Fonctionnalisation de surfaces d'électrodes par un film de poly(3,4-éthylènedioxythiophène) PEDOT pour l'élaboration de microcapteur spécifique des acides ascorbique et urique : application à l'étude des propriétés antioxydantes du sérum sanguin

Les acides ascorbique (AA) et urique (AU) sont d'un grand intérêt biologique vu les différents rôles qu'ils jouent dans l'organisme (agents antioxydant, cofacteur d'hydroxylation, marqueur du métabolisme des purines). Chez les cliniciens, le dosage de ces deux molécules aide à l'établissement de diagnostics et aux suivis thérapeutiques. Face aux méthodes classiques actuellement utilisées (la chromatographie liquide à haute performance et la spectrométrie), qui nécessitent souvent des étapes de prétraitement de l'échantillon, l'objectif de ce travail est de mettre au point un microcapteur voltammétrique fonctionnalisé par un polymère conducteur électrogénéré le poly (3,4-éthylènedioxythiophène) PEDOT. Le capteur a permis de doser sélectivement et simultanément les deux acides. L'étude des conditions d’élaboration du film (paramètres d'électrolyse, épaisseur du dépôt) et des paramètres de mesure électrochimique a permis d'optimiser les performances analytiques du capteur (sensibilité, seuil limite de détection, domaine de linéarité) dans des solutions modèles. L'étude a mis en évidence un mécanisme EC' de régénération de l'acide urique par l'acide ascorbique au voisinage de l'électrode. Le capteur a ensuite été éprouvé directement dans le sérum sanguin sans aucune préparation de l'échantillon. Les résultats des dosages électrochimiques des deux acides sont en très bonne adéquation avec ceux des méthodes chromatographiques et enzymatiques.Ascorbic (AA) and uric (UA) acids are of a great biological interest considering the various physiological roles they play (antioxidants, cofactor of hydroxylation, marker of the purins metabolism). In medicine, the assay of both molecules contributes to the establishment of diagnosis and therapies. In alternative to the traditional methods currently used (high performance chromatography liquid and spectrometry), which are generally time consuming and often require costly materials, complex experimental protocols and sample pretreatment, the aim of this work is to develop a voltammetric microsensor functionalized by a electrogenerated conducting polymer (3,4-ethylenedioxythiophene) PEDOT. This sensor made possible a selective and sensitive simultaneous detection of both acids. The study of the electropolymerization parameters (PEDOT film thickness, electropolymerization potential range, monomer concentration) and of the electrochemical measurements parameters (potential scan rate) allows the optimization of the analytical performances of the microsensor (sensitivity, limit of detection and linear range). The study highlighted also an EC’ mechanism of regeneration of uric acid by ascorbic acid in the vicinity of the electrode. Electrochemical assay of the two acids was finally performed in the human blood serum without any preparation of the sample. The results are in very good agreement with those of the standardized chromatographic and enzymatic methods

Evidence of an EC' mechanism occurring during the simultaneous assay of ascorbic and uric acids on poly(3,4-ethylenedioxythiophene) modified gold microsensor.

A voltammetric microsensor has been developed for the simultaneous assay of ascorbic (AA) and uric (UA) acids in aq. soln. The electrode surface has been modified by means of electropolymd. conductive poly(3,4-ethylenedioxythiophene) (PEDOT) org. films. The anodic peak potential sepn. between both acids was more than 300 mV. The sensitivity of the microsensor for UA was found to be dependent on the presence of AA in the mixt. By using square wave voltammetry (SWV), it increased from 77.5 mA mM-1 cm-2 without AA to 86.2 mA mM-1 cm-2 with AA 1 mM. An EC' catalytic mechanism was highlighted, inducing the regeneration of reduced UA by AA at the vicinity of the electrode surface

Simultaneous assay of ascorbate and urate antioxidants in human blood serum using PEDOT-based electrocehmical microsensor

An electrochemical microsensor has been developed for the simultaneous assay of ascorbate (AA) and urate (UA) antioxidants in human blood serum. The electrode surface was modified by means of electropolymerized conductive poly(3,4-ethylenedioxythiophene) PEDOT organic films. Highly selective responses (detection potential difference more than 250 mV) were obtained for both biomarkers by optimizing the PEDOT thickness. Contrary to most previous studies the analytical performances were recorded by testing the microsensor directly in blood serum without any pretreatment of the samples. Using differential pulse voltammetry (DPV) the sensitivity and detection limit were 0.481 µA µM-1 cm-2 and 4.2 µmol L-1 for AA and 1.815 µA µM-1 cm-2 and 2.0 µmol L-1 for UA. The calibration curves were linear in the concentration range 5–200 µmol L-1 for AA and 3–700 µmol L-1 for UA. The accuracy of the sensor was satisfactorily compared with the reference analytical methods provided that the synergic effect between both antioxidants was considered. The sensor response was unmodified in the presence of the major biochemical interfering species

