Integrated electrochemical biosensor based on algal metabolism for water toxicity analysis Aliki Tsopela, Ahmet Lale, Emilie Vanhove, Olivier Reynes, Isabelle Séguy, Pierre Temple-Boyer, Philippe Juneau, R Izquierdo, Jérôme Launay To cite this version: Aliki Tsopela, Ahmet Lale, Emilie Vanhove, Olivier Reynes, Isabelle Séguy, et al.. Integrated electro- chemical biosensor based on algal metabolism for water toxicity analysis. Biosensors and Bioelectron- ics, Elsevier, 2014, 61, pp.290 - 297. <10.1016/j.bios.2014.05.004>. <hal-01504978> HAL Id: hal-01504978 https://hal.archives-ouvertes.fr/hal-01504978 Submitted on 10 Apr 2017 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Integrated electrochemical biosensor based on algal metabolism for water toxicity analysis A. Tsopela1,2, A. Lale1,2, E. Vanhove1,2, O. Reynes3, I.Séguy1,2, P. Temple-Boyer1,2, P. Juneau4, R. Izquierdo4, J. Launay1,2 1 CNRS, LAAS, 7 avenue du colonel Roche, F-31400 Toulouse, France 2 Université de Toulouse, UPS, LAAS, F-31400 Toulouse, France 3 Université de Toulouse, UPS, LGC, 31062 Toulouse, France 4 Université du Québec à Montréal ; 201 Président Kennedy ; Montréal, Canada Abstract An autonomous electrochemical biosensor with three electrodes integrated on the same silicon chip dedicated to the detection of herbicides in water was fabricated by means of silicon- based microfabrication technology. Platinum (Pt), platinum black (Pt Bl), tungsten/tungsten oxide (W/WO ) and iridium oxide (Pt/IrO ) working ultramicroelectrodes were developed. 3 2 Ag/AgCl and Pt electrodes were used as reference and counter integrated electrodes respectively. Physical vapor deposition (PVD) and electrodeposition were used for thin film deposition. The ultramicroelectrodes were employed for the detection of O , H O and pH 2 2 2 related ions H O+/OH-, species taking part in photosynthetic and metabolic activities of algae. 3 By measuring the variations in consumption-production rates of these electroactive species by algae, the quantity of herbicides present at trace level in the solution can be estimated. Fabricated ultramicroelectrodes were electrochemically characterized and calibrated. Pt Black 1 ultramicroelectrodes exhibited the greatest sensitivity regarding O and H O detection while 2 2 2 Pt/IrO ultramicroelectrodes were more sensitive for pH measurement compared to W/WO 2 3 ultramicroelectrodes for pH measurement. Bioassays were then conducted to detect traces of Diuron herbicide in water samples by evaluating disturbances in photosynthetic and metabolic activities of algae caused by this herbicide. Keywords: ultramicroelectrodes; oxygen; hydrogen peroxide; pH measurement; algal metabolism; pesticides detection 2 1. 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 (Barbash et al., 2001). 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 (Schellin et al., 2004) and liquid (Jáuregui et al., 1997) 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 (Eltzov and Marks, 2011). 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 (Lagarde and Jaffrezic-Renault, 2011; Grieshaber et al., 2008; Li et al., 2013). 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 (Jang et al., 2011). 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 3 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 (Ben-Yoav et al., 2009). Amperometric biosensors have been widely used for the oxygen monitoring in wastewaters (Chan et al., 2000), detection of organophosphates (Mulchandani et al., 2001), phenols (Mulchandani et al., 2005), cyanide (Ikebukuro et al., 1996) and heavy metal ions (Lehmann et al., 2000). 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 (Lagarde and Jaffrezic-Renault, 2011). Potential values are then correlated to the concentration of the analyte (Lei et al., 2006). Although pH electrodes have been widely used for detection of pesticides in agriculture (Mulchandani et al., 2001; Gaberlein et al., 2000), other ion selective electrodes, such as chloride ion selective electrode, have also generated major interest in wastewater treatment domain (Han, 2002). Conductometric biosensors record variations in conductivity of the electrolyte containing charged species in contact with the electrode (Lagarde and Jaffrezic-Renault, 2011). These variations result from the production or consumption of charged species during different reactions and can therefore indicate a biological process taking place (Lawrence and Moores, 1972; Jaffrezic-Renault and Dzyadevych, 2008). They have found various applications in environmental monitoring for detection of heavy metal ions and pesticides (Chouteau et al., 2004; Chouteau et al., 2005). 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 (Giardi and Pace, 2005; Brayner et al., 2011; Moreno- Garrido, 2008; Carrilho et al., 2003). Due to their high sensitivity to toxic substances, even 4 lower detection limits (LOD) can be reached (Brayner et al., 2011). 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 (Kasai and Hatakeyama, 1993; Lagarde and Jaffrezic-Renault, 2011). 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 (Pandard and Rawson, 1993). 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 (Mittler, 2002), 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 (Mittler et al., 2004). Along with O and H O , pH monitoring can also provide information on 2 2 2 metabolic activity modifications induced by herbicides and other inhibitors based on changes in medium basification and acidification rates (Schubnell et al., 1999). As a matter of fact, different sensors have been developed for the detection of the species mentioned above, viewed as indicators of water pollution (O , H O , H O+/OH- ions). Two basic 2 2 2 3 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 (Lee et al., 2007). Platinum is the most suitable candidate for oxygen detection as it is physically stable (Zhang and Wilson, 1993), 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. Desic, 2005). Platinum microdiscs (Sosna et al., 2007) and array of platinum 5 microelectrodes (Wittkampf et al., 1997) 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 (Qiang et al., 2010; Lee and Park, 2011). Although platinum is the most commonly applied material in oxygen sensors, gold electrodes have been designed to measure dissolved oxygen in environmental samples (Liu et al., 2007), bacterial films (Lee et al., 2007) or during cell culture (Pereira Rodrigues et al., 2008). Carbon nanotubes integrated on a screen-printed algal biosensor have also been used to detect oxygen produced by algae (Shitanda et al., 2009). As already stated, hydrogen peroxide is an essential species to monitor as it is taking part in various biological processes (Li et al., 2013). Gold (Brett et al., 1997), platinum(Zhang and Wilson, 1993; Yokoyama et al., 1997), carbon based (Cho et al., 2010; Nowall and Kuhr, 1997), enzyme modified (Ruzgas et al., 1996) and Prussian Blue modified (Karyakin et al., 2009) electrodes are situated in the main stream of research interest towards suitable working electrode materials for the detection of H O . Platinum is still the preferred option as it provides a high 2 2 catalytic surface for H O oxidation (Arbault et al., 1995) and it is even possible to increase 2 2 active surface area of the electrodes by platinization. Indeed, sensors with platinized working electrodes yield greater sensitivity due to their nanoscale structure (Vaddiraju et al., 2010) and the large number of electroactive sites that facilitates electron transfer (Li et al., 2013). Furthermore, on platinum black (Pt Bl) microelectrodes, H O oxidation is taking place at a 2 2 lower potential compared to bare Pt electrodes, minimizing interferences induced by other electroactive species (Arbault et al., 1995). Platinum black microelectrodes have been widely used to study ROS species released during cellular oxidative bursts (Li et al., 2013) and monitor the production of H O by mitochondria during aerobic metabolism (Ben-Amor et al., 2013). 2 2 They have also found various applications as amperometric glucose sensors (De Corcuera et al., 2005). 6 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 (Ruffien-Ciszak et al., 2008; Huang et al., 2011) giving way to metal/metal oxide based pH sensors that are easily miniaturized and provide a well- defined metal/metal oxide interface (Daomin Zhou, 2008). More specifically, iridium oxide films can be formed by various physical and chemical methods (Ges et al., 2005) and exhibit many advantages among which high pH sensitivity, biocompatibility, chemical stability and fast response time (Daomin Zhou, 2008; Marzouk et al., 1998). IrO films have therefore found 2 applications in cell metabolism monitoring in cell culture volumes (Ges et al., 2005) and microenvironmental studies (Bezbaruah and Zhang, 2002). 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 (Ruffien-Ciszak et al., 2008). W/WO 3 sensors have been developed for skin pH monitoring (Ruffien-Ciszak et al., 2008), determination of extracellular pH values for cultured cells (Yamamoto et al., 2003) and pH measurement of environmental water samples (Dimitrakopoulos et al., 1998). 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 (O ), hydrogen 2 peroxide (H O ) and pH related ions (H O+/OH-). The electrochemical microsystem is further 2 2 3 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 7 caused by herbicides. These disturbances are represented by variations in consumption- production 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. 2. Experimental 2.1 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 (Christophe et al., 2013). 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 H O and dissolved O measurements whereas tungsten/tungsten 2 2 2 oxide (W/WO ) and iridium oxide (Pt/IrO ) electrodes were used for pH monitoring. The 3 2 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. 8 2.2 Fabrication of the electrochemical microcells Electrochemical microcells were fabricated on a thermally oxidized silicon wafer (SiO ) 2 (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 SiO substrate. Silver was deposited 2 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 (Si N ) insulation layer (100 nm) (Figure 1a). ICP-CVD process 3 4 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 Si N film was 3 4 optimized accordingly (Vanhove et al., 2013). 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 (Vanhove et al., 2013). 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). 9