Aliki Tsopela

Ahmet Lale

Emilie Vanhove

Olivier Reynes

Isabelle Séguy

Pierre Temple-Boyer

Philippe Juneau

R Izquierdo

Jérôme Launay

Integrated electrochemical biosensor based on algal metabolism for water toxicity analysis

Keywords: ultramicroelectrodes, oxygen, hydrogen peroxide, pH measurement, algal metabolism, pesticides detection

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

Introduction

Water quality assessment has been an issue of particular concern over the past few years and is actually generating major interest. Contamination of water resources brought about by agricultural and industrial practices can pose possible threats and have a long-term impact on the environment and all living organisms. Pesticides are frequently employed in agriculture to protect crops and control non-crops and can easily leach from the soil in underground water supply and be often detected in rivers, lakes and later in consumption water [START_REF] Barbash | Major herbicides in ground water: results from the National Water-Quality Assessment[END_REF]. It is therefore a major challenge to rapidly detect contamination by monitoring the quantities of different toxicants present in water.

Conventional analysis methods like gas [START_REF] Schellin | Determination of organophosphorus pesticides using membrane-assisted solvent extraction combined with large volume injection-gas chromatography-mass spectrometric detection[END_REF] and liquid [START_REF] Jáuregui | Liquid chromatography-atmospheric pressure ionization mass spectrometry for the determination of chloro-and nitrophenolic compounds in tap water and sea water[END_REF] chromatography performed in highly equipped laboratories do still provide the most accurate and sensitive means of detecting water contamination but they encounter considerable limitations as they are expensive and time consuming [START_REF] Eltzov | Whole-cell aquatic biosensors[END_REF].

Electrochemical detection systems are a potential alternative to these methods as they are easily miniaturized and portable enabling on-site analysis and providing rapid, real-time feedback information on the presence of pollutants [START_REF] Lagarde | Cell-based electrochemical biosensors for water quality assessment[END_REF][START_REF] Grieshaber | Electrochemical Biosensors -Sensor Principles and Architectures[END_REF][START_REF] Li | Highly Sensitive Platinum-Black Coated Platinum Electrodes for Electrochemical Detection of Hydrogen Peroxide and Nitrite in Microchannel[END_REF]. More specifically, lab on chip based, electrochemical biosensors meet the requirement for a rapid, cost-effective, accurate and sensitive analytical system and give information on water quality by detecting wide range of chemical species [START_REF] Jang | State-of-the-art lab chip sensors for environmental water monitoring[END_REF].

They convert a biochemical phenomenon into a detectable and measurable electrical signal that is proportional to the analyte concentration and provide an early indication by sorting the samples needed to be further analyzed by conventional techniques.

Electrochemical biosensors can be amperometric, potentiometric and conductometric.

The operating principle of amperometric biosensors lies in applying a potential and recording the current corresponding to oxidation and reduction of electroactive species on electrode surface. Current variations result from variations in consumption-production rates of electroactive species and reflect the biochemical phenomenon taking place [START_REF] Ben-Yoav | A whole cell electrochemical biosensor for water genotoxicity bio-detection[END_REF]. Amperometric biosensors have been widely used for the oxygen monitoring in wastewaters [START_REF] Chan | Designing an amperometric thick-film microbial BOD sensor[END_REF], detection of organophosphates [START_REF] Mulchandani | Biosensors for direct determination of organophosphate pesticides[END_REF], phenols [START_REF] Mulchandani | Amperometric microbial biosensor for p-nitrophenol using Moraxella sp.-modified carbon paste electrode[END_REF], cyanide [START_REF] Ikebukuro | Flow-type cyanide sensor using an immobilized microorganism[END_REF] and heavy metal ions [START_REF] Lehmann | Amperometric measurement of copper ions with a deputy substrate using a novel Saccharomyces cerevisiae sensor[END_REF].

Potentiometric biosensors are designed to measure the difference in potential between working electrode and a stable reference electrode with no current flowing through the system [START_REF] Lagarde | Cell-based electrochemical biosensors for water quality assessment[END_REF]. Potential values are then correlated to the concentration of the analyte [START_REF] Lei | Microbial biosensors[END_REF]. Although pH electrodes have been widely used for detection of pesticides in agriculture [START_REF] Mulchandani | Biosensors for direct determination of organophosphate pesticides[END_REF][START_REF] Gaberlein | Microbial and cytoplasmic membranebased potentiometric biosensors for direct determination of organophosphorus insecticides[END_REF], other ion selective electrodes, such as chloride ion selective electrode, have also generated major interest in wastewater treatment domain [START_REF] Han | Flow injection microbial trichloroethylene sensor[END_REF].

Conductometric biosensors record variations in conductivity of the electrolyte containing charged species in contact with the electrode [START_REF] Lagarde | Cell-based electrochemical biosensors for water quality assessment[END_REF]. These variations result from the production or consumption of charged species during different reactions and can therefore indicate a biological process taking place [START_REF] Lawrence | Conductimetry in Enzyme Studies[END_REF][START_REF] Jaffrezic-Renault | Conductometric Microbiosensors for Environmental Monitoring[END_REF]. They have found various applications in environmental monitoring for detection of heavy metal ions and pesticides [START_REF] Chouteau | Development of novel conductometric biosensors based on immobilised whole cell Chlorella vulgaris microalgae[END_REF][START_REF] Chouteau | A bi-enzymatic whole cell conductometric biosensor for heavy metal ions and pesticides detection in water samples[END_REF].

As far as biological recognition elements are concerned, algae are often used in biochemistry for trace detection of environmental toxic compounds as they are sensitive to changes in their environment induced by the presence of toxicants and can therefore provide information on toxic effect of pollutants on living organisms [START_REF] Giardi | Photosynthetic proteins for technological applications[END_REF][START_REF] Brayner | Micro-algal biosensors[END_REF][START_REF] Moreno-Garrido | Microalgae immobilization: Current techniques and uses[END_REF][START_REF] Carrilho | The use of silica-immobilized brown alga (Pilayella littoralis) for metal preconcentration and determination by inductively coupled plasma optical emission spectrometry[END_REF]. Due to their high sensitivity to toxic substances, even lower detection limits (LOD) can be reached [START_REF] Brayner | Micro-algal biosensors[END_REF]. Microalgae and photosynthetic cyanobacteria are used in biosensors industry as they are capable of providing information on pollution levels based on modifications in photosynthetic or metabolic activity [START_REF] Kasai | Herbicide susceptibility in two green algae, Chlorella vulgaris and Selenastrum capricornutum[END_REF][START_REF] Lagarde | Cell-based electrochemical biosensors for water quality assessment[END_REF]. Monitoring of species such as oxygen, hydrogen peroxide and pH related ions that participate in algae's metabolism and photosynthesis can indicate the presence of herbicides. For instance, amperometric microalgae sensors are designed to monitor photosynthetic oxygen production and can therefore indicate pollutant related photosynthetic activity inhibition [START_REF] Pandard | An amperometric algal biosensor for herbicide detection employing a carbon cathode oxygen electrode[END_REF].

Furthermore, hydrogen peroxide, the concentration of which is related to potential stress exerted on algae, could be useful indicator of the presence of herbicides. As reported by Mittler [START_REF] Mittler | Oxidative stress, antioxidants and stress tolerance[END_REF], reactive oxygen species (ROS) can be stress indicators and signals for defense pathways activation. Different types of stress (pathogens, extreme temperatures, light, heavy metals, salt, pollutants) can cause metabolic disruptions resulting in an increase in the production of ROS [START_REF] Mittler | Reactive oxygen gene network of plants[END_REF]. Along with O2 and H2O2, pH monitoring can also provide information on metabolic activity modifications induced by herbicides and other inhibitors based on changes in medium basification and acidification rates [START_REF] Schubnell | An ISFET-algal (Chlamydomonas) hybrid provides a system for eco-toxicological tests[END_REF].

As a matter of fact, different sensors have been developed for the detection of the species mentioned above, viewed as indicators of water pollution (O2, H2O2, H3O + /OH -ions). Two basic detection methods, amperometry and potentiometry have been discussed and various electrode materials have been mentioned.

As far as oxygen monitoring is concerned, noble metals (Pt or Au) are primarily used in literature as working electrode materials so that the electrode surface does not participate in the chemical reaction [START_REF] Lee | Needle-type dissolved oxygen microelectrode array sensors for in situ measurements[END_REF]. Platinum is the most suitable candidate for oxygen detection as it is physically stable [START_REF] Zhang | Electrochemical oxidation of H2O2 on Pt and Pt + Ir electrodes in physiological buffer and its applicability to H2O2-based biosensors[END_REF], mechanically robust (Ben-Amor et al., 2013) and compared to gold and other metals is the best electrocatalyst for the oxygen reduction reaction (Maja N. [START_REF] Desic | Study of gold-platinum and platinum-gold surface modification and its influence on hydrogen evolution and oxygen reduction[END_REF]. Platinum microdiscs [START_REF] Sosna | Development of a reliable microelectrode dissolved oxygen sensor[END_REF] and array of platinum microelectrodes [START_REF] Wittkampf | Silicon thin film sensor for measurement of dissolved oxygen[END_REF] have been employed as dissolved oxygen sensors in environmental monitoring. Different techniques have been followed to enhance sensitivity by increasing electro-active surface area and porosity of platinum electrodes and therefore enhancing mass transport of species towards electrode surface [START_REF] Qiang | Highly sensitive and reusable Pt-black microfluidic electrodes for long-term electrochemical sensing[END_REF][START_REF] Lee | A Highly Miniaturized Dissolved Oxygen Sensor Using a Nanoporous Platinum Electrode Electroplated on Silicon[END_REF]. Although platinum is the most commonly applied material in oxygen sensors, gold electrodes have been designed to measure dissolved oxygen in environmental samples [START_REF] Liu | An Innovative Microelectrode Fabricated Using Photolithography for Measuring Dissolved Oxygen Distributions in Aerobic Granules[END_REF], bacterial films [START_REF] Lee | Needle-type dissolved oxygen microelectrode array sensors for in situ measurements[END_REF] or during cell culture [START_REF] Pereira Rodrigues | Cell-based microfluidic biochip for the electrochemical real-time monitoring of glucose and oxygen[END_REF]. Carbon nanotubes integrated on a screen-printed algal biosensor have also been used to detect oxygen produced by algae [START_REF] Shitanda | Amperometric screenprinted algal biosensor with flow injection analysis system for detection of environmental toxic compounds[END_REF].

As already stated, hydrogen peroxide is an essential species to monitor as it is taking part in various biological processes [START_REF] Li | Highly Sensitive Platinum-Black Coated Platinum Electrodes for Electrochemical Detection of Hydrogen Peroxide and Nitrite in Microchannel[END_REF]. Gold [START_REF] Brett | Thin-film gold electrodes produced by magnetron sputtering. Voltammetric characteristics and application in batch injection analysis with amperometric detection[END_REF], platinum [START_REF] Zhang | Electrochemical oxidation of H2O2 on Pt and Pt + Ir electrodes in physiological buffer and its applicability to H2O2-based biosensors[END_REF][START_REF] Yokoyama | Hydrogen peroxide augmentation in a rat striatum after methamphetamine injection as monitored in vivo by a Pt-disk microelectrode[END_REF], carbon based [START_REF] Cho | Kinetics determination of electrogenerated hydrogen peroxide (H2O2) using carbon fiber microelectrode in electroenzymatic degradation of phenolic compounds[END_REF][START_REF] Nowall | Detection of hydrogen peroxide and other molecules of biologicl importance at an electrocataltic surface on a carbon fiber microelectrode[END_REF], enzyme modified [START_REF] Ruzgas | Peroxidasemodified electrodes: Fundamentals and application[END_REF] and Prussian Blue modified [START_REF] Karyakin | Diffusion controlled analytical performances of hydrogen peroxide sensors: Towards the sensor with the largest dynamic range[END_REF] electrodes are situated in the main stream of research interest towards suitable working electrode materials for the detection of H2O2. Platinum is still the preferred option as it provides a high catalytic surface for H2O2 oxidation [START_REF] Arbault | Monitoring an oxidative stress mechanism at a single human fibroblast[END_REF] and it is even possible to increase active surface area of the electrodes by platinization. Indeed, sensors with platinized working electrodes yield greater sensitivity due to their nanoscale structure [START_REF] Vaddiraju | Emerging synergy between nanotechnology and implantable biosensors: A review[END_REF] and the large number of electroactive sites that facilitates electron transfer [START_REF] Li | Highly Sensitive Platinum-Black Coated Platinum Electrodes for Electrochemical Detection of Hydrogen Peroxide and Nitrite in Microchannel[END_REF]. Furthermore, on platinum black (Pt Bl) microelectrodes, H2O2 oxidation is taking place at a lower potential compared to bare Pt electrodes, minimizing interferences induced by other electroactive species [START_REF] Arbault | Monitoring an oxidative stress mechanism at a single human fibroblast[END_REF]. Platinum black microelectrodes have been widely used to study ROS species released during cellular oxidative bursts [START_REF] Li | Highly Sensitive Platinum-Black Coated Platinum Electrodes for Electrochemical Detection of Hydrogen Peroxide and Nitrite in Microchannel[END_REF] and monitor the production of H2O2 by mitochondria during aerobic metabolism (Ben-Amor et al., 2013).

They have also found various applications as amperometric glucose sensors [START_REF] De Corcuera | Improved platinization conditions produce a 60-fold increase in sensitivity of amperometric biosensors using glucose oxidase immobilized in poly-o-phenylenediamine[END_REF].

Potentiometry is widely applied for pH monitoring and glass membrane electrodes are commonly used. However, glass fragility and size limitation impede in situ measurement in complex configurations and mediums [START_REF] Ruffien-Ciszak | Electrochemical microsensors for cutaneous surface analysis: Application to the determination of pH and the antioxidant properties of stratum corneum[END_REF][START_REF] Huang | A flexible pH sensor based on the iridium oxide sensing film[END_REF] giving way to metal/metal oxide based pH sensors that are easily miniaturized and provide a welldefined metal/metal oxide interface [START_REF] Zhou | Chapter 10 -Microelectrodes for in-vivo determination of pH[END_REF]. More specifically, iridium oxide films can be formed by various physical and chemical methods [START_REF] Ges | Thin-film IrOx pH microelectrode for microfluidic-based microsystems[END_REF] and exhibit many advantages among which high pH sensitivity, biocompatibility, chemical stability and fast response time [START_REF] Zhou | Chapter 10 -Microelectrodes for in-vivo determination of pH[END_REF][START_REF] Marzouk | Electrodeposited iridium oxide pH electrode for measurement of extracellular myocardial acidosis during acute ischemia[END_REF]. IrO2 films have therefore found applications in cell metabolism monitoring in cell culture volumes [START_REF] Ges | Thin-film IrOx pH microelectrode for microfluidic-based microsystems[END_REF] and microenvironmental studies [START_REF] Bezbaruah | Fabrication of anodically electrodeposited iridium oxide film pH microelectrodes for microenvironmental studies[END_REF]. Tungsten/tungsten oxide sensors represent another attractive alternative for pH monitoring as tungsten is cheaper compared to other metals such as Pt or Au. Additionally, stable tungsten oxides can be easily formed by electrochemical oxidation of the metal at low potential [START_REF] Ruffien-Ciszak | Electrochemical microsensors for cutaneous surface analysis: Application to the determination of pH and the antioxidant properties of stratum corneum[END_REF]. W/WO3 sensors have been developed for skin pH monitoring [START_REF] Ruffien-Ciszak | Electrochemical microsensors for cutaneous surface analysis: Application to the determination of pH and the antioxidant properties of stratum corneum[END_REF], determination of extracellular pH values for cultured cells [START_REF] Yamamoto | Solid-state pH ultramicrosensor based on a tungstic oxide film fabricated on a tungsten nanoelectrode and its application to the study of endothelial cells[END_REF] and pH measurement of environmental water samples [START_REF] Dimitrakopoulos | A tungsten oxide coated wire electrode used as a pH sensor in flow injection potentiometry[END_REF].

Thus, elaboration of detection materials and analytical methods led to the development of different systems applied for detection of water pollution. In this study, an autonomous fully integrated three-electrode electrochemical microsystem is developed by means of microfabrication technology on a silicon chip. The performance of the microsystem is evaluated through electrochemical characterization and calibration is performed by amperometric and potentiometric methods for the three species of interest: dissolved oxygen (O2), hydrogen peroxide (H2O2) and pH related ions (H3O + /OH -). The electrochemical microsystem is further used in bioassays, serving as screening prior to lab-analysis, to detect traces of toxicants in water samples by evaluating their effect on a biological sensing element, such as algae. Pollution level was estimated by measuring disturbances in photosynthetic and metabolic activities of algae caused by herbicides. These disturbances are represented by variations in consumptionproduction rates of the three electro-active species (dissolved oxygen, hydrogen peroxide and pH related ions) by algae and could therefore be detected by the electrochemical microsensor. A variation of oxygen production during algal photosynthesis is evidenced in the presence of Diuron herbicide.

Experimental

Electrode materials and configuration

Electrochemical microcells designed for this work consist of three integrated electrodes (working, reference, and counter electrodes) on the same silicon chip [START_REF] Christophe | Elaboration of integrated microelectrodes for the detection of antioxidant species[END_REF].

Working electrode materials were determined according to previously mentioned electro-active species needed to be detected. Platinum and functionalized Platinum Black (Pt Bl) working electrodes were fabricated for H2O2 and dissolved O2 measurements whereas tungsten/tungsten oxide (W/WO3) and iridium oxide (Pt/IrO2) electrodes were used for pH monitoring. The autonomous, miniaturized electrochemical cell was completed by integrating a platinum counter electrode and a silver based electrode in the structure. The latter was further electrochemically oxidized to form the Ag/AgCl reference electrode. In order to determine the arrangement and form of the three integrated electrodes, different electrode configurations were patterned; parallel and concentric arrangements were tested. For concentric arrangement, working electrode is encircled by the reference and counter electrodes that have a ring shape while for parallel arrangement, all electrodes are placed in parallel with reference electrode placed in the middle of the structure to reduce ohmic drop. Both single ultramicroelectrodes and ultramicroelectrode arrays were evaluated in terms of detection properties. In this way, favorable properties of ultramicroelectrodes and planar array configurations were used to enhance the efficiency of the sensors. Dimensions and distance between electrodes in the array were determined so that diffusion profiles of adjacent electrodes will not overlap.

Fabrication of the electrochemical microcells

Electrochemical microcells were fabricated on a thermally oxidized silicon wafer (SiO2) (oxide layer thickness: ~ 1 µm). All thin metal layers were evaporated on the oxidized silicon substrate (Figure 2a) and conventional photolithography followed by lift-off technique was conducted in order to obtain metal patterns. Platinum (Pt) (150 nm) and tungsten (W) (500 nm) layers correspond to working electrodes while silver (Ag) layer (400 nm) represents the future reference electrode. Platinum (Pt) (150 nm) also serves as the counter electrode. The underlying titanium (Ti) layer (20 nm) assures adhesion of platinum on SiO2 substrate. Silver was deposited on a platinum layer, which in this case serves as a barrier layer, in order to avoid diffusion of titanium atoms in the silver layer. All layer thicknesses were selected so that no mechanical stress is exerted on the silicon wafer. Following the development of all metal layers, electrode's active surface area needed to be precisely defined as it will be later used to correlate current intensity recorded to the quantity of species to be detected. Low temperature Inductively Coupled Plasma Chemical Vapour Deposition (ICP-CVD) was conducted for the deposition of high quality silicon nitride (Si3N4) insulation layer (100 nm) (Figure 1a). ICP-CVD process yields conformal deposition that enables perfect covering of the whole structure -even the lateral parts of metal layers. However, in order to enable patterning of a conformal coating, a specific double layer lift-off process has been developed and the thickness of the Si3N4 film was optimized accordingly [START_REF] Vanhove | Final capping passivation layers for long-life microsensors in real fluids[END_REF]. This insulation layer also contributes to long-term stability of electrochemical microcells in contrast to organic insulation layers that do not provide sufficient stability through time in complex, corrosive mediums [START_REF] Vanhove | Final capping passivation layers for long-life microsensors in real fluids[END_REF]. After fabrication, devices were diced and attached on a printed circuit board (PCB). Wedge bonding was subsequently carried out to complete packaging procedure and wire bonds were covered in a silicon glob-top to assure mechanical protection and avoid electrical short circuit (Figure 1b).

Surface functionalization of ultramicroelectrodes

In order to attain high sensitivity of platinum working electrodes, porosity and surface area were enhanced by forming "black platinum" electrodes. Platinization procedure was carried out by reducing a solution of hexachloroplatinic acid (32 mM in phosphate buffer solution) which contains lead acetate (1.23 mM) at -60 mV versus Ag/AgCl/KClsat (Ben-Amor et al., 2013). The amount of Pt deposited was controlled by the coulometric charge versus time plots recorded during platinization so that a nanostructured surface will be obtained. Platinization charge deposited on single Pt ultramicroelectrodes (d = 50 µm) was 0.5 µC.µm -2 compared to 0.25 µC.µm -2 on the arrays of Pt ultramicroelectrodes (25 disks of 10 µm diameter).

Regarding pH electrodes, tungsten W surface was oxidized to form a WO3 film by cyclic voltammetry in a 2 M H2SO4 solution, from +1.0 to +2.0 V vs Ag/AgCl/KClsat, at the scan rate of 20 mV.s -1 for 20 cycles [START_REF] Yamamoto | Solid-state pH ultramicrosensor based on a tungstic oxide film fabricated on a tungsten nanoelectrode and its application to the study of endothelial cells[END_REF]. For the deposition of IrO2 on Pt electrodes, an iridium tetrachloride based solution was prepared as described elsewhere [START_REF] Marzouk | Improved electrodeposited iridium oxide pH sensor fabricated on etched titanium substrates[END_REF]. A constant potential (0.6 V/ECS) was applied and the duration of the deposition was indicated by the total charge transferred (0.5 C.cm -2 ) [START_REF] Ges | Thin-film IrOx pH microelectrode for microfluidic-based microsystems[END_REF].

In order to form the Ag/AgCl reference electrode and obtain a fully integrated electrochemical cell, Ag surface was electrochemically oxidized. Silver surface was first examined by conducting cyclic voltammetry in acidified potassium nitrate (KNO3) solution of 0.1 M. The electrode was then oxidized in 10 -2 M potassium chloride solution (KCl) by linear sweep voltammetry within the potential range of 0.1-0.25 V/ECS and a low scan rate of 1 mV.s -1 which yields stable and homogeneous Ag/AgCl structure [START_REF] Christophe | Elaboration of integrated microelectrodes for the detection of antioxidant species[END_REF]. Indeed, higher scan rates could result in porous layers and electroactive species trapped between pores induce changes in reference electrode's potential [START_REF] Christophe | Elaboration of integrated microelectrodes for the detection of antioxidant species[END_REF].

Electrochemical characterization and calibration

All electrochemical experiments were conducted using a potentiostat (VMP3, BioLogic) with a low-current option controlled by EC-Lab software, at room temperature.

Cyclic voltammetry in a zero oxygen 0.5 M H2SO4 solution was conducted to characterize the quality of fabricated Pt and Pt Bl electrodes. Potential range scanned was -0.2 to +1.1 V vs Ag/AgCl/KClsat at a scan rate of 100 mV. Material properties were tested in order to validate that all thin layers developed by microfabrication technology gave results similar to the ones obtained with bulk materials.

Bioassays

As far as bioassays are concerned, green micro algae Chlamydomonas reinhardtii (CC-125) were cultivated at 25°C with time regulated light/dark (16h/8h) cycles in High Salt Medium (HSM) [START_REF] Sueoka | Deoxyribonucleic acid replication in meiosis of Chlamydomonas reinhardi. I. Isotopic transfer experiments with a strain producing eight zoospores[END_REF]. The same solution was used through all our tests. The main goal of this study being to determine the relation between the change in metabolic activities of algae and the presence of herbicides, measurements were conducted in model HSM solution in order to eliminate possible interferences found in real water samples. Nevertheless, for final application, we plan to validate this concept in consumption water obtained from groundwater, rivers or lakes). Growth parameters were controlled so that algae's growth rate is constantly kept at the exponential phase. Considering that the quantity of species produced or consumed depends on the number of cells in the solution, initial algal solutions were centrifuged to obtain a 5-fold density increase, in order to yield the maximum possible signal. Concentration of cells per mL of solution was always kept constant through different experiments and was measured by Multisizer Coulter Counter (Beckman Coulter). Bioassays using the fabricated electrochemical microsensors were performed in the dense algal solutions and current evolution was observed during light and dark periods in the presence and absence of Diuron (3(3,4-dichlorophenyl)-1,1dimethylurea) herbicide, the toxic chemical to be detected. Chronoamperometry was conducted and the potential applied corresponds to the O2 reduction potential which was estimated through cyclic voltammetry at -0.7 V vs integrated Ag/AgCl. All reagents were purchased from Sigma-Aldrich.

Results and discussion

Electrochemical characterization -calibration of ultramicroelectrodes

The voltammograms obtained for fabricated Pt and Pt Bl thin film electrodes in 0.5 M H2SO4 (voltammogramms not shown) presented peaks related to the formation and reduction of platinum oxides as well as the electrosorption and electrodesorption of protons indicating a behaviour similar to the one of Pt polycrystalline bulk material [START_REF] Tilak | The real condition of oxidized pt electrodes: Part III. Kinetic theory of formation and reduction of surface oxides[END_REF]. The radius of fabricated single and array Pt and Pt Bl electrodes was then evaluated in Fe(CN)6 4-solution and compared to the dimensions initially designed. The value was determined through the theoretical equation relating Ilim current (value of the oxidation plateau) to the radius of the electrode (Equations 1 and 2) [START_REF] Saito | A Theoretical Study on the Diffusion Current at the Stationary Electrodes of Circular and Narrow Band Types[END_REF]). Equation 2 is given for an array of microdisk electrodes widely spaced where there is no overlapping of diffusion layers and Ilim represents the sum of currents obtained for each ultramicroelectrode as they are connected in parallel. As expected, platinum black porous structure yielded well-defined oxidation plateaus and steeper waves indicating rapid electron transfer reactions. Platinization induces a 6-fold increase in electrode's Ilim value.

𝐼 𝑙𝑖𝑚 = 4𝑛𝐹𝐷𝐶𝑟

Equation 1

𝐼 𝑙𝑖𝑚 = 4𝑛𝐹𝐷𝐶𝑟 𝑧 Equation 2
Where F is the Faraday constant (F = 96500 C.mol -1 ), n is the number of electrons exchanged per mole (n = 1), C is Fe(CN)6 4-ion concentration (0.01 M), D represents the diffusion coefficient (6.3 x 10 -6 cm 2 .s -1 ) [START_REF] Konopka | Diffusion coefficients of ferri-and ferrocyanide ions in aqueous media, using twin-electrode thin-layer electrochemistry[END_REF] and z is the number of electrodes in the array.

Regarding Ag electrode it is important to note that the obtained Ag/AgCl is a quasi-reference electrode as it is in direct contact with the examined solution without any internal electrolyte [START_REF] Simonis | New concepts of miniaturised reference electrodes in silicon technology for potentiometric sensor systems[END_REF]. Quasi-reference electrode's stability was evaluated in 0.01 M KCl solution. Ag/AgCl electrode's potential compared to external Ag/AgCl/KClsat electrode was plotted versus time (results not shown). Potential deviation in time was estimated around 0.16 µV.s -1 , value that is an acceptable for the present application given the fact that short-time measurements are conducted.

Following validation of electrochemical performance of different electrode materials, microelectrochemical cells were used to detect electroactive species of interest (H2O2, O2, pH related ions H3O + /OH -).

H2O2 detection was firstly performed by following current increase after successive injections of H2O2 in PBS (10 mM) solution (Figure 2a). Concentration range tested was between 10nM and 10µM so that it can be verified that the detection of low H2O2 quantities (range of interest in biological applications) is feasible. An anodic potential of 0.6 V and 0.3 V vs Ag/AgCl/KClsat was applied to initiate the oxidation of H2O2 present in the solution for Pt and Pt Bl electrodes respectively. The potential applied corresponds to the plateau potential of the oxidation wave determined by cyclic voltammetry in 10 mM H2O2 solution in PBS. The lower potential value applied for Pt Bl compared to the one applied for Pt electrodes is ascribed to electrocatalytic activity of platinum black towards H2O2 reactions [START_REF] Arbault | Monitoring an oxidative stress mechanism at a single human fibroblast[END_REF].

Calibration curves for H2O2 microsensors were obtained by plotting current increase recorded for each injection (Figure 2b). Platinum and platinum black single electrodes and arrays were compared in terms of sensitivity and limits of detection (LOD).

Linear response was observed through the entire concentration range tested (R² ≥ 0.99) for all Pt and Pt Bl single electrodes and arrays. Black platinum electrode arrays exhibited the greatest sensitivity for H2O2 detection with a slope of 17 pA.mm -2 .nM -1 and a limit of detection of 10 nM. As a matter of fact, arrays hold the advantages of microelectrodes while generating a higher current signal. Their high signal-to-noise ratio combined with the high electrocatalytic activity of platinum black surface lead to enhanced electrode performance regarding detection properties, detection limits and sensitivities. Pl Bl single electrodes also yielded higher sensitivity than Pt electrodes with a slope of 12.4 pA.mm -2 .nM -1 but 1.4 times lower than the one provided by Pt Bl arrays.

Prior to validation of O2 microsensors in algal solutions it was important to verify that the sensor yields a response correlated to O2 concentration. The electrode potential was therefore set at -0.7 V vs Ag/AgCl/KClsat, (potential corresponding to the plateau value of oxygen reduction current) and output current was recorded for a saturated and a zero oxygen CaCl2 (0.1 M) solution at 22 °C. As expected, O2 concentration variation induces a variation in current intensity generated by the O2 reduction reaction on platinum or platinum black electrodes (results not shown).

Overall, Pt Bl ultra-microelectrode arrays present the greatest sensitivity for O2 and H2O2

reactions due to the electrocatalytic activity of platinum black towards these reactions [START_REF] Arbault | Monitoring an oxidative stress mechanism at a single human fibroblast[END_REF][START_REF] Ben-Amor | Enhanced Detection of Hydrogen Peroxide with Platinized Microelectrode Arrays for Analyses of Mitochondria Activities[END_REF]. Indeed, Pt Bl electrodes exhibit increased electro-active surface area and porosity compared to platinum electrodes and therefore enhanced mass transport of species towards electrode surface. Their nanoscale structure [START_REF] Vaddiraju | Emerging synergy between nanotechnology and implantable biosensors: A review[END_REF] and large number of electroactive sites enhance electron transfer [START_REF] Li | Highly Sensitive Platinum-Black Coated Platinum Electrodes for Electrochemical Detection of Hydrogen Peroxide and Nitrite in Microchannel[END_REF]. This potential is given by Nernst equation and for metal/metal oxide electrodes depends only on the pH of the solution. As pointed out by the graph in Figure 3a, the Pt/IrO2 pH sensor presented no hysteresis. Both electrodes demonstrated an almost linear response over a wide pH range

(2.0-12.0). The sensitivity of Pt/IrO2 electrode (60 mV/pH) was superior to that of W/WO3 electrode (42 mV/pH) (Figure 3b). Pt/IrO2 exhibits a supra-Nernstian response due to the oxidation states present on the surface as already reported in literature [START_REF] Kim | Fabrication method and characterization of electrodeposited and heat-treated iridium oxide films for pH sensing[END_REF][START_REF] Olthuis | pH sensor properties of electrochemically grown iridium oxide[END_REF]. On the other hand, W/WO3 electrodes demonstrate a sub-Nernstian response that can be due to defects in the metal-metal oxide interface produced through oxidation of the tungsten layer. What is more, if the oxidation of the tungsten layer deposited through PVD is not optimized, native tungsten oxides such as WO2 or W2O5 [START_REF] Pasti | Switching between voltammetry and potentiometry in order to determine H+ or OH-ion concentration over the entire pH scale by means of tungsten disk electrode[END_REF] can be also created on the surface along with WO3 which leads to a lower sensitivity of the final layer. In order to improve characteristics of the W/WO3 sensors, optimization of the electrooxidation procedure is in progress.

Bioassays

Special emphasis was placed on O2 detection as it was considered to be the main pollution indicator, given the fact that O2 evolution reflects photosynthetic activity of algae and that it is largely altered by pollutants' existence. Photosystem II (PSII) is a complex located in the thylakoid membranes of algae where light energy is used to produce oxygen from water oxidation through a series of electron transfer reactions during photosynthesis [START_REF] Varsamis | Development of a photosystem II-based optical microfluidic sensor for herbicide detection[END_REF][START_REF] Giardi | Photosynthetic proteins for technological applications[END_REF]. Urea-based pollutants, such as Diuron, disrupt the electron flow in PSII because of their high-affinity binding to the PSII complex [START_REF] Pfister | Photoaffinity labeling of an herbicide receptor protein in chloroplast membranes[END_REF]. They bind to the D1 protein of PSII within its plastoquinone QB binding pocket blocking photosynthetic electron transport and therefore inhibiting O2 production [START_REF] Tischer | Relationship between inhibitor binding by chloroplasts and inhibition of photosynthetic electron transport[END_REF].

At first, the performance of the sensor for O2 detection was evaluated with algae in the absence of toxicants in order to demonstrate the relationship between the photosynthetic activity and the current signal measured. Light and dark periods were therefore altered to obtain equal periods of daylight illumination and darkness and current variations were recorded through chronoamperometry. Under illumination, amperometric responses recorded, exhibited an increase in the oxygen reduction current due photosynthesis. The decrease was evident when light was turned off as a result of respiration process during which algae consume oxygen and produce CO2 (Figure 4). Initial duration of light and dark cycles was six minutes. We then reduced cycle duration to two minutes in order to validate that similar response is obtained even by applying shorter time intervals. It is important to notice that the change in current evolution is immediately evident when light is switched on and off.

Variations in photosynthetic activity of algae were then determined in presence of Diuron herbicide (Figure 5). As ethanol 70% is the solvent used to prepare Diuron solutions, its volume was kept constant through different injections to prevent solvent influence on algal response.

Current evolution through respiration and photosynthesis with and without Diuron is presented in Figure 5a. The impact of the herbicide on electron flow and therefore photosynthetic O2

production is confirmed by comparing the steep slope corresponding to oxygen production rate (nA/min) during photosynthesis obtained for the blank algal solution with the smoother one obtained for Diuron contaminated algal solution.

It is yet demonstrated that there is no toxicantinduced inhibition on respiration procedure as the two slopes corresponding to oxygen consumption for blank and contaminated algal solution are identical.

Oxygen production rates for different pollutant concentrations (0.2-1 µM) were compared in order to demonstrate concentration dependent inhibition effect of the herbicide on photosynthesis (Figure 5b). The response of a blank algae solution was first compared to the one of an algal solution with ethanol to validate that ethanol has no inhibitory effect on O2 production. Oxygen production rate obtained with our Pt Bl ultramicroelectrode array (25 disks of diameter d=10 µm) for a blank algae solution (given strain: Chlamydomonas reinhardtii with a concentration 6 *10 6 cells/mL) is 10nA/min. We then compared the latter rate to the rate obtained for algal solution containing different concentrations of Diuron herbicide. The rate corresponding to the limit of maximum acceptable concentration of this pollutant implemented by governments (0.6 µM of Diuron) was finally calculated at 4 nA/min.

The inhibition on the oxygen production rate percent induced by herbicides was estimated from the oxygen production rate calculated before and after the herbicide injection and it is defined as followed:

𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑖𝑜% = 𝑟𝑎𝑡𝑒 𝑏𝑙𝑎𝑛𝑘 -𝑟𝑎𝑡𝑒 𝑖 𝑟𝑎𝑡𝑒 𝑏𝑙𝑎𝑛𝑘
Where rateblank represents oxygen production rate during photosynthesis for blank algal solution and ratei represents the rate for different concentrations of Diuron injected in algal solutions.

The Diuron calibration graph, given in Figure 6, demonstrates that inhibition increases along with Diuron concentration. Variation in production rate obtained was 65% inhibition per µM of Diuron with LOD of around 0.2 µM lower than maximum acceptable concentration in drinking water implemented by European governments (0.64 µM). It is therefore demonstrated that the fabricated electrochemical biosensor can be an efficient indicator of water pollution.

Although aforementioned fabricated systems aim for environmental applications that require disposable devices in compliance with regulations, the characteristics of our prototypes were tested in order to examine their reproducibility. The devices were used through several tests with algae and different concentrations of Diuron pollutant without significant variation in their response (around 60 tests with one device). A cleaning step in 0.5 M H2SO4 before each series of measurements was applied in order to reactivate the electrode surface and recycle the system.

The stability of the insulation layer that defines active areas as well as the stability of different active electrode layers can thus be validated. We can therefore also target other applications that require repetitive and/or online measurements.

CONCLUSION

We have presented above the development of a micro-biosensor for the detection of environmental pollution. Electrochemical microcells were fabricated for the detection of O2, H2O2 and H3O + /OH -ions, species taking part in metabolic activities of algae. Electrochemical microcells consist of three integrated electrodes (working, reference, and counter) on silicon chip. Electrode materials were chosen according to the species to be detected: Pt and black Pt electrodes were therefore used for O2 and H2O2 detection while W/WO3 and Pt/IrO2 electrodes were selected for pH monitoring. Electrodes were characterized, calibrated and further used in bioassays in algal solutions. Given the fact that this paper aims at demonstrating that pollutants' detection can be simply carried out by monitoring changes in algal metabolism induced by presence of herbicides, we firstly focused on O2 monitoring (i.e photosynthetic activity) as indicator of pollution as it is the element the most representative of variations in metabolic activities. O2 evolution was therefore measured in algal solutions and Diuron herbicide detection was achieved with a sufficiently low LOD of 0.2 µM. However, we also plan to determine in future work the increase in H2O2 and OH -production caused by presence of herbicides. To the best of our knowledge, the safety level of H2O2 and OH -in Chlamydomonas reinhardtii algae cells in non-contaminated water is not already determined but it is clearly mentioned that herbicides cause metabolic disruptions resulting in significant increase in the production of ROS [START_REF] Fedtke | Plant Toxicology[END_REF]. Similar to O2 monitoring, the measurement will be conducted by comparing the production of these two species for a blank solution and a herbicide contaminated solution using the H2O2 and pH sensors we fabricated that exhibit high sensitivity and satisfactory LOD.

These aforesaid results may form the basis for the integration of the three independent This final multi-detection structure will be completed by integrating an optical sensor on the same µfluidic platform [START_REF] Lefèvre | Algal fluorescence sensor integrated into a microfluidic chip for water pollutant detection[END_REF]. Thus, combining amperometric and optical detection, improved sensor's performance towards herbicide detection can be attained.

Figures caption

For

  Pt and Pt Bl working electrodes, active surface area was determined by linear sweep voltammetry in solutions of potassium hexacyanoferrate (II) trihydrate (10 mM) in 0.1 M KCl used as a supporting electrolyte. Calibration curves for H2O2 measurements were then obtained by recording current responses to different concentrations. pH sensors calibration was performed by monitoring the potential of the two metal oxide electrodes in solutions of different pH through open circuit potential (OCP) mode. O2 sensors performance was pre-validated in saturated in oxygen and zero oxygen CaCl2 (0.1 M) solutions by comparing current values obtained.

  pH measurements were conducted by the open circuit potential technique for calibration of W/WO3 and Pt/IrO2 electrodes. Equilibrium potential was recorded for different pH solutions.

  Photosystem II, with a direct impact on O2 production, while herbicides like Paraquat target

Figure 1 .

 1 Figure 1. (a) Schematic representation of all layers comprising the fabricated electrochemical sensor (b) Autonomous electrochemical micro-sensor.

Figure 2 .

 2 Figure 2. (a) Current measurement after successive injections of H2O2 for a Pt Bl array electrode. The potential applied is 0.3 V vs Ag/AgCl/KClsat Inset: Current response for 10 nM H2O2 injection. (b) H2O2 calibration curves obtained for Pt Bl and Pt single electrodes and arrays.

Figure 3 .

 3 Figure 3. pH monitoring with OCP measurement. a) IrO2 response for different pH values, b) Calibration curves for IrO2 and W/WO3 electrodes.

Figure 4 .Figure 5 .Figure 6 .Figure 1 .Figure 2 .Figure 3 .

 456123 Figure 4. Current measurement through algal respiration and photosynthesis with Pt Bl array electrode

Figure 4 .

 4 Figure 4. Current measurement through algal respiration and photosynthesis with Pt Bl array electrode.

Figure 6 .

 6 Figure 6. Calibration curves (inhibition percent in oxygen production rate during photosynthesis versus Diuron concentration) for Pt Bl array electrode.

Acknowledgement

The authors would like to thank the French "Agence nationale de la Recherche" (ANR, project DOLFIN, n° ANR-13-JS03-0005-01). Special thanks should also be addressed to Céline Christophe (LAAS-CNRS), Stephane Arbault, Salem Ben Amor and Damien Quinton of the Institute of Molecular Sciences (ISM, University of Bordeaux) as well as Florent Lefèvre and Annie Chalifour (Department of biological science, UQAM). Furthermore, microfabrication procedure was partly supported by the French RENATECH network.


Salah Edine Naimi
email: snaimi@ensa.ump.ma

Bekkay Hajji

Iryna Humenyuk

Jérôme Launay

P Temple-Boyer

Temperature influence on pH-ISFET sensor operating in weak and moderate inversion regime: Model and circuitry

Keywords: pH-ISFET, EKV-model, Weak inversion, Thermal drift, Readout circuit

come   L'archive ouverte pluridisciplinaire

Introduction

Since its development in the 1970 [START_REF] Bergveld | Development of an ion-sensitive solid-state device for neurophysiological measurements[END_REF], the Ion-Sensitive field effect transistors (ISFET) has been receiving more attention especially in areas of environmental monitoring applications and biomedical analysis. As a result, it has been used for the detection of multiple ions due to its many ion-sensitive membranes [START_REF] Yin | Characteristics of silicon nitride after O 2 plasma surface treatment for pH-ISFET applications[END_REF][START_REF] Lee | Development of a highly-sensitive acetylcholine sensor using a charge-transfer technique on a smart biochip[END_REF]. However, many industrial and laboratory applications require the precise measurements of pH. Commercially available sensors are generally very expensive devices, especially if reasonable precision and reliability are requested. Several investigations have demonstrated that the pH-ISFET sensor presents some drawbacks related to thermal dependency and long-term drift [START_REF] Janicki | Ion sensitive field effect transistor modelling for multidomain simulation purposes[END_REF][START_REF] Chen | An intelligent ISFET sensory system with temperature and drift compensation for long-term monitoring[END_REF][START_REF] Jamasb | A physical model for threshold voltage instability in S i 3 N 4 -gate H + -sensitive FETs (pH ISFETs)[END_REF]. These factors limit the performances of pH-ISFET sensors, leading to unacceptable results in critical measurements. In order to improve the pH-ISFETs stability, a lot of temperature compensation techniques have been reported in the literature such as the proper choice of biasing current for isothermal operating point to reduce ISFET temperature coefficient, the use of a p-n junction diode for temperature compensation on a single ISFET device, an ISFET based operational amplifier employment that improves temperature stability or the combination of several methods [START_REF] Chan | A CMOS ISFET interface circuit with dynamic current temperature compensation technique[END_REF][START_REF] Chunga | New ISFET interface circuit design with temperature compensation[END_REF][START_REF] China | A novel pH sensitive ISFET with on chip temperature sensing using CMOS standard proces[END_REF]. At the same time a lot of work has been done in the last thirty years, including simulations of its temperature characteristics according to theoretical models. They show that the ISFET temperature dependence is complicated in nature. Generally, these models were based on the so-called site-binding model and the MOSFET theory. The temperature influence is relatively complicated since it's related to the reference electrode, electrolyte/insulator potential and ISFET based MOS transistors [START_REF] Barabash | Analysis of the threshold voltage and its temperature dependence in Electrolyte-Insulator-Semiconductor Field-Effect Transistors (EISFET's)[END_REF]. Additionally, the IS-FET sensor is used, classically, in his strong inversion regime, this implies high power consumption, while the trend towards low-voltage operation, both for reasons of compatibility with digital technology, and to meet the needs of battery-operated equipment. Another strategy is to consider the weak-inversion operating. Previous studies have shown this possibility [START_REF] Shepherda | Weak inversion ISFETs for ultra-low power biochemical sensing and real-time analysis[END_REF]. Sub-threshold MOS operation, or weak inversion, is most often required by the low-power applications in modern electronics.

This article reviews our recent successful modeling of the temperature influence on pH-ISFET sensors by using MOSFET theory and site-binding model. In this work we implemented a simplified Enz-Krummenacher-Vittoz (EKV) model to explain the characteristics of ISFET sensor [START_REF] Enz | An analytical MOS transistor model valid in all regions of operation and dedicated to low-voltage and low-current applications[END_REF], in weak and moderate inversion regime, and discuss how the temperature can affect the behavior of the sensor. This paper deals with temperaturedependent ISFET drift modeling development and to its validation by comparison with experimental measurements. We have, also, investigated an alternative circuit operating in a subthreshold regime to have a linear relation between an output and the pH, and an improved thermal stability of this output.

ISFET electrical properties : background review

pH sensitive film/electrolyte interface modeling

Figure 1 shows a simplified schematic representation of the pH-ISFET sensor using p-type silicon substrates. The basic

n + n + p -typeSi Source Drain Bulk V ds V gs Ref erence electrode SiO2 SiO2

Si3N4

Si3N4 SiO2

Si3N4

Encasulation Encapsulation structure that serves as the basis for the ISFET is fabricated in standard CMOS chip technology. In the following and based on already existing theories, we combined the well-known site binding model and the MOSFET model to describe the behavior of an ISFET sensor. In many devices of this kind, it is assumed that protons interact with the insulator according to sitebinding theory. Whereas in the case of silicon nitride membrane, several studies have demonstrated that when the S i 3 N 4 surface is exposed to water, a sensitive surface becomes oxygen rich [START_REF] Künhold | Modeling the ph response of silicon nitride isfet devices[END_REF]. Consequently the silicon nitride surface contains sites in two forms, silanol (S iOH) and amine groups (S iNH 2 ), previous works have mentioned that all other sites can be neglected. More details on the site-binding theory can be found in [START_REF] Yates | site-binding model of the electrial double layer at the oxide/water interface[END_REF][START_REF] Siu | Basic properties of the Electrolyte-S iO 2 -S i system: Physical and theoretical aspects[END_REF][START_REF] Harame | Ion-sensing devices with silicon nitride and borosilicate glass insulators[END_REF]. A first-order model simplification suggests that if the electrolyte concentration is reasonable, the hypothesis of mono-sites is plausible. With this simplification the response of the sensor to the pH is given by the surface potential ψ 0 and dependent on the pH value of the electrolyte, it can be expressed as:

ψ 0 = 2.303 KT q β 0 1 + β 0 (pH pzc -pH) ( 1
)
where T is the temperature of the system, K is the Boltzmann's constant, q the electron charge, pH pzc is the non zero pH, and β 0 is a parameter which reflects the chemical sensitivity of the gate insulator and is dependent on the density of surface sites N s and the double layer capacitance C DL . The parameter β 0 is given by:

β 0 (T ) = 2q 2 N s √ Ka.Kb KTC DL (2)
where Ka, Kb are dissociation constants for the chemical reactions at the insulator interface. The double layer capacitance C DL consists of the two series capacitances C Gouy and C Helm , where C Gouy represent the equivalent Gouy-Chapman capacitance and C Helm the Helmholtz capacitance, which has already been developed by site-binding theory and the electrical doublelayer theory [START_REF] Fung | A generalized theory of an electrolyte-insulator-semiconductor field-effect transistor[END_REF][START_REF] Martinoia | A behavioral macromodel of the ISFET in SPICE[END_REF]. It should be noted that C Gouy and C Helm are temperature dependent, however, since these are secondorder effects, we will assume C Gouy and C Helm to be independent of temperature [START_REF] Chen | An intelligent ISFET sensory system with temperature and drift compensation for long-term monitoring[END_REF].

Thus, it has been possible to investigate the pH-ISFET flatband voltage shift with pH and the variation of the potential at

D S G V pH G' C Helm C Gouy E ref ψ 0 isolant/SC solution
∆V FB = V pH = E re f + χ sol + ∆ϕ l j -ψ 0 ( 3
)
where E re f is the potential of Ag/AgCl reference electrode, ∆ϕ l j is the potential drop between the reference electrode and the solution, which typically has a value of 3mV, χ sol is the electrolyte-insulator surface dipole potential, with a typical value of 50mV [START_REF] Barabash | Analysis of the threshold voltage and its temperature dependence in Electrolyte-Insulator-Semiconductor Field-Effect Transistors (EISFET's)[END_REF]. Both ∆ϕ l j and χ sol are pH independent. ψ 0 is the surface potential from (1). In equation ( 3), ψ 0 is the only parameter responsible for ISFET pH sensitivity, as previously explained by the site binding theory. Hence the macro-model represented schematically in Fig. 2.

MOSFET part modeling : EKV Model

The working principle of the ISFET is based on the standard MOSFET structure. Therefore, a robust model, of the MOS-FET part, is suitable for performing robust and valid model in a large interval of temperatures and pH. Another critical point might be the number of parameters for each model, who must be smallest. EKV model is particularly suitable as it provides accurate modeling with a small set of parameters [START_REF] Enz | An analytical MOS transistor model valid in all regions of operation and dedicated to low-voltage and low-current applications[END_REF]. This section describes the equations and parameters used for the computer simulation version of the EKV-MOSFET model. The set of equations includes modeling of basic physical effects, geometrical aspect, substrate effect, modeling of mobility reduction due to vertical field and modeling of thermal behavior. The EKV model proposes the following empirical expression of source-drain current (I DS ) valid for all regimes of operating.

I DS = 2nβU T 2 [ln 2 (1+e V GS -V T 0 -n.V S 2nU T )-ln 2 (1+e V GS -V T 0 -n.V D 2nU T )](4)
where U T is the thermodynamic voltage, V T 0 is the threshold voltage, n is the body effect factor and β the transfer parameter, V GS is the gate to source voltage, V S is the source potential and V D is the drain potential. For long channel model implementation, we consider only parameters accounting for first order effects. The model used is based on the ideal MOS structure of long and large channel with uniform substrate doping. Notice that the current expression in (4) is let as a difference between forward and reverse current using standard interpolation function, thus a better fitting is obtained in weak and moderate inversion. The expressions of EKV parameters V T 0 , n and β are:

V T 0 = V FB + φ F + γ φ F (5) n = 1 + γ 2 V P + φ F + 4U T (6) β = K P W L 1 1 + θ.V P (7)
where V FB is the flat-band voltage, φ F is the bulk Fermi potential, γ is the body effect parameter, K P is the transconductance parameter, W is the channel width and L is the channel length. The mobility reduction modeling introduces only one parameter, θ. Finally V P is the pinch-off voltage.

Basically, V P and n factors are specific to EKV model, although a possible physical link between EKV model and charge-sheet model seem to exist [START_REF] Sallese | Inversion charge linearization in MOSFET modeling and rigorous derivation of the EKV compact model[END_REF]. The V P term expression is:

V P = V GS -V FB -φ F -γ( V GS -V FB + ( γ 2 ) 2 - γ 2 ) (8)

ISFET Temperature characteristic

It seems that the pH-ISFET sensor, sometimes, do not follow the theory of operation. The data obtained show that the temperature dependence is complicated in nature and the user should beware of using the sensors during variable environmental conditions e.g. field tests. In such case an additional electronic circuit is needed to reduce the temperature drift of the sensor response. In order to explore a pH-ISFET parameters, subject to temperature changes, this section describe a simplified temperature-dependent model of MOSFET part of the pH-ISFET as well as the reference electrode temperature influence. We show, later, that these compact models provide accurate representation of measured DC component performance.

MOSFET temperature effect

The EKV model introduces a few parameters for intrinsic parameter's temperature dependence, we consider two of them here, TCV the temperature coefficient of the threshold voltage and BEX the mobility temperature exponent [START_REF] Bucher | The EPFL-EKV MOSFET model equations for simulation[END_REF].

V T 0 (T ) = V T 0 -TCV(T -T nom ) (9)
K P (T ) = K P ( T T nom ) BEX (10)
The temperature dependence of the bulk Fermi potential φ F is as :

φ F (T ) = φ F T T nom -3 KT q ln( T T nom ) -Eg(T nom ) T T nom + Eg(T ) ( 11
)
Where Eg is the energy gap of silicon, which should obey :

Eg(T ) = 1.16 -0.000702. T 2 T + 1108 ( 12
)
Where T is the absolute temperature and T nom is the nominal temperature chosen as a reference temperature (T nom = 300.15C). The intrinsic model parameters γ, φ F and K P are related to process parameters like oxide thickness and channel doping. Generally it is preferable to avoid to link such parameters for better fitting of experimental measurements.

Temperature coefficient of the reference electrode

The liquid solution, which acts as the gate ion-conductor, is connected to the gate voltage source by means of an Ag/AgCl reference electrode. The potential of the reference electrode E re f is related to the temperature according to the expression [START_REF] Barabash | Analysis of the threshold voltage and its temperature dependence in Electrolyte-Insulator-Semiconductor Field-Effect Transistors (EISFET's)[END_REF]:

E re f (T ) = E abs ( H + H 2 ) + E re f ( Ag AgCl ) + ( dE re f dT )(T -298.16) (13)
Where E abs (H + /H 2 ) is the normalized hydrogen potential, E re f (Ag/AgCl) is the relative potential of the reference electrode, the two potential are temperature independent (their values are respectively 4.7V and 0.205V), the temperature coefficient of AgCl/Solution junction is practically negligible, d(E re f )/dT is the temperature coefficient equal to 140µV/K [START_REF] Cobbold | Transducers for Biomedical Measurements: Principles and Applications[END_REF][START_REF] Gomer | An experimental determination of absolute halfcell EMF's and single ion free energies of solvation[END_REF][START_REF] Bousse | The chemical sensitivity of Electrolyte/Interface/Silicon structures[END_REF].

Fabrication and testing

The N-type ISFET fabrication was carried out at LAAS/CNRS clean room facilities. Standard N+ implantation (dopant: arsenic, energy: 50 keV, dose: 10 16 at/cm 2 ) was used to realize the source and drain N+ regions into the boron-doped (10 15 at/cm 3 ) P-type silicon substrate. The gate dielectric is based on a S iO 2 /S i 3 N 4 sensitive membrane structure [START_REF] Temple-Boyer | Study of front-side connected chemical field effect transistors for water analysis[END_REF]. It is obtained by a thermal oxidation, in dry oxygen, to grow a thin layer of S iO 2 (thickness: 50nm) followed by a low pressure chemical vapor deposited (LPCVD) process to form on top a thin S i 3 N 4 /S iO 2 film (thickness: 50nm) acting as ion-sensitive layer as well as wafer-level passivation layer. The length of the canal is 30µm and its width is 800µm. To measure the current-voltage characteristics of the pH-ISFET, a source and substrate are grounded and the source to drain voltage was fixed to 1V. To measure pH sensitivity, the pH buffer solutions were changed from pH 2 to pH 10. To investigate the stability and reliability of the pH sensors, the effects of temperature on the responses of an ISFET are evaluated.

Temperature can change the characteristics of a sensor, the potential of a reference electrode, the pH of electrolyte and the whole measuring system. For latter changes, constant temperature is maintained inside the room. To keep the electrode, electrolyte, ISFET temperature constant, a heat regulating loop including a temperature-sensing device and a heating device have been used. Also, experimental results can only be compared validly to theory, if the temperature during measurements is fixed. We use a temperature controller to set the temperature in the range of 23 -50 • C.

M4 M3 M2

ISFET The potential Vbias is set to V CC (it may vary from 2.5V to 3V). The transistor M2 is used in conjunction with the transistor M4 to control the variation of V 1 , thus the ration 70/3 of M2 don't match the ration 800/30 of the pH-ISFET.

I 1 I 1 Vcc (3V ) Vcc Vee Vee (-3V ) Vcc Vcc I 2 R=30kΩ V out M6 M5 M8 M7 M10 M12 M11 M9

The readout circuit

Since the purpose of this work is to study the benefit of using an ISFET sensor in weak-inversion regime, we propose a simple readout IC with low temperature dependence. From (4) it can be easily shown that the source-drain current I DS , in weak inversion region, must comply with the following requirement (V S = 0, V DS ≫ V T ):

I DS = 2nβU T 2 e V GS -V T 0 -V pH nU T ( 14
)
With V pH = ∆V FB , thus the macro-model of the ISFET in Fig. 2 is still available in weak inversion. This section explores some ideas on how to use such results in a simple circuit.

Basic principle of the circuit

For V pH we get from equations (1)(3) and ( 13):

V pH = 2.303U T (pH -pH pzc ) + E re f ( Ag AgCl ) + ( dE re f dT )(T -298.16) + χ sol + ∆ϕ l j (15)
The absolute potential of the standard hydrogen electrode E abs ( H + H 2 ) is compensated by the work function of the metal back contact/electronic charge [START_REF] Martinoia | A behavioral macromodel of the ISFET in SPICE[END_REF]. Let us consider the expression in [START_REF] Siu | Basic properties of the Electrolyte-S iO 2 -S i system: Physical and theoretical aspects[END_REF], it can be rewritten as:

V pH = V 1 -V 2 = a pH U T + b (16) With        a pH = 2.303(pH -pH pzc ) + q k dE re f dT b = E re f ( Ag AgCl ) -298.
16

dE re f dT + χ sol + ∆ϕ l j ( 17
)
These two terms, a pH and b, being to the first approximation independent of the temperature. The temperature-dependency of the first term of V pH (a pH U T ) can be eliminated if the pH-ISFET is operating in weak inversion. The problem, however, will still not be solved; the resulting term b/U T cannot be eliminated easily (as by a differential structure). In the following analysis, we focus on this second term and try to minimize its influence. In equation ( 16), V pH is expressed as a differential potential (V 1 -V 2 ), if each potential passes through an attenuator circuit, the output of the attenuator stage is (V 3 -V 4 ), with:

V 3 -V 4 = α 1 V 1 -α 2 V 2 ( 18
)
yielding

V 3 -V 4 U T = α 1 V 1 -α 2 (V 1 -b) U T + α 2 a pH ( 19
)
If α 1 and α 2 are chosen as

α 1 V 1 ≈ α 2 (V 1 -b)
, the expression in equation 19 becomes :

V 3 -V 4 U T ≈ α 2 a pH = α 2 (2.303(pH -pH pzc ) + q k dE re f dT ) (20)
The last expression in equation ( 20) is a function of pH but temperature independent (we suppose that the effect of the temperature on pH pzc and dE re f /dT is negligible). However, the term α 2 must be temperature independent. To achieve the earlier condition,

α 1 V 1 ≈ α 2 (V 1 -b), the potential V 1 does
not have to vary too much. In the next subsections we propose a circuit able to extract the potential V pH and then divide it by the thermal potential U T . This illustrates the general principle of the proposed circuit. The basic topology of this circuit is given in Fig. 3, it has three stages as shown in the figure. As suggested previously, once the tension V pH is extracted (with the first stage), the two components of a differential potential are passed through two attenuators in the second stage, the third stage allows to divide the output by a thermal voltage U T . In the previous considerations, the voltage V 1 , can obviously be fixed by a reference voltage, placed in the gate of the transistor M13. However, this tension is used actually to keep for each stage, the tension resulting from the sensor as a differential voltage. This will allow a better linearization of the output.

First stage : modified Caprio's Quad

The first stage of the circuit in Fig. 3 is based on a Caprio's Quad, which is originally a V-I converter. The circuit is useful for moderate input amplitudes, the voltage swing at the input cannot be very large (±500mV at most). The equivalent voltage source V pH is accurately replicated across the source connections of M3 and M4. The current sources I 1 allow limiting the currents in a desired region (sub-threshold or moderate inversion regions). Obviously the pH-ISFET and the transistor M2 should have a perfectly matched ration W/L. As previously mentioned, the potential V 1 must be constant or variable in reasonable limits, this can be achieved by adjusting the width of the transistor M4. Consequently the greatest variation of the voltage V 1 was found to be around 6% for large variations of the temperature and the pH (T = 20 -60 • C, pH = 2 -12). Thus, the voltage swing, V 2 -V 1 is caused by the variation of V 2 . In the next section we will discuss this results.

Second stage : attenuators

The output of each attenuator (M5, M6, M7, M8 or M9, M10, M11, M12) is linearly proportional to inputs. One of these attenuators can be used to have

α 1 V 1 ≈ α 2 (V 1 -b)
as mentioned previously. In fact we need this stage to reduce the potentials values for the entry of the third stage too. It is assumed that good layout techniques will help reduce errors due to process and thermal variations. These circuits are very immune to temperature for great variations of the entry.

Third stage : source coupled circuit

A differential amplifier composed of the two matched transistors M13 and M14 in addition to the source current I 2 , provides a simple method of realizing a division of the input voltage by the thermal potential U T . The basic equation of the circuit is :

I out = I DM13 -I DM14 = I 2 × tanh( V d 2ηU T ) ( 21
)
where I DM13 , I DM14 are the drains current of M13 and M14, V d is the differential input voltage (V d = V 3 -V 4 ) and η is the sub-threshold slope factor. Equation [START_REF] Cobbold | Transducers for Biomedical Measurements: Principles and Applications[END_REF] assumes that M13 and M14 are operating in weak inversion. If the circuit operates under the restriction, |V d | << U T , the output current of the circuit is given by :

I out ≈ I 2 V d 2ηU T (22)
The circuit uses the current mirror as an active load (M15-M16). Note that the output of the circuit is a current, and if it is required to have an output voltage, a differential current to voltage converter is needed. To simplify this can be realized by using a resistor (R) as shown, in this case, the output voltage is given by:

V out ≈ RI 2 V d 4ηU T ( 23
)
As the differential-pair (M13-M14) does not operate correctly for large signal, therefore we are constrained to make the Caprio's quad signals pass through two attenuator circuits to reduce the input voltages. In order to demonstrate the feasibility of the proposed circuit and to show an improvement of the temperature independent output, we simulate this circuit by using the MOSIS CMOS (AMI 1.0µm) minimum channel length technology and SPICE parameters. The electronic circuit simulation program HSPICE, which is one of the simulators that can be suitably adapted to design and simulate the ISFET transducers has been used.

Simulation results and discussion

ISFET characteristics

The equations provided in the previous sections are used to simulate the ISFET behavior under different operating conditions. The EKV parameters and site-binding coefficients are determined from experimental data. In order to obtain an optimized set of coefficients, it is desirable to take into account all types of experimental data, e.g. temperature, pH and gatesource voltage (Vgs) changes. The parameters are determined from the experimental data by an efficient mathematical, least squares minimization, methods.

Drain to source current versus gate to source voltage (Ids -Vgs) curves, measured in buffer solutions of different pH, are shown in Fig. 4-5-6. We present the results as a series of plots. The curves are shifted by 54.7mV/pH at 23 • C to a positive voltage for the samples immersed in higher-pH buffer solutions, which can be explained by the site binding theory. The experimental results were found to be in agreement with the theoretical predictions. As can be seen from Fig. 7, the fitted curves accurately match the measured values at pH 4, 10 and specific value of pH 7 (Fig. 9-b). In a second experiment, the pH-ISFET was operating in a buffer solution at pH 5.2 with temperature varied in from 10 to 52 • C. Taking into account that the model is relatively simple, the simulation results are satisfactory. The fit is excellent, for all ranges of pH and temperature in weak inversion and remain good for moderate inversion. Obviously, for long and wide device we would expect the best agreement, with a few MOSFET part parameters.

Process I Process II

N A ≈ 1e 17 (cm -3 ) N A ≈ 1e
Gate to Source Voltage Vgs(V)

Drain to Source Current Ids(A)

T=21.5°C to 41°C The model parameters (EKV and site binding) were obtained through a nonlinear regression process, by the least squares method. The mean value, as well as the standard deviation of each parameter are given in table 1. Data processing was done for 23 devices tested under various pH and temperature conditions. As we used a nonlinear regression, the reliability factor (RF) defined in equation 24 is used to measure of the goodness of fit instead of the coefficient of determination (R 2 ). In equation 24, Ids exp (i) is the experimental measurement number i, while Ids th (i) is the calculated values.