Elaboration of integrated microelectrodes for the detection of antioxidant species

(Pt–Pt–Ag/AgCl) and (Au–Pt–Ag/AgCl) electrochemical microcells (ElecCell) were developed for the detection of redox species by cyclic voltammetry. A special emphasis was placed on the SU-8 waferlevel passivation process in order to optimize the electrochemical properties of the different “thin film” metallic layers, i.e. gold or platinum for the working electrode, platinum for the counter electrode and silver/silver chloride for the reference electrode. (Au–Pt–Ag/AgCl) microcells were applied for the detection of antioxidant species such as ascorbic and uric acids in phosphate buffer solution, evidencing high sensitivity but low selectivity. Works were extended to skin analysis, demonstrating that a good electrical contact with the skin hydrolipidic film allowed the effective evaluation of the skin global antioxidant capacity

Fonctionnalisation de surfaces d'électrodes par un film de poly (3,4-éthylènedioxythiophène) PEDOT pour l'élaboration de microcapteur spécifique des acides ascorbique et urique (application à l'étude des propriétés antioxydantes du sérum sanguin)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

PEDOT-modified electrochemical microsensors: a versatile probe for the detection of antioxidant biomarkers

International audienceThe development of low-cost biosensors that enable a sensitive, selective and reliable determination of analytes has received considerable attention. In this context, electrochemistry represents a powerful tool for dealing with the detection of chemical species. Indeed, microelectrodes can be integrated using silicon-based technologies, and are compatible with the use of electrocatalytic conductive polymers. One of them, the poly(3,4-ethylenedioxythiophene) (PEDOT), was shown to exhibit optimized properties for bioanalysis: low oxidation potential, good stability, high electrical conductivity, good detection selectivity and biocompatibility. As a result, PEDOT was used for the determination of different molecules of biological interest such as antioxidant biomarkers. Among them, ascorbic (AA) and uric (UA) acids were widely studied since they are associated with oxidative stress and related pathologies. In previous works, we demonstrated the integration of (Au-Pt-Ag/AgCl) electrochemical microcells (ElecCell) for the bioelectrochemical detection, as well as the catalytic behaviour of PEDOT towards the simultaneous determination of AA and UA acid in aqueous solutions and blood serum. This paper presents the integration of (Au/PEDOT-Pt-Ag/AgCl) ElecCell microdevices for the detection of antioxidant markers (fig. 1). First, it deals with the optimization of the PEDOT electrodeposition process on integrated gold microelectrodes. Then, more attention is brought to the dopamine (Dop) analysis, a well-known neurotransmitter coexisting usually with AA and UA in biological samples. Thus, using PEDOT-modified ElecCell microdevices, the selective determination of dopamine was achieved in presence of both ascorbic and uric acids (fig. 2). Excellent catalytic properties were thus evidenced for their simultaneous detection with high sensibilities (around 0.85, 3.0 and 1.65 pA.cm-2 .M-1 for AA, UA and Dop respectively) and low detection limits (< 500 nM) (fig. 3 and 4). Such results intend to demonstrate that silicon-based, PEDOT-modified electrochemical microsensors are convenient probes for the practical determination of antioxidant biochemical markers in biological samples. Fig. 1: Details of the (Au/PEDOT-Pt-Ag/AgCl) Fig 2: Differential pulse voltammogramm of ElecCell microdevice an equimolar solution (1 mM) of AA, UA and Dop Fig. 3: Differential pulse voltammogramms for Fig. 4: Calibration curves of AA, UA and Dop different concentrations of dopamine (0.1-300 M

Investigations of ring nanoelectrodes integrated into microwell arrays for the analysis of isolated mitochondria at the microscale

