMMA plugs used as standards for the calibration have a thickness on the order of a few millimetres, the porosities calculated from the calibration process are underestimated.

Table 2 provides an overview of comparison of 14 C-PMMA porosity method with more popular imaging methods, such as Scanning Electron Microscope (SEM) and X-Ray Computed Microtomography (XRCT). Other class of methods provide pore size distribution (Hg-porosimetry and adsorption methods), or provide merely bulk porosity value (TW method and He-pycnometry). The main advantage of 14 C-PMMA method consists on his ability to provide quantitative porosity maps which are easy to analyse, and which account all connected pores of the studied section, regardless of pore size (it is why this method is sometimes called multi-scale). For instance, concerning microcracks, the method is able to map at core scale all cracks, whatever their aperture [START_REF] Mazurier | Development of a fracture network in crystalline rocks during weathering: Study of Bishop Creek chronosequence using X-ray computed tomography and 14C-PMMA impregnation method[END_REF][START_REF] Bonnet | Determining crack aperture distribution in rocks using the 14C-PMMA autoradiographic method: experiments and simulations[END_REF]. This is not the case for XRCT or SEM, which does not provide porosity maps, but where pore can be detected by image segmentation. The smaller pores are not detected using these methods because their size are smaller than pixel or voxel size. In fact, for these conventional imaging methods, the resolution is a function of the sample size, controlling the pixel or voxel size. 14 C-PMMA has his own drawbacks, such as the need to manipulate open radioactive sources, and the fact that it is not 3D (Table 2). However, the cost remains the same than XRCT, and the total analysis time is also quite comparable to other porosimetric methods. As emphasized by Table 2, the bulk methods have not to be ignored, because they provided reference values for porosity calculation.

Intrinsic Water Permeability

We used here the same device employed by [START_REF] Sardini | Quantification of microscopic networks by image analysis and measurement of the permeability of the Soultz-sous-Forêts granite (Alsace, France)[END_REF], except that core samples with a diameter of 35 mm were covered on their side with LOCTITE EA 9483 epoxy resin to avoid any lateral leakage of water during the permeability experiment. The high viscosity of this epoxy resin prevents intrusion in the pore space of the samples. The thicknesses of the core samples range from 10 to 15 mm. The sample thicknesses were large compared to rock grain size in order to have a measurement representative of the whole rock.

Large phenocrysts of feldspars or hornblende can be however the same size as the sample thickness used for these measurements. It was not possible to use thicker samples because the maximal thickness acceptable by the used device is 2.5 cm. Figure 2 shows the experimental setup for the permeability measurement.

Permeability experiments were performed in steady state flow conditions, using a pressure gradient (ΔP) kept between 1 and 2 bar across the samples. Two nitrogen reservoirs imposed the up and downstream pressure into the H2O vessel (Figure 2). These reservoirs were isolated from the water circuit by an impervious membrane; the pressures imposed by the inflation of these N2 reservoirs provided the pressure gradient necessary to the flow of water through the rock sample. The differential pressure was recorded during the experiment, and the flow rate of water was measured using a capillary (Figure 2). The experimental flow rate being very low (ranged between 0.7 to 4x10 -4 mm 3 /s), the permeability was calculated assuming a laminar flow regime, using Darcy's law:

= !" (5)
where k is the intrinsic permeability (m²), Q is the measured flow rate (m 3 s -1 ), η is the dynamic viscosity (Pa.s), S is the surface area of the plug sample corrected from the impregnation resin (surface efficient for the water flux) (7.07 10 -4 m²) and l is the length of the core sample.

Electrical conductivity

Electrical measurements were performed with a Solartron Impedance Analyser 1260 using 6 NaCl brines with increasing salinity levels of 0.01; 0.05; 0.1; 0.5; 1 and 2 wt %. Note that the range of salinities used encompasses the range of total salt concentrations commonly found for geothermal fluids (0.1 -1 wt %) [START_REF] Henley | Geothermal systems ancient and modern: a geochemical review[END_REF]. Sample saturation was performed at 20°C by immersion in NaCl brines for two weeks.

A resistivity cell of Teflon was designed to receive the plug samples used for the permeability measurements (Figure 3). After placing the plug sample in the resistivity cell, NaCl brine was added through the two upper apertures of the cell to ensure electrical conduction between the electrodes and the surface of the plug sample. Acquisition of the impedance spectra was performed with the two-electrode configuration in the 0.1 -3•10 6 Hz frequency range. The conductivity magnitude |$| was obtained with following Equation ( 6):

|$| = % |&| (6)
where the impedance amplitude |Z| has been selected at a frequency close to 1 kHz (e.g., [START_REF] Lévy | The role of smectites in the electrical conductivity of active hydrothermal systems: electrical properties of core samples from Krafla volcano, Iceland[END_REF] for which the imaginary part of the complex impedance Z was minimum. For simplicity in writing, the conductivity magnitude, |$|, will be simply named the electrical conductivity, σ, hereafter.

Results and discussion

Progression of hydrothermal alteration

The hydrothermal alteration exhibited by the sample series has already been discussed thoroughly in a previous article [START_REF] Delayre | Couplages altérations hydrothermales du caprock / propriétés pétrophysiques des roches en contexte géothermal de haute enthalpie[END_REF]. In this article, we report the main results and petrographic features of the samples that we believe play a significant role in the evolution of the petrophysical properties investigated.

Figure 4 presents selected areas of the mineral maps obtained in the QEMSCAN® analysis of samples PA 13 and PA 4 and PA 7.2, PA 10.1, PA 12 and PA 11. For the whole sample series, the mineral phases identified in the QEMSCAN® analysis consist of feldspar minerals belonging to the plagioclase series (andesine, labradorite, oligoclase and bytownite), hornblende and quartz, corresponding to primary minerals, while alteration paragenesis is represented by clay minerals (montmorillonite and kaolinite), iron oxides, carbonates and sulphide minerals. The matrix of the rock with the above-mentioned primary minerals embedded corresponds to a mesostasis where feldspar microlites and devitrification of glass have been observed using optical microscopy in a previous work [START_REF] Delayre | Couplages altérations hydrothermales du caprock / propriétés pétrophysiques des roches en contexte géothermal de haute enthalpie[END_REF]. The alteration paragenesis exhibited by the samples is very common in hydrothermally altered volcanic rocks and can be identified as argillic alteration [START_REF] White | Epithermal environments and styles of mineralization: variations and their causes, and guidelines for exploration[END_REF][START_REF] Pirajno | Hydrothermal processes associated with meteorite impacts[END_REF]). From a more petrographic point of view, the size of the feldspar and hornblende crystals ranges from the sub-millimetre to the millimetre and frequently exhibit rectangular and sub-hexagonal sections, respectively (Figure 4). The lowest alteration intensity is observed in sample PA13, which has been discriminated as two different samples, PA13.1 and PA13.2, where the latter corresponds to the wall-rock area of small fractures characterized by higher abundances of silica and iron oxides, as observed during field sampling (arrows 1 and 2, respectively, sample PA13.2 in Figure 4). Crystallization of montmorillonite produces a patchy alteration pattern in the mesostase along with a moderate alteration of hornblende crystals, as shown in the PA 13.1 sample (arrow 3, Figure 4).

Intragranular alteration of hornblende crystals is clear in the PA 4 sample, where montmorillonite crystallizes in association with the iron oxide phases along hornblende cleavages intersecting at 120° (Figure 4). Among the samples investigated, a particular feature is found for PA7.2, where a vein of predominantly montmorillonite is observed and crosses the entire sample (Annex 1). In Figure 4, the PA10.1 sample exhibits a higher alteration intensity revealed by the stronger pervasiveness of alteration of montmorillonite and silica present throughout the mesostase and hornblende crystals, which, for this sample, are completely replaced by montmorillonite and sulphide minerals (± silica ± iron oxides)

(arrows 1 and 2, respectively; Figure 4). With regard to the alteration intensity, the end member of this sample series is represented by PA 11, where intense pervasive alteration is observed (Figure 4). In addition to the total replacement of the hornblende crystals by montmorillonite and silica (± kaolinite) (arrow 3, PA 11, Figure 4), the PA 11 sample presents frequently open cavities resulting from the quasi-total dissolution of feldspar crystals in which few remnants can still be observed (arrow 2, PA 11, Figure 4). This dissolution vugs are often completely or partially filled with kaolinite and silica and to a lesser extent with montmorillonite (arrow 1, PA 11, Figure 4). In this last sample, such a strong occurrence of kaolinite could be attributed to advanced argillic alteration resulting from the circulation of more acidic (low pH) fluids. However, the absence of minerals such as alunite, typical of advanced argillic alteration, and the low crystallinity of kaolinite revealed by X-ray diffraction (XRD) analysis could indicate a weathering origin [START_REF] Hughes | Crystallinity of kaolin minerals and their weathering sequence in some soils from Nigeria, Brazil and Colombia[END_REF][START_REF] White | Epithermal environments and styles of mineralization: variations and their causes, and guidelines for exploration[END_REF][START_REF] Delayre | Couplages altérations hydrothermales du caprock / propriétés pétrophysiques des roches en contexte géothermal de haute enthalpie[END_REF].

Quantitative evaluation of hydrothermal alteration

The quantitative results obtained in the QEMSCAN® analysis are presented in Table 3, in which samples were ranked from left to right with increasing clay mineral abundances.

Overall, good agreement is found between the quantitative results and the qualitative characterization performed in the previous section. The abundance of feldspar minerals is relatively stable throughout the sample series, varying between 30 and 40 %, except for the PA12 and PA11 samples, which display abundances of 23.31 and 4.27 %, respectively, due to the partial or total dissolution of feldspar crystals. Feldspars are the main source of alumina for montmorillonite and kaolinite. This explain their decreasing amount as the alteration degree increases. This replacement is confirmed by the shape of montmorillonite aggregates as observed in PA11 (rectangular sections in Figure 4).The raw quantitative results do not seem to reflect the replacement or dissolution of the mesostase. However, a general decreasing trend is found when abundance recalculation is performed considering the white pixels in the mineral maps.

As observed in the mineral maps, only the PA 13.2 sample, the least argillized of the sample series, shows a high abundance (13.9 %) of hornblende, where due to the higher alteration intensity, this value falls below 1 %. Nevertheless, no significant trend of hornblende abundance can be found with increasing amounts of clay mineral phases, as depicted by the PA10.1 sample exhibiting a hornblende abundance of 0.1 %, considered moderately argillized. The abundances of carbonates and iron oxides are low (i.e., < 3 %) and erratic throughout the sample series even though PA13.2 presents a slightly higher iron oxide abundance. With regard to the major hydrothermal alteration products, both kaolinite and montmorillonite are ubiquitous in all samples, where for the latter, an increasing trend from 4.69 to 22.74 % is observed. For the former, the abundances are systematically below 1 %, with the exception of samples PA 12 and PA 11, which display abundances of 3.92 and 14.82 %, respectively.

As mentioned in the materials and methods section in this study, only certain samples of the whole series were selected for QEMSCAN® analysis. Thus, to include the remaining samples in the petrophysical analysis, the abundance of structural water in each sample was determined in view of using its value as a proxy for the clay mineral abundance. The results are presented in Table 4 and Figure 5, where the total clay mineral abundances are plotted against the H2O + values obtained from the bulk-rock chemical analysis.

A good and linear correlation is found (R²= 0.9854) between the total clay mineral abundances and crystal lattice water proportions, which therefore confirms the assumption established previously. However, such indicators should be handled carefully when used for the semi-quantitative evaluation of clay minerals. Indeed, several hydrothermal minerals (e.g., the zeolite group, alunite, epidote, pyrophyllite) can also exhibit embedded water in their crystalline structures and therefore may bias the results if used without caution. For the first two samples, PA12 and PA14, no significant infilling or evidence of fluid-rock interactions in the vicinity of the microfractures was observed in the mineral maps. This could indicate that the generation of the observed microfractures was posterior to hydrothermal alteration. Conversely, deposition of iron oxides as well as carbonates was observed in the main fracture and in the dissolution vugs. The PA10.1 sample revealed that these structures were used as pathways for hydrothermal fluid circulation. In the autoradiographs, such crystallization of carbonates is associated with low porosity areas, as observed in the PA10.1 and PA12 samples (arrow 3 and arrow 2 in Figure 6). Additionally, deposition of silica (and presumably of iron oxides) tends to have a similar impact by clogging the connected pores and therefore reducing the overall average porosity of the rock. This process is particularly notable for the PA13 sample, where, in the autoradiographs, a large porosity contrast is found between PA13.1 and PA13.2, displaying silica abundances of 4.91 and 8.64 %, respectively (Table 3). The sulphide minerals are also congruent with the low porosity areas when infilling dissolution vugs or in pseudomorphosed hornblende crystals (arrow 1, PA10.1, Figure 6).

Comparison of the mineral maps and autoradiographs from the 14 C-PMMA method

Overall, an increasing trend of the connected porosity is found for the whole sample series when calculated from the autoradiographs (Table 4), although underestimated, as explained in section 3.6. In the autoradiographs presented, the porous areas are associated mainly with the clay mineral phases, dissolution vugs and fractures and are visible as dark areas in the autoradiograph. Notably, montmorillonite crystallizing along hornblende cleavages is associated with porous areas (darker) as clearly revealed by the autoradiograph of the PA 4 sample (arrow 1, Figure 6). Destabilization of the mesostase through dissolution and replacement by mineral paragenesis leads to a global porosity increase of the rock matrix where even the pseudomorphosed hornblende crystals are difficult to discriminate from the rock matrix (arrows 3 and 1 for PA12 and PA11, respectively, Figure 6). Conversely, the dissolution vugs associated with feldspar crystals in PA11 are clearly distinguished from the rock matrix (arrow 2, PA 11, Figure 6). Infilling of kaolinite, observed in the same feldspar crystal, seems to reduce the porosity. Only quartz crystals remain unaffected by hydrothermal alteration and can be easily observed due to their lower porosity (arrow 3, PA11, Figure 6). revealing that in the rock series investigated, the dominant process of porosity creation is related to the crystallization of clay minerals (i.e., montmorillonite and kaolinite). Moreover, the silicification and crystallization of iron oxides reported above for sample PA13.2 compared to PA13.1 seems to have a great impact on the connected porosity, inducing a decrease from 10.5 to 4.48 %. The moderate scattering of porosity data at low H2O + values is attributed to the presence of fractures and dissolution vugs causing an increase in connected porosity, as observed for the PA10.1 sample.

Evolution of the bulk connected porosity and intrinsic permeability

The intrinsic permeabilities measured for the sample series, ranging from 7.7 10 -20 to 5.4 10 -17 m², show an increasing trend with the connected porosities measured by the TW method (Figure 7,B). The highest permeability value is found for the PA11 sample and seems unexpected since this sample is the most argillized among the samples in the present series.

This finding should be related to the strong destabilization of the rock matrix as well as to the dissolution vugs developed at the expense of the feldspars, as observed in the mineral maps and autoradiographs. Numerous data of permeability reported in the literature for effusive andesites [START_REF] Wright | Pore structure of volcanic clasts: measurements of permeability and electrical conductivity[END_REF][START_REF] Farquharson | Permeability and porosity relationships of edifice-forming andesites: a combined field and laboratory study[END_REF][START_REF] Mielke | Effect of hydrothermal alteration on rock properties in the Tauhara Geothermal Field, New Zealand[END_REF][START_REF] Heap | Exploring the scale-dependent permeability of fractured andesite[END_REF], Heap et al., 2018[START_REF] Mordensky | Physical and mechanical property relationships of a shallow intrusion and volcanic host rock, Pinnacle Ridge, Mt. Ruapehu, New Zealand[END_REF] among others) exhibit a range of permeability from 10 -17 to 10 -11 m². Nevertheless lower values down to 10 -19 -10 -20 m 2 are also mentioned [START_REF] Wollentz | Volcanology and geothermal energy[END_REF][START_REF] Petrov | Deformation of metavolcanics in the Karachay Lake area, Southern Urals: petrophysical and mineralchemical aspects[END_REF][START_REF] Navelot | Petrophysical properties of volcanic rocks and impacts of hydrothermal alteration in the Guadeloupe Archipelago (West Indies)[END_REF]. It should be noted that most of the values reported in the literature are gas permeabilities. Other data can be found in the literature on lavas or lavas block of various compositions [START_REF] Bernard | Transport Properties of Pyroclastic Rocks from Montagne Pelée Volcano (Martinique, Lesser Antilles)[END_REF][START_REF] Dobson | Porosity, permeability, and fluid flow in the Yellowstone geothermal system, Wyoming[END_REF][START_REF] De Maisonneuve | Characterization of juvenile pyroclasts from the Kos Plateau Tuff (Aegean Arc): insights into the eruptive dynamics of a large rhyolitic eruption[END_REF]among others) or on pyroclastic materials [START_REF] Degruyter | Controls on magma permeability in the volcanic conduit during the climactic phase of the Kos Plateau Tuff eruption (Aegean Arc)[END_REF][START_REF] Heap | A multidisciplinary approach to quantify the permeability of the Whakaari/White Island volcanic hydrothermal system (Taupo Volcanic Zone, New Zealand)[END_REF][START_REF] Heap | Quantifying the role of hydrothermal alteration in creating geothermal and epithermal mineral resources: the Ohakuri ignimbrite (Taupō Volcanic Zone, New Zealand)[END_REF]. The permeability data reported in the literature are 2 to 4 orders of magnitude higher than the permeabilities measured in this work. Part of this shift could be explained by the fact that permeability values found in the literature are mainly gas permeability while in the present study we determined water permeability. This latter value is probably closer to real permeability in natural systems affected by smectitic alteration. Indeed smectitic material consists in expandable clay phases which can strongly affect the permeability in water saturated conditions. As an illustration in Krafla system, Escobedo (2018) observed 2-3 orders of magnitude between gas and water permeabilities in hyaloclastic formations affected by smectitic alteration. From this observation, we deduce that hydrothermal alteration induces a consequent decrease in the permeability (at least one order of magnitude) due to the formation of expandable clay minerals. The subsequent dissolution of the rock matrix and pre-existing minerals, however, counteracts this process by opening new pathways for fluid circulation, thereby enhancing permeability. Intermediate clay-rich facies that would complete the data set between PA10.1 and PA11 would be needed to increase the robustness of the relationship observed.

Unfortunately, such samples were not recognized at the investigated scale due to the sharpness of the transition between very altered to poorly altered zones.

Impact of argillic alteration on the electrical conductivity

Figure 8 shows the variation in bulk conductivity measured for each sample depending on the electrical conductivity of the saturating fluid on a log-log scale. For all samples, the bulk conductivity increases with increasing electrical conductivity and presents the typical evolution observed for argillized rocks containing clay minerals with a significant cation exchange capacity (CEC) [START_REF] Revil | Electrical properties of zeolitized volcaniclastic materials[END_REF][START_REF] Flóvenz | The role of electrical interface conduction in geothermal exploration[END_REF][START_REF] Komori | Vertical mapping of hydrothermal fluids and alteration from bulk conductivity: simple interpretation on the USDP-1 Site, Unzen Volcano, SW Japan[END_REF]. Indeed, the electrical conduction in saturated rocks typically involves two different mechanisms: electrolytic conduction, related to the migration of cations and anions through the connected pore space, and surface conduction, enabled by the presence of high-CEC minerals (e.g., smectite), present at the surface as weakly adsorbed cations. The latter mechanism is generally justified through the formation of an electrical double layer at the mineral surface when put in contact with an electrolyte [START_REF] Gouy | Sur la constitution de la charge électrique à la surface d'un électrolyte[END_REF][START_REF] Chapman | LI. A Contribution to the Theory of Electrocapillarity[END_REF][START_REF] Stern | Zur theorie der elektrolytischen doppelschicht[END_REF]. Both mechanisms are represented in Figure 8. At a low solution conductivity (from 200 to ~2000 µS cm -1 ), surface conduction is the dominant electrical conduction mechanism and results in the apparent flat part of the plot, especially notable for the PA 12 sample, while at a higher solution conductivity (from ~10,000 to 30,000 µS cm -1 ), electrolytic conduction dominates, resulting in the linear increasing trend (log-log scale) observed.

As expected, the two most argillized samples (i.e., PA12 and PA11) have significantly higher bulk electrical conductivities than the rest of the samples due to the higher abundances of montmorillonite. However, the PA 10.1 sample, which was considered to be moderately argillized (clay mineral abundance: 8.09 %), exhibited an excess electrical conductivity. From the QEMSCAN® analysis, a strong association of montmorillonite with sulphide minerals was observed, unique among the samples investigated. If these sulphide minerals constitute a connected network, they could be the reason for the high electrical conductivity. A third mechanism of electrical conduction could therefore be involved in the PA 10.1 sample corresponding to electronic conduction, in which the charge carriers are the valence electrons located in the outer shell of metallic atoms.

In the area of Petite-Anse, the magnetotelluric survey performed during the 2012-2013 exploration phase by [START_REF] Coppo | Exploration géophysique (magnétotellurique et gravimétrie) du potentiel géothermique de La Martinique (Montagne Pelée, Anses d'Arlet, Pitons Du Carbet et Lamentin)[END_REF] revealed electrical resistivities < 10 Ohm.m at shallow depths. This value is very similar to the electrical resistivities measured in this work for the most argillized samples PA11 and PA12 exhibiting resistivities (after conversion from conductivities) ranging from 9.6 to 1.8 Ohm.m and from 8.0 to 2.7 Ohm.m, respectively, which are in the range of the saturating solution salinities used.

In Figure 9 (A), the bulk conductivities of the samples saturated with the 2 wt % NaCl solution are plotted against the structural water proportions and methylene blue values (A and B, respectively). An increasing trend is observed between the bulk conductivities and structural water proportions with a relatively good correlation (R²= 0.8591), as expected from the increasing contribution of the surface conductivity resulting from the increasing montmorillonite abundances. From this figure, it seems nevertheless difficult to discriminate the samples with low H2O+ values (low clay mineral abundances). A better correlation coefficient is found (R²= 0.9579) when the electrical conductivities are plotted against the methylene blue values (Figure 9,B). This results from the fact that the methylene blue value is mainly representative of the clay mineral abundance with a high CEC. On the other hand, structural water proportion measurement integrates the kaolinite abundance, a clay mineral known to have a low CEC and therefore contributes to a limited extent to electrical conduction, hence the lower correlation coefficient.

Several indicators have been used in the literature to evaluate the contribution of the surface conductivity arising from clay minerals, especially smectite. The surface or interface conductivity is generally derived from electrical conductivity models, such as the one of [START_REF] Waxman | Electrical conductivities in oil-bearing shaly sands[END_REF] or more recently, the one of [START_REF] Revil | Nature of Surface Electrical Conductivity in Natural Sands, Sandstones, and Clays[END_REF]. In this work, to evaluate the contribution of surface conductivity, we used the isoconductivity point, as defined by [START_REF] Shainberg | Effect of exchangeable sodium percentage, cation exchange capacity, and soil solution concentration on soil electrical conductivity[END_REF] corresponding to the conductivity value for each sample where its bulk conductivity and solution conductivity are equal.

Figure 10 presents the plot of this indicator against the methylene blue value. As shown in Figure 13, we were able to determine the isoconductivity point only for 5 samples (i.e., PA 3.2, PA 5.1 PA 10.1 PA 12 and PA 11) due to the low bulk conductivities of the remaining samples. A strong and linear correlation is found (R²= 0.9806) between the isoconductivity point and methylene blue value, revealing the strong relation between this indicator and the abundance of montmorillonite.

Conclusion

In this work, the evolution of the petrophysical properties (i.e., connected porosity, permeability and electrical conductivity) of the samples collected at the Petite Anse-Diamant hydrothermal system was investigated on the basis of the quantitative evaluation of minerals by scanning electron microscopy (QEMSCAN®) method. The crystal lattice water abundance was found to be a relevant indicator of the clay mineral abundance for the sample series. This work suggests the need for further investigations in two directions. First, this work has accounted for classical petrophysical properties (bulk connected porosity, permeability and electrical conductivity), but recent studies carried out in similar contexts have shown the possibility of using new non-intrusive indicators based on low-frequency electromagnetic measurements (e.g., the quadrature conductivity and the normalized chargeability; see [START_REF] Revil | Alteration of volcanic rocks: A new non-intrusive indicator based on induced polarization measurements[END_REF][START_REF] Ghorbani | Complex conductivity of volcanic rocks and the geophysical mapping of alteration in volcanoes[END_REF][START_REF] Lévy | Electrical resistivity tomography and time-domain induced polarization field investigations of geothermal areas at Krafla, Iceland: comparison to borehole and laboratory frequency-domain electrical observations[END_REF]. These new indicators can be useful in supplementing the information provided by conventional electrical resistivity measurements and should also be compared to mineralogical and petrographic data.

Second, the methodology used in this work based on the coupling of innovative techniques (mineral mapping, 14 C-PMMA method and petrophysical properties) could be applied to investigate the impact of HT hydrothermal alteration (200-300°C) located at the reservoir level of high-enthalpy hydrothermal systems. These studies would clearly require an adaptation of the experimental conditions through measurement of the same properties as a function of the temperature and pressure. 5. Conductivities of the solutions saturating the samples and bulk conductivities (expressed in µS.cm -1 ).

Figure captions

  The strong association of montmorillonite and sulphide minerals is not systematic throughout the sample series investigated and should reveal either a chemical variation in fluid composition affecting the rocks or local variation in the pre-existing lithology. Such variation in fluid composition is supported by the crystallization of carbonates, with calcite as the major mineral phase, manifested as infilling of fractures and dissolution vugs in sample PA10.1.Strong pervasive alteration is observed in the PA 12 sample, where at this stage, complete replacement of the hornblende crystals by montmorillonite, strongly associated with iron oxides, is observed (arrow 1, PA 12, Figure4). Dissolution of the mesostase as well as of feldspar minerals is revealed by the increased number of white pixels corresponding to porosity in the mineral maps obtained in the QEMSCAN® analysis. At this stage, only quartz crystals remain unaffected by hydrothermal alteration.

Figure 6

 6 Figure 6 presents the mineral maps of the PA13, PA4, PA10.1, PA12 and PA11 samples in parallel with autoradiographs of the same areas. Notably, several fractures undetected by microscopy are clearly revealed by the autoradiographs of samples PA13, PA4 and PA10.1.

Figure 7 (

 7 Figure 7 (A) presents the evolution of the connected porosity estimated from the triple weight method as a function of the structural water proportion (H2O + ), used in this study as a proxy of the clay mineral abundance. The connected porosities of the samples range from 3.74 to 35.35 % and show strong linear relationships with the H2O + values (R²= 0.8835),

From

  the 14 C-PMMA autoradiograph analysis, the development of argillic alteration, sometimes superimposed by weathering alteration (i.e., PA 11), leads to an increase in the connected porosity, primarily due to the replacement of the rock matrix and pre-existing minerals by clay minerals, identified here as montmorillonite and kaolinite. Dissolution vugs and fracturing were found to be significant in several samples and contribute to the increases in connected porosities observed. The crystallization of silica, iron oxides and carbonates counteracts this process, leading to a decrease in the connected porosity. The permeability measurements revealed a significant decrease of 2 to 4 orders of magnitude when compared to the permeability data acquired for andesites in the literature. No clear relation to the increase in clay mineral abundance was observed; however, the low permeabilities classically observed for rocks belonging to the caprock formation should be reached during the first steps of argillic alteration development. The electrical conductivity measured for the whole sample series shows a linear relationship with the montmorillonite abundance, represented in this work by the methylene blue value, which is easy to acquire in the laboratory and in the field.Consequently, these new data obtained by coupling different techniques provide three results in a practical and modeling viewpoint. First, they confirm the interest of using geophysical methods based on resistivity measurement (e.g., Direct Current resistivity method and low-frequency electromagnetic methods) to locate caprock formations especially rich in montmorillonite. Let us recall that these geophysical techniques referred as electrical methods share the advantages of being fast, non-intrusive and covering wide areas of investigation in comparison with conventional methods (e.g., drilling, field sampling). Second, our data show that there exist no clear relationship between permeability and clay abundance and thus between permeability and electrical resistivity. Consequently, there is little hope to characterize quantitatively in the field the hydraulic properties of caprock formations by electrical methods. Third, the combination of mineral maps and spatial distribution of the connected porosity observed in autoradiographs allows to clearly discriminate two types of porosity existing in caprock formations: porosity associated with clay mineral phases and that related to dissolution vugs and fractures. Thus our approach provides a quantitative petrographycal basis for modeling the hydraulic properties of caprock formations considered as dual-porosity systems.

Figure 1 :

 1 Figure 1: Locations of the Petite Anse-Diamant area on Martinique Island (A) and the samples investigated in this study (B). The red point (E-F) indicates the location of the Eaux-Ferrées thermal source (source: IGN, French institute of geography).

Figure 2 :

 2 Figure 2: (A) Experimental setup for the permeability measurements (Sammartino et al. 1999). (B) Cross-section of the permeability cell with (1) the pressure vessels of stainless steel, (2) the confining cell and (3) the plug sample. (C) Perspective view of the plug sample with the diameters of the core sample and plug opening (mm).

Figure 3 :

 3 Figure 3: Resistivity cell used for acquisition of the impedance spectra.

Figure 4 :

 4 Figure 4: QEMSCAN® mineral maps of samples PA13.2, PA13.1, PA4, PA 7.2, PA10.1, PA12 and PA 11. White areas are pixels detected as porosity. Sample PA13 is the freshest sample of andesite, presenting moderate alteration of hornblende to montmorillonite at the bottom of the section (arrow 3), and silica and oxides abundance in the mesostase (arrows 1and 2). In sample PA4, intragranular alteration of hornblende crystals is underscored by montmorillonite and oxides in cleavage planes. Sample PA10.1 exhibits pervasive alteration of the mesostase (presence of montmorillonite, silica, and sulphide minerals) (arrows 1,2). In sample PA12, hornblende crystals are totally replaced by montmorillonite and iron oxides (arrow 1); carbonate veins are also detected (arrow 2). Sample PA11 is extremely altered; hornblende is totally replaced by silica and montmorillonite (arrow 3), and feldspars phenocrysts are partially dissolved, containing dissolution voids( arrow 2), which are partially filled with kaolinite and silica (arrow 1). In this sample, quartz grains remains unaltered.

Figure 5 :

 5 Figure 5: Correlation plot between the abundances of the clay phases identified by the QEMSCAN® system and the crystal lattice water proportions obtained from bulk-rock chemical analysis. Mt = montmorillonite and K = kaolinite.

Figure 6 :

 6 Figure6: QEMSCAN® Mineral maps (left) and related autoradiographs (right) from the 14 C-PMMA method of samples PA 13, PA 4, PA 10.1, PA 12 and PA 11. In autoradiographs, the darker the grey level is, the higher the porosity is. In the freshest sample (PA13) the most porous mineral is hornblende, partially replaced by montmorillonite in PA 13.1 side (arrow 1). In PA4 sample, porosity is well developed in hornblende cleavage planes (arrow 1), and corresponds to montmorillonite; feldspars remains lowly porous (arrow 2). In sample PA 10.1, low porosity area are initial feldspar phenocrysts (arrow 2), but are also in sulphide of pseudomorphosed hornblende (arrow1) and in carbonate cements. Montmorillonite-rich areas and in a less extent mesostase present an higher porosity. In PA12, Feldspars remain unaltered and lowly porous (arrow 1), as well carbonate. High porosity of pseudomorphosed hornblende cannot be differentiate from the porosity of the mesostase (arrow 3). Sample PA11, which is the most affected by hydrothermal alteration, presents a totally different porosity pattern than sample PA12. In this sample, quartz grains remains the only lowly porous mineral (arrow 3). The connected mesostase matrix is porous, but pseudomorphosed hornblende (arrow 1) and altered/dissolved feldspars associated with dissolution voids (arrow 2) are more porous.

Figure 7 :

 7 Figure 7: (A) Plot of the connected porosity of the samples against the structural water content (H2O + ) value obtained with the triple weight (TW) method and from bulk-rock chemical analysis. The H2O + values are used as indicators of the clay mineral abundances (see section 2.2). (B) Darcian permeability against the connected porosity.

Figure 8 :

 8 Figure 8: Bulk conductivities versus conductivities of the solutions saturating the samples. The 1:1 line is used to estimate the isoconductivity point of each sample.

Figure 9 :

 9 Figure 9: Correlation plots of the bulk conductivity measured for the samples saturated with the 2 wt % NaCl solution depending on the structural water proportion (H2O + ) (A) and blue methylene (BM) value (B).

Figure 10 :

 10 Figure 10: Evolution of the isoconductivity point depending on the blue methylene (BM) value.

Figure 1 :

 1 Figure 1: Locations of the Petite Anse-Diamant area on Martinique Island (A) and the samples investigated in this study (B). The red point (E-F) indicates the location of the Eaux-Ferrées thermal source (source: IGN, French institute of geography).

Figure 2 :

 2 Figure 2: (A) Experimental setup for the permeability measurements (Sammartino et al. 1999). (B) Cross-section of the permeability cell with (1) the pressure vessels of stainless steel, (2) the cell core (in PEEK) and (3) the plug sample. (C) Perspective view of the plug sample with the diameters of the core sample and plug opening (mm).

Figure 7 :

 7 Figure 7: (A) Plot of the connected porosity of the samples against the structural water content (H2O + ) value obtained with the triple weight (TW) method and from bulk-rock chemical analysis. The H2O + values are used as indicators of the clay mineral abundances (see section 2.2). (B) Intrinsic permeability against the connected porosity.

Figure 9 :

 9 Figure 9: Correlation plots of the bulk conductivity measured for the samples saturated with the 2 wt % NaCl solution depending on the structural water proportion (H2O + ) (A) and blue methylene (BM) value (B).

Table captions Table 1 :

 captions1 Description of color and consolidation of each samples.

Table 2 .

 2 Comparison of 14 C-PMMA porosity method with conventional imaging methods.

Table 3 :

 3 Quantitative evaluation of rock mineral composition from QEMSCAN® analysis.

Table 4 :

 4 Crystal lattice water proportions (H2O + ) from bulk-rock chemical analysis; connected porosities ϕtw and ϕFA obtained by triple-weighted method (TW) and from film autoradiography (FA) respectively and Darcian permeabilities k of samples.

Table 5 .

 5 Conductivities of the solutions saturating the samples and bulk conductivities (expressed in µS.cm -1 ).

	1044
	1045
	1046
	1047
	1048
	1049
	1050
	1051
	1052
	1053
	1054
	1055
	1056
	1057

Table 1 :

 1 Description of color and consolidation of each samples.

		Consolidation	Color
	PA 1	good	grey
	PA 2	good	grey
	PA 3.1	good	grey
	PA 3
	PA 3.2	good	red wall-rock (FeOx)
	PA 4	good	grey
	PA 5.1	moderate	grey yellowish
	PA 7.2	moderate	whitish grey
	PA 10.1	good	dark grey
	PA 11	poorly consolidated	yellowish
	PA 12	poorly consolidated	greenish
	PA 13.1	good	whitish grey
	PA13
	PA 13.2	very hard	red wall-rock (FeOx)

Table 4 :

 4 Crystal lattice water proportions (H2O + ) from bulk-rock chemical analysis; connected porosities ϕtw and ϕFA obtained by triple-weighted method (TW) and from film autoradiography (FA) respectively and Darcian permeabilities k of samples.

	Solution

Acknowledgements:

The authors are grateful to Juuso Sammaljärvi, Eveliina Muuri and Marja Siitari-Kauppi for the fruitful discussions on the 14 C-PMMA method and for the preparation of the samples. This work was partly carried out with the financial support of the ANR Agence Nationale de la Recherche (French National Research Agency), project ANR-17-CE06-0012-03 ExCiTING (Exploration des ressources géologiques et des reservoirs par prospection géophysique des propriétés des argiles). The authors also acknowledge financial support from the European Union (ERDF) and Région Nouvelle Aquitaine. We would like to thank Kristian Bär and Jamie Farquharson for their thorough review which helped us improve the manuscript. Appendix A. Supplementary data. QEMSCAN® maps.

Water porosity by Triple Weighting

Bulk value 3D Connected dm 3 to mm 3

• Fast and cheap.

• Essential for controlling porosity from imaging methods

• Only bulk method.

• Speed of water intrusion depend on rock water permeability, and can be long.

He-Pycnometry bulk value 3D Connected cm3 to mm3

• Fast intrusion of He.

• Cheap.

• Essential for controlling porosity from imaging methods

• Only bulk.

• Apparent volume should be known to determine porosity. Montmorillonite-rich areas and in a less extent mesostase present an higher porosity. In PA12, Feldspars remain unaltered and lowly porous (arrow 1), as well carbonate. High porosity of pseudomorphosed hornblende cannot be differentiate from the porosity of the mesostase (arrow 3).

Sample PA11, which is the most affected by hydrothermal alteration, presents a totally different porosity pattern than sample PA12. In this sample, quartz grains remains the only lowly porous mineral (arrow 3). The connected mesostase matrix is porous, but pseudomorphosed hornblende (arrow 1) and altered/dissolved feldspars associated with dissolution voids (arrow 2) are more porous. The 1:1 line is used to estimate the isoconductivity point of each sample.


Priyanka Dutta

Dongni Han

Bertrand Goudeau

Dechen Jiang

Danjun Fang

Neso Sojic
email: neso.sojic@enscbp.fr

Reactivity mapping of luminescence in space: Insights into heterogeneous electrochemiluminescence bioassays

Keywords: electrochemiluminescence, heterogeneous bioassays, bead-based immunoassays, electrochemistry, imaging

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

Introduction

Electrochemiluminescence (ECL) is an electrochemical process in which light is emitted by the excited state of the luminophore. [START_REF] Bard | Electrogenerated Chemiluminescence[END_REF][START_REF] Sojic | Analytical Electrogenerated Chemiluminescence: From Fundamentals to Bioassays[END_REF] The phenomenon is triggered by an initial electron-transfer reaction occurring at the electrode surface. [START_REF] Hesari | Review-Electrogenerated Chemiluminescence: Light Years Ahead[END_REF][START_REF] Hu | Applications and trends in electrochemiluminescence[END_REF] Then, it continues by a chemical reaction between highly reactive intermediates that produces the excited state of the luminophore. Finally, it ends with its relaxation to the ground state and the photon emission. The luminophores are excited by the application of an external potential instead of a light source, which removes the possibility of background signals from scattered light and provides improved sensitivity to the detection system. [START_REF] Dolci | Development of a New Device for Ultrasensitive Electrochemiluminescence Microscopy Imaging[END_REF][START_REF] Qi | Electrogenerated Chemiluminescence Biosensing[END_REF][START_REF] Valenti | Transparent Carbon Nanotube Network for Efficient Electrochemiluminescence Devices[END_REF] Controlling the applied potential provides a temporal control and better selectivity to the system. [START_REF] Doeven | Potential-Resolved Electrogenerated Chemiluminescence for the Selective Detection of Multiple Luminophores[END_REF][START_REF] Guo | Potential-Resolved Multicolor Electrochemiluminescence for Multiplex Immunoassay in a Single Sample[END_REF][START_REF] Li | Spatially-resolved multicolor bipolar electrochemiluminescence[END_REF][START_REF] Voci | Selfenhanced multicolor electrochemiluminescence by competitive electron-transfer processes[END_REF]) These characteristics are the main reasons behind the fact that ECL has turned out today as a highly significant detection method in the field of analytical chemistry as well as an imaging method. [START_REF] Cao | Immuno-Electrochemiluminescent Imaging of a Single Cell Based on Functional Nanoprobes of Heterogeneous Ru(bpy)32+@SiO2/Au Nanoparticles[END_REF][START_REF] Ding | Imaging Cell-Matrix Adhesions and Collective Migration of Living Cells by Electrochemiluminescence Microscopy[END_REF][START_REF] Guo | Potential-Resolved Multicolor Electrochemiluminescence for Multiplex Immunoassay in a Single Sample[END_REF][START_REF] Ma | Luminol Electrochemiluminescence for the Analysis of Active Cholesterol at the Plasma Membrane in Single Mammalian Cells[END_REF][START_REF] Marquette | Electrochemiluminescent biosensors array for the concomitant detection of choline, glucose, glutamate, lactate, lysine and urate[END_REF][START_REF] Theakstone | Cathodic' electrochemiluminescence of [Ru(bpy)3] 2+ and tri-n-propylamine confirmed as emission at the counter electrode[END_REF][START_REF] Xu | Imaging Latent Fingerprints by Electrochemiluminescence[END_REF][START_REF] Zanut | Insights into the mechanism of coreactant electrochemiluminescence facilitating enhanced bioanalytical performance[END_REF]Zhang et al. 2019b;[START_REF] Zhou | Electrochemiluminescence Imaging for Parallel Single-Cell Analysis of Active Membrane Cholesterol[END_REF][START_REF] Zhu | Electrogenerated Chemiluminescence Imaging of Electrocatalysis at a Single Au/Pt Janus Nanoparticle[END_REF] The model system involving [Ru(bpy)3] 2+ as the luminophore and tripropylamine (TPA) as the coreactant forms the basis of a large number of commercially available bead-based ECL immunoassays. [START_REF] Ma | Recent Progress in Electrochemiluminescence Sensing and Imaging[END_REF][START_REF] Qi | Electrogenerated Chemiluminescence Biosensing[END_REF] In heterogeneous ECL, the luminophore is immobilized on a non-conductive surface (or object) and is not directly oxidized at the electrode surface. [START_REF] Habtamu | A sensitive electrochemiluminescence immunosensor for celiac disease diagnosis based on nanoelectrode ensembles[END_REF][START_REF] Miao | Electrogenerated Chemiluminescence 69: The Tris(2,2'bipyridine)ruthenium(II), (Ru(bpy)32+)/Tri-n-propylamine (TPrA) System RevisitedA New Route Involving TPrA•+ Cation Radicals[END_REF][START_REF] Sentic | Mapping electrogenerated chemiluminescence reactivity in space: mechanistic insight into model systems used in immunoassays[END_REF]) Heterogeneous ECL has been widely studied over the last few decades and has found applications in a large number of immunoassays, such as tumor biomarkers, for detection of cardiac and other infectious diseases. [START_REF] Blackburn | Electrochemiluminescence detection for development of immunoassays and DNA probe assays for clinical diagnostics[END_REF][START_REF] Muzyka | Current trends in the development of the electrochemiluminescent immunosensors[END_REF][START_REF] Zhou | Magnetic beads-based electrochemiluminescence assay for rapid and sensitive detection of telomerase activity[END_REF]) Such heterogeneous assays are based on non-conductive beads (e.g. polystyrene, PS) decorated with a specific capture antibody. As in the present work, a sandwich immunoassay is formed in presence of the target analyte with the detection antibody conjugated to a [Ru(bpy)3] 2+ label (Figure 1a). [START_REF] Deiss | Multiplexed Sandwich Immunoassays using Electrochemiluminescence Imaging Resolved at the Single Bead Level[END_REF][START_REF] Habtamu | A sensitive electrochemiluminescence immunosensor for celiac disease diagnosis based on nanoelectrode ensembles[END_REF][START_REF] Zhou | Synthesis, labeling and bioanalytical applications of a tris(2,2′-bipyridyl)ruthenium(II)-based electrochemiluminescence probe[END_REF] The remarkable sensitivity of such bead-based bioassays is due to the particular reactivity of the freely-diffusing TPA coreactant . [START_REF] Miao | Electrogenerated Chemiluminescence 69: The Tris(2,2'bipyridine)ruthenium(II), (Ru(bpy)32+)/Tri-n-propylamine (TPrA) System RevisitedA New Route Involving TPrA•+ Cation Radicals[END_REF][START_REF] Zanut | Insights into the mechanism of coreactant electrochemiluminescence facilitating enhanced bioanalytical performance[END_REF]) Indeed, in this heterogeneous format, the labels (denoted PS--[Ru(bpy) ] * ), which are immobilized on the PS beads are located at micrometric distances from the electrode generate ECL and are not directly oxidized at the electrode surface. [START_REF] Habtamu | A sensitive electrochemiluminescence immunosensor for celiac disease diagnosis based on nanoelectrode ensembles[END_REF][START_REF] Sentic | Microscopic imaging and tuning of electrogenerated chemiluminescence with boron-doped diamond nanoelectrode arrays[END_REF][START_REF] Zanut | Insights into the mechanism of coreactant electrochemiluminescence facilitating enhanced bioanalytical performance[END_REF] The mechanism proposed can be described by the following reactions:

TPAH + TPA + H + (1) TPA -e TPA •+ (2) TPA •+ TPA • + H + (3) TPA • + PS--[ ( ) ] Im + + PS--[ ( ) ] (4)
TPA •+ + PS--[ ( ) ] TPA + PS--[ ( ) ] * (5) PS--[ ( ) ] * PS--[ ( ) ] + hν (6)
where Im + is the iminium product. [START_REF] Miao | Electrogenerated Chemiluminescence 69: The Tris(2,2'bipyridine)ruthenium(II), (Ru(bpy)32+)/Tri-n-propylamine (TPrA) System RevisitedA New Route Involving TPrA•+ Cation Radicals[END_REF][START_REF] Qiu | Mass spectrometric snapshots for electrochemical reactions[END_REF]) PS--[Ru(bpy) ] *

represents the ECL label bound to the PS bead.

Only the freely-diffusing coreactant TPA is oxidized at the electrode surface generating TPA •+ cation radical (reaction 2). This later deprotonates rapidly to form the neutral radical TPA • (reaction 3). Both TPA •+ and TPA • radicals diffuse over micrometric distances and react with the luminophore to generate its excited state. Indeed, TPA • is a strong reductant and reduces the immobilized PS--Ru(bpy)3 2+ to PS--Ru(bpy)3 + (reaction 4). Then, the cation radical TPA •+ oxidizes PS--Ru(bpy)3 + to form the excited state PS--Ru(bpy)3 2+* (reaction 5).

The excited state then deactivates by emitting a photon (reaction 6) that generates ECL. This ECL route is the predominant pathway for such bioassays. [START_REF] Miao | Electrogenerated Chemiluminescence 69: The Tris(2,2'bipyridine)ruthenium(II), (Ru(bpy)32+)/Tri-n-propylamine (TPrA) System RevisitedA New Route Involving TPrA•+ Cation Radicals[END_REF] In addition, it is noteworthy that similar heterogeneous mechanism operates for ECL microscopy of cells, where the ECL labels are immobilized also on a non-conductive object (i.e. the cell membrane). [START_REF] Ma | Recent Progress in Electrochemiluminescence Sensing and Imaging[END_REF][START_REF] Valenti | Single Cell Electrochemiluminescence Imaging: From the Proof-of-Concept to Disposable Device-Based Analysis[END_REF][START_REF] Voci | Surface-Confined Electrochemiluminescence Microscopy of Cell Membranes[END_REF]Zhang et al. 2019a) Many factors affecting the light intensity and properties of ECL in such formats still remains unexplored. [START_REF] Sentic | Mapping electrogenerated chemiluminescence reactivity in space: mechanistic insight into model systems used in immunoassays[END_REF][START_REF] Valenti | Essential Role of Electrode Materials in Electrochemiluminescence Applications[END_REF][START_REF] Zanut | Insights into the mechanism of coreactant electrochemiluminescence facilitating enhanced bioanalytical performance[END_REF]) For example, a major drawback is the important decrease of the ECL intensity over time, even if this behavior is generally not mentioned or detailed in the ECL reports. However, it limits significantly the development of ECL-based microscopy and bioassays. This work aims at investigating the effects responsible for the decrease of the light intensity in heterogeneous ECL. Such a decrease could be due either to the initial electrochemical process or to the final photophysical step. In other words, the evolution of the electrochemical and/or photophysical properties of the electrode surface or the ECL label might be involved in this signal loss. For that, we labeled the non-conductive polystyrene (PS) beads with the [Ru(bpy)3] 2+ complex via a sandwich immunoassay or a peptide bond and used them as the (bio)sensing platform for heterogeneous ECL studies. We mapped the photoluminescence (PL) and ECL reactivities of single PS [Ru(bpy)3] 2+ -decorated beads using two optical configurations (Figure 1c). We showed a remarkable photophysical stability of the ECL label, even in presence of the very reactive coreactant radicals. A direct correlation of the ECL decrease with the lower TPA oxidation current is also demonstrated.

The effects of very cathodic pulses on the ECL signals were then studied. This 3D imaging approach provides valuable information on the photophysical stability of the labels and on the parameters controlling the ECL signals.

Materials and Methods

Materials

All the reagents were purchased from Sigma-Aldrich unless otherwise noted. PS beads

2.2.Instrumentation

The electrochemical cell was a 3-electrode system where the working electrode was glassy carbon (GC) or gold electrodes. A platinum wire was used as the counter electrode and Ag/AgCl/KCl (3M) electrode was used as the reference electrode. The experiments were performed using a µ-Autolab type Ⅲ potentiostat. The PL and ECL images were recorded using an epifluorescence microscope from Leica (DMI6000, Leica Microsystems) and an Electron Multiplying Charge Coupled Device (EM-CCD 9100-13) Camera from Hamamatsu.

The working electrode was placed in two different configurations inside the electrochemical cell for top-view and side-view study (Figure 1c). PL and ECL experiments were performed in PBS containing 200 mM TPA.

Immunoassay with PS beads

The PS beads were labeled with the [Ru(bpy)3] 2+ complex either via a sandwich immunoassay or via a peptide bond. In the first case, antigen storage aliquots were prepared in PBS 1x/BSA 0.1% and detection antibody storage aliquots were prepared in tris-buffered saline (TBS StartingBlock). Each washing step was done in 100 µL of TBS with 1% Tween 20. The assay was performed by incubating for 2 hours the microbeads functionalized with a capture antibody (anti-IL-8) first in a sample containing antigen (dilute to the appropriate concentration with PBS Starting Block) and washed. Then they were incubated for 30 min. in 50 µL of the biotinylated detection antibodies solution (3µg/mL of antibody in PBS StartingBlock) and washed. Finally, the ECL label was attached to form immunocomplex by exposing the beads to a solution containing a streptavidin-modified [Ru(bpy)3] 2+ (SA-Ru) label (Figure 1a).

2.4.Functionalization of the PS beads with the ruthenium label via a peptide bond

An alternative labeling procedure based on the formation of a peptide bond was also employed to functionalize the PS beads with the [Ru(bpy)3] 2+ dye. 10 μL of beads suspension

(2.5%) was washed with PBS (pH 7.4) and re-suspended in 1 mL of PBS. In the same time, 1 mg of Ru(bpy)3 2+ -NHS ester (bis(2,2'-bipyridine)-4'-methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester-bis(hexafluorophosphate) was dissolved in 100 μL of dimethyl sulfoxide and this solution was added to the beads suspension. This mixture was incubated on +4°C for 3 hours with continuous stirring. After the incubation the beads were washed from reaction solution with PBS 15 times by the centrifugation for 10 min at 10000 rpm to separate the beads from the solution. Finally, beads were suspended in 1 mL PBS and kept at 4°C. Figure 1b shows a functionalized bead with the [Ru(bpy)3] 2+ label via formation of amide bond.

Beads were then washed for the last time and immobilized on the electrode surface for PL and/or ECL mapping.

Results and Discussions

PS beads were first functionalized with the [Ru(bpy)3] 2+ labels by sandwich immunoassay (Figure 1a). The surface of the microbeads was modified with a capture antibody (anti-IL-8). The beads were incubated first in a sample containing the corresponding antigen and then in a solution of biotinylated detection antibody. The final step was to attach the ECL labels by exposing the beads to a solution containing a streptavidin-modified

[Ru(bpy)3] 2+ complex (SA-Ru). We used beads with a 12 µm diameter in order to obtain wellresolved PL and ECL patterns. The PS--Ru(bpy)3 2+ -modified beads were allowed to deposit on the surface of glassy carbon (GC) or Au electrodes. PL imaging of the bead modified by the sandwich immunoassay procedure was first performed under the microscope in the topview configuration (denoted (i) in Figure 1c) in the epifluorescence mode. The PL image

shows that the distribution of the ruthenium label throughout the bead is homogeneous (Figure 1d). Similar ECL and PL images and behaviors were observed for the beads functionalized with both procedures (e.g. sandwich immunoassay and amide-type reaction).

Therefore, since similar PL and ECL results were obtained for both kinds of beads, we used the beads functionalized by the amide-type reaction to simplify the experimental protocol.

Figure 2a-c shows a sequence of successive ECL images of the bead in the top-view configuration; the ECL signal decreases over time. ECL intensity measured over the entire bead decreased almost by a factor 3 after 30 s on GC electrode and is almost negligible after 60 s (Figure 2d). A similar behavior is observed on Au electrode but the drop of the ECL signal is even stronger with a decrease by a factor 9 after 30 s (Figure 2e). On the Au electrode, the formation of an oxide layer at the very anodic potentials required to generate ECL [START_REF] Valenti | Essential Role of Electrode Materials in Electrochemiluminescence Applications[END_REF][START_REF] Zu | Electrogenerated chemiluminescence. 66. The role of direct coreactant oxidation in the ruthenium tris(2,2')bipyridyl/tripropylamine system and the effect of halide ions on the emission intensity[END_REF] may contribute to the decrease of the signal. The top-view imaging of the bead highlights the decrease of the ECL intensity.

However, this configuration does not give precise information on the PL or ECL reactivity along the axis normal to the electrode surface (i.e. z-axis on Figure 1c). To investigate the photophysical stability of the [Ru(bpy)3] 2+ label immobilized on the bead, we changed the optical configuration and the angle of observation of the ECL-emitting bead. An orthogonal side-view configuration (denoted (ii) in Figure 1c) was used to obtain a 2D mapping normal to the electrode surface. Therefore, further studies of the labeled beads were performed in the side-view configuration. Figure 3a-c shows a sequence of PL/ECL/PL images of the same bead. The lower part of the image corresponds to the real bead and the upper part to its mirror image formed by the light reflection on the GC surface. The PL images allow precisely defining the position of the bead and its interface with the electrode. As already mentioned, the heterogeneous mechanism operating in these conditions is described by the reactions 1-6. Using the side-view configuration, PL images of the same bead (Figures 2a and2c)

were taken before and after the generation of ECL, respectively. A potential of 1.1 V was applied for 65 s in the PBS solution containing the TPA coreactant and Figure 2b displays the corresponding first ECL image of the single functionalized bead. ECL is generated just in the region close to the electrode. As expected, it means that the labels located closed to the electrode, i.e. in ROI-1, were exposed to both TPA • and TPA •+ radicals. On the other hand, the labels located in ROI-2 were as in the bulk and not affected by the reactions with the coreactant radicals. After one and two anodic pulses of 1.1 V for 65 s each, the PL intensity decreases with the same drop in both regions (Figure 2d). No greater decrease was observed in the region near the electrode (i.e. ROI-1) than far from the electrode (i.e. ROI-2). In the top-view configuration, a similar decrease was evidenced in the PL mode over the entire bead (Figure 2e). The same behavior was obtained on Au electrode. This PL decrease is only due to the photobleaching of the [Ru(bpy)3] 2+ complex during the illumination, which is required to collect the successive PL images. This study demonstrates a remarkable stability of the photophysical properties of this model label even when exposed for 1-2 minutes to strongly reactive redox radicals. It shows also that the ECL process, and in particular the initiating electrochemical step, does not affect the photophysical properties of this luminophore.

Therefore, we can conclude that the observed ECL decrease is not related to an alteration of the photophysical properties of the ECL label. Since the ECL labels are photochemically stable under the application of the anodic potential, we investigated then the influence of the electrochemical oxidation step on the ECL signal and its stability. The anodic current was monitored as a function of time for six successive pulses of 1.1 V for 65 s on GC electrode (Figure 4a). It shows the gradual decrease in current after each potential pulse. For example, the current measured 5 s after the application of the first potential pulse (i.e. at a time when the capacitive component becomes negligible) was 275 µA; it decreased to ~200 µA at the 2 nd pulse and was just 62 µA at the 6 th anodic potential pulses, a cathodic potential of -2 V was applied for 65 s to the GC electrode.

Top-view ECL images of the same labeled bead c) before and d) after applying -2 V for 65 s.

ECL emission was generated by applying a constant potential of 1.1 V. Scale bar: 10 µm.

Experiments were performed in a PBS solution containing 200 mM TPA (pH 7.4).

Since electrochemistry is responsible for this progressive ECL loss, we tested different treatments of the electrode surface to regenerate the initial electrochemical properties of the GC surface. Recently, Liu and co-workers reported a drastic enhancement of the ECL of ligand-stabilized Au nanoclusters by on-electrode pre-oxidation with triethylamine coreactant. [START_REF] Peng | Pre-oxidation of Gold Nanoclusters Results in a 66 % Anodic Electrochemiluminescence Yield and Drives Mechanistic Insights[END_REF] The electrochemical treatment, the nature of the luminophore and the ECL processes are different from our heterogeneous work. Herein, we obtained the optimal results by applying negative potential of -2 V for 65 s. Figure 4b shows the variations of the faradaic current during a series of six successive pulses of 1.1 V for 65s on GC electrode. We imposed -2 V for 65 s between each anodic pulse. One can observe that this cathodic treatment of the electrode surface regenerated efficiently the properties of the GC surface. Indeed, the oxidation current of TPA increased in the ECL experiment, which followed the application of the cathodic pulse (Figure 4b). In fact, the anodic current recorded after this regeneration step is even stronger than the one measured during the first anodic pulse. We obtained reproducible and strong current for at least 6 successive ECL experiments.

It means that this pretreatment step created functional groups on the GC surface that efficiently oxidize TPA. On the contrary, the oxidation potential required to generate ECL degrades progressively the electrochemical properties of the GC surface for the TPA oxidation. Future works will be directed towards the identification of the chemical groups created on the electrode surface by the anodic potential and the regenerative cathodic treatment. We imaged the ECL emission of the beads before and after this regeneration step (Figure 5 c-d). ECL images revealed that we recovered the ECL pattern with this simple negative potential. It is also visible when we calculated the corresponding mean ECL intensity after the application of this cathodic potential (Figure 5a). The ECL emission of the beads could be successfully regenerated at least 6 times after applying this negative potential. Figure 5b showed a good correlation between the regeneration of the oxidation current and the ECL intensity. Finally, the restoration of ECL signals confirmed that the electrogenerated TPA radicals did not damage the photophysical properties of the ECL label and its remarkable stability in presence of highly oxidizing and reducing radicals.

Conclusion

In summary, heterogeneous ECL bioassays were investigated by taking single Ru(bpy)3 2+ -decorated PS beads as the analytical platform. With two optical configurations, mapping the photoluminescence and ECL reactivities allowed deciphering the processes responsible for the significant decrease of the ECL intensity over time in heterogeneous assays using the model TPA coreactant. Photoluminescence intensity remained constant in the region close to the electrode where ECL is generated. It showed the remarkable photophysical stability of the Ru(bpy)3 2+ labels in presence of highly oxidizing and reducing TPA radicals electrogenerated locally. We demonstrated that the ECL vanishing is only due to the lower TPA oxidation current. Indeed, the correlation of the ECL variation with the anodic current confirmed that the oxidation step of the TPA coreactant is the one decreasing the ECL signal.

We tested a simple electrochemical treatment, which allowed us to regenerate the electrode surface and thus to recover several times the strong initial ECL signal. The present study helps in understanding several significant factors behind the complex ECL mechanism in heterogeneous bioassays. The reactivity imaging approach provides insights into the ECL mechanism and the main factors governing the stability of the emission, which should find promising applications to improve the optical readout and its reproducibility in biological and chemical sensing, new detection schemes and microscopy.

Figure 1 .

 1 Figure 1. a) Sandwich bead-based immunoassay for interleukin 8 (IL-8) detection with ECL

(

  radius: 6 µm) were obtained from Kisker Biotech GmbH & Co. Phosphate buffer solution (pH 7.4, 0.1 M) was prepared by mixing 0.1 M sodium phosphate monobasic monohydrate and 0.1 M sodium phosphate dibasic heptahydrate solution with degassed solution. TPA was dissolved in PBS and the pH was adjusted to 7.4 with phosphoric acid. Capture antibody specific for interleukin 8 (IL-8), the complementary biotinylated detection antibody and IL-8 recombinant protein were obtained from R&D Systems Inc. The streptavidin-modified ruthenium complex used as a label in the immunoassay experiments was synthesized according to the procedure described by Deiss et al.[START_REF] Deiss | Multiplexed Sandwich Immunoassays using Electrochemiluminescence Imaging Resolved at the Single Bead Level[END_REF]

Figure 2 .

 2 Figure 2. a-c) Sequence of successive ECL images of a single labeled bead recorded in the

  ECL generation requires explicitly the sequential reactions of both reducing and oxidizing TPA radicals with PS--[Ru(bpy)3] 2+ at the same location on the bead. The corresponding concentration profiles of both coreactant radicals diffusing from the electrode and around the bead were simulated previously.[START_REF] Sentic | Mapping electrogenerated chemiluminescence reactivity in space: mechanistic insight into model systems used in immunoassays[END_REF] The spatial extension of this ECL reactive layer is confined in the first 2-3 µm height of the bead next to the electrode surface.[START_REF] Sentic | Mapping electrogenerated chemiluminescence reactivity in space: mechanistic insight into model systems used in immunoassays[END_REF]) It means that only the [Ru(bpy)3] 2+ labels located in this micrometric region close to the electrode are exposed to the actions of the TPA • and TPA •+ radicals. To study the effects of both highly oxidizing and reducing TPA radicals on the photophysical properties of the PS--[Ru(bpy)3] 2+ labels, two regions of interest (ROI) were considered: ROI-1 is the region of the bead which is close to the electrode (i.e. z < 3 µm) whereas ROI-2 is the region located far from the electrode (i.e. z > 3 µm). In other words, ROI-1 is located in the reaction layer where TPA • and TPA •+ radicals coexist and ROI-2 can be considered as a control ROI, which is not impacted by the reactivity of the TPA radicals.

Figure 3 .

 3 Figure 3. Side-view images of the same labeled bead in PL and ECL modes. a) PL image

Figure 4 .

 4 Figure 4. Successive chronoamperometric curves obtained when applying a sequence of 6

Figure 5 .

 5 Figure 5. Effect of the application of the cathodic potential of -2 V on the ECL emission,

Acknowledgements

PD acknowledges the Agence Nationale de la Recherche (Neocastip ANR-15-CE09-0015-03). DH thanks the Nanjing Medical University for financial support. NS wishes to acknowledge the support from the Sino-French international research network IRN0876 (Centre National de la Recherche Scientifique, CNRS) "New nanostructured materials and biomaterials for renewable electrical energy sources" for providing facilities.