Reliability Factor = ( n i=1 (Ids exp (i) -Ids th (i)) 2 n i=1 (Ids exp (i)) 2 ) (24)

Temporal drift and hysteresis experiments

Drift phenomena in pH-ISFETs typically appear as relatively slow, monotonic, and temporal changes in the threshold voltage of the device. Figure 8 shows the gate to source (Vgs) voltage drift when Ids and Vds were fixed to 100µA and 1V respectively, while the buffer solution is at pH 10. The test was made for approximately 4h. A heating/cooling cycle has been performed to estimate the influence of the temperature on the pH-ISFET. The experiment of the illumination influence has been performed in the same test (by switching on the light, the room illumination has been found to increase from 70 to 1500Lux). The shift of ∆Vgs is around 5mV (equivalent to 0.1 pH measurement error). Firstly, the pH-ISFETs responses are characterized by a decrease which should be related to their adaptation to the watery medium. The increase of temperature from 22 • C to 40 • C is responsible for an increase of the output voltage around 25mV. This phenomenon is related to the decrease of the ISFET threshold voltage. The initial period being apart, the complete experiment has also allowed estimating the temporal drift of the pH-ISFETs around 7.5mV/hour, i.e. 0.15pH/hour. When the heating/cooling cycle is no longer, the temporal drift is still evidenced but is strongly reduced. The drift can be estimated to 1mV/hour, i.e. 0.02pH/hour. Like the time drift effect, the hysteresis also limits the accuracy of ISFET pH measurements. Hysteresis tests were performed by varying the pH All series of measurements have been devoted to characterizing the static sensor response, i.e. the dependence of the sourcedrain current versus source-grid tension on the pH and temperature changes in weak and moderate inversion regime. Based on these results, the behavioural thermal drift of the pH-ISFET in the sub-threshold region can be modeled by a combination of basic EKV model for the MOSFET part of the ISFET and a simplistic version of the site-binding model using Martinoia macro-model [START_REF] Martinoia | Modeling ISFET microsensor and ISFET-based microsystems: a review[END_REF] presented in Fig. 2. On the other hand, the electrolyte-membrane part plays an important role in the static characteristics of the sensors. The thermal effect of the electrode/electrolyte/membrane cannot be neglected and limiting the performance of the sensor (E re f drift is around 0.14mV/ • C, the threshold voltage thermal drift of the MOS part is around 9mV/ • C and the surface potential drift is around 1mV/ • C).

Circuit simulation

Figure 10 shows the temperature and the pH actions on the first stage outputs (V 1 and V 2 ). As can be seen from the figure, the pH influences significantly the potential V 2 instead of the potential V 1 . The maximal variation of V 1 can be estimated around 19mV (6%), while that of V 2 is approximately 410mV, for pH varying from 2 to 12. As an analytical calculation of the attenuation α 1 (α 2 ) is very difficult (especially to take into account of the effect of the temperature variation). The influence of the temperature, the canal width of the transistor M10 and the pH on the terms

α 1 V 1 , α 2 (V 1 -b) and α 1 V 1 -α 2 (V 1 -b)
was simulated. The results of these simulations are shown in figure 11. The circuit displayed a minimum change of the term

α 1 V 1 -α 2 (V 1 -b)
under several conditions such as the change of temperature or the potential V 1 . In figure 11 the change of the potential V 1 is imposed by the variation of the pH from 1 to 12, the potential V 1 was extracted from the simulation of the V (V) V (V)

1 α 1 V 1 (mV) (a)
1 α 2 (V 1 -b) (mV) (b)
W M10 = 3µm W M10 = 4µm W M10 = 5µm
V (V) 1 α 1 V 1 - α 2 (V 1 -b) (mV) (c)
W M10 = 3µm W M10 = 4µm W M10 = 5µm
Figure 11: simulations showing the variation of (a)

α 1 V 1 , (b) α 2 (V 1 -b) and (c) α 1 V 1 -α 2 (V 1 -b), for T =
1 V 1 -α 2 (V 1 -b
) can be adjusted to a value equal or close to zero by acting on the canal width of the transistor M10. It has to be noted that in Fig. 11-c, each of the three curves includes the results of simulations for T = 20, 40 and 60 • C. In the above analysis, the value of the attenuation α 1 is chosen and then the attenuator circuit sized according to the constraints of stage 3. The resulting values of α 1 and α 2 are around -0.5 and -0.9 respectively. As illustrated by figure 12, the circuit simulation shows a linear response across a large range of pH, as well as a very insensitive temperature behavior. We show that this compact circuit provides accurate response, although the temperature varies between 20 and 60 • C. The thermal compensation scheme provides an improved thermal stability by eliminating the temperature effect on the circuit output. The width of the transistor M4 of the Caprio's-quad was adjusted to keep the potential V 1 constant. The simulation results confirm the theoretical considerations presented in the paper. The circuit output presents a maximum voltage drift, with temperature, around 28µV/ • C for pH=12 and 3.7µV/ • C for pH=5. The maximum power consumption of the readout circuit was estimated to be around 18.6mW. In integrated IC application perspective, the power supply and the biasing currents can be further minimized by reducing the aspect ratios of ISFET device and all transistors for lowering the total power consumption.

Conclusions

In this study, we analyzed the behavior of a pH-ISFET sensor shown by changing the temperature and the pH of the electrolyte solution. The simulation results show that the combination of a simplified EKV model with basic description of a surface potential can fit experimental results with good accuracy in weak and moderate regimes.

The model takes into account a temperature dependency of reference electrode, the S iO 2 /S i 3 N 4 electrolyte/insulator inter-face and the semiconductor part of the pH-ISFET. The model was able to explain the experimental measurements of threshold voltage drift, estimated around 9mV/C (at T=300K). The extracted value of pH pzc (4.2 at T=300K) is in good agreement with the reported values for S i 3 N 4 ion sensitive membrane. The temporal drift combined to the thermal drift shows the limits of the pH sensors, the drift was estimated to 0.02 pH/h. If the thermal hysteresis is performed, this value increase to 0.15 pH/h for a shift of approximately 20 • C. The study introduces, also, an original architecture that implements a low-power readout circuit for a pH-ISFET featuring thermal compensation, based on MOS transistors operated in sub-threshold. The ISFET thermal model is used to simulate a proposed readout circuit. As a result, the circuit output voltage drift, with temperature, is less than 3.7µV/ • C, around pH=5. The main advantage of the proposed solution consists in the absence of a temperature sensor, resulting in a greater simplicity of the circuit.

Figure 1 :

 1 Figure 1: Schematic of the pH-ISFET structure (N-Type).

Figure 2 :

 2 Figure 2: Equivalent circuit for the pH-ISFET macro-model

Figure 3 :

 3 Figure 3: Proposed readout circuit to provide high thermal stability and linearity, stage 1: Caprio's quad, stage 2 : attenuators, stage 3 : differential amplifier. The ratio values indicate W/L in µm.The potential Vbias is set to V CC (it may vary from 2.5V to 3V). The transistor M2 is used in conjunction with the transistor M4 to control the variation of V 1 , thus the ration 70/3 of M2 don't match the ration 800/30 of the pH-ISFET.

Figure 4 :Figure 5 :

 45 Figure 4: Drain-Source current (Ids) vs Gate-Source voltage (Vgs) for pH = 2, T = 21.5 • C -31 • C -41 • C

Figure 6 :

 6 Figure 6: Drain-Source current (Ids) vs Gate-Source voltage (Vgs) for pH = 10, T = 23 • C -37 • C -50 • C

Figure 7 :Figure 8 :Figure 9 :Figure 10 :

 78910 Figure 7: Effect of temperature (a) or pH (b) variations on Drain-Source current (Ids) vs Gate-Source voltage (Vgs) curves. The value of RF indicate the reliability factor of the simulation

Figure 12 :

 12 Figure 11: simulations showing the variation of(a) α 1 V 1 , (b) α 2 (V 1 -b) and (c) α 1 V 1 -α 2 (V 1 -b),for T = 20, 40 and 60 • C and the canal width of the transistor M10, W M10 = 3, 4 and 5µm, as the potential V 1 is varied. The interval of variation of V 1 is deduced from the simulation of the first stage for pH varying from 2 to 12.

Table 1 :

 1 Extracted model parameters for MOSFET part and surface potential parameters values with regression statistics for a total of 23 tested devices. (N A is the substrate p-type doping concentration, RF is the Reliability Factor (RF was calculated for each device), (*) no temperature variations were made for this process).

	15 (cm -3 )

J. Launay was born on

 March 11, 1975. He received the degree in electronic engineering from the "Institut National des Sciences Appliquées de Toulouse" (France) in 1998. He joined the "Laboratoire d'Architecture et d'Analyse des Systèmes" from the French "Centre National de la Recherche Scientifique" (LAAS-CNRS) in 1998 and received the PhD degree from the "Institut National des Sciences Appliquées de Toulouse" (France) in 2001. In 2002, he became lecturer at the University of Toulouse (France). His research activities include the development of electrochemical micro-sensors for the detection in liquid phase. October 25, 1966. He received his Engineers Degree in electronic engineering from the "Ecole Supérieure d'Electricité" (Paris-France) in 1990 and his Masters Degree in microelectronics from the University of Toulouse (France) in 1992. He joined the "Laboratoire d'Architecture et d'Analyse des Systèmes" (LAAS) from the French "Centre National de la Recherche Scientifique" (CNRS) in 1992 and received the PhD degree from the "Institut National des Sciences Appliquées de Toulouse" (France) in 1995. Since then, as a CNRS researcher, he has worked at LAAS on the development of physical and chemical micro-sensors.

P. Temple-Boyer was born on