International audienceMitochondria are known to be the major cellular source for ATP (through the oxidative phosphorylation pathway) but also to play main roles into cell apoptosis and related disorders (ageing, neurological diseases, cancers,...). Consequently, new methodological approaches are resuired in order to decipher mitochondria metabolisms. In this context, we are investigating the ElecWell (electrochemical microwell) technological platform based on the integration of ring nanoelectrodes into microwell arrays. Similar to ultra-microelectrodes, they exhibit high current density, fast response time, reduced charging current and high signal to noise ratio. In addition, the use of small analysis volumes (< 1 pL) makes them well suited for the detection of exocytotic events or for the analysis of single mitochondrion. Consequently, platinum ring nanoelectrodes (RNE) (surface: 10-15 µm 2) were integrated into SiO2-based microwells (radius: 3 and 4.5 µm, depth: 5.2 µm, total volume: < 1 pL, figures 1 and 2). These electrochemical devices were characterized by cyclic voltammetry in Fc(MeOH) solutions using single well or arrayed configurations, and optimised according to COMSOL™ simulations. Steady-state sigmoidal responses were shown for RNE devices in agreement with theoretical models and design laws were defined for RNE arrays in the frame of mitochondria statistical analysis. Finally, the ElecWell platform was tested for the mitochondrial analysis (figure 3), allowing the electrochemical monitoring of oxygen consumption rate in response of specific drugs (ethanol, ADP, antimycine A) for several thousands of mitochondria (figure 4). Fig. 1: Integration of platinum ring nanoelectrodes Fig 2: Fabrication of RNE-based devices into a SiO2-based microwell (radius: 3 m) onto glass substrates Fig. 3: Fluorescent analysis of mitochondria Fig. 4: amperometric monitoring of mitochondrial deposited on a RNE-based microwell array metabolisms during an EtOH/ADP/AA cycl

Investigations of ring nanoelectrodes integrated into microwell arrays for the analysis of isolated mitochondria at the microscale

International audienceMitochondria are known to be the major cellular source for ATP (through the oxidative phosphorylation pathway) but also to play main roles into cell apoptosis and related disorders (ageing, neurological diseases, cancers,...). Consequently, new methodological approaches are resuired in order to decipher mitochondria metabolisms. In this context, we are investigating the ElecWell (electrochemical microwell) technological platform based on the integration of ring nanoelectrodes into microwell arrays. Similar to ultra-microelectrodes, they exhibit high current density, fast response time, reduced charging current and high signal to noise ratio. In addition, the use of small analysis volumes (< 1 pL) makes them well suited for the detection of exocytotic events or for the analysis of single mitochondrion. Consequently, platinum ring nanoelectrodes (RNE) (surface: 10-15 µm 2) were integrated into SiO2-based microwells (radius: 3 and 4.5 µm, depth: 5.2 µm, total volume: < 1 pL, figures 1 and 2). These electrochemical devices were characterized by cyclic voltammetry in Fc(MeOH) solutions using single well or arrayed configurations, and optimised according to COMSOL™ simulations. Steady-state sigmoidal responses were shown for RNE devices in agreement with theoretical models and design laws were defined for RNE arrays in the frame of mitochondria statistical analysis. Finally, the ElecWell platform was tested for the mitochondrial analysis (figure 3), allowing the electrochemical monitoring of oxygen consumption rate in response of specific drugs (ethanol, ADP, antimycine A) for several thousands of mitochondria (figure 4). Fig. 1: Integration of platinum ring nanoelectrodes Fig 2: Fabrication of RNE-based devices into a SiO2-based microwell (radius: 3 m) onto glass substrates Fig. 3: Fluorescent analysis of mitochondria Fig. 4: amperometric monitoring of mitochondrial deposited on a RNE-based microwell array metabolisms during an EtOH/ADP/AA cycl

Microwell Array Integrating Ring Nanoelectrodes for The Monitoring of Metabolic Responses at Isolated Mitochondria

International audienceMitochondria are major cell organelles since they are the main source of ATP owing to the oxidative phosphorylation pathway. They also play an important role into several other metabolic pathways (Krebs cycle, lipid synthesis…) and when defective, they are involved into severe pathologies (myopathies, neurological diseases, cancers...). Consequently, new methodological approaches [1, 2] are required in order to decipher mitochondria metabolisms and to provide efficient tools for diagnosis. In this context, we have developed microsystems, namely ElecWell platforms, which combine electrochemical [1] and optical [2] sensing abilities. These are based on the integration of platinum ring nanoelectrodes (RNE, surface: 10-15 µm 2) into SiO2-based microwell arrays (well radius: 3 to 4.5 µm, depth: 5 µm, individual volume < 1 pL), as shown on figure 1. Similarly to ultramicroelectrodes, RNE exhibit high current density, fast response time, reduced charging current and high signal-to-noise ratio. These electrochemical devices were characterized by cyclic voltammetry using single well or array configurations, and optimised according to COMSOL™ simulations. In addition, the glass substrate of the microsystems allows the observation by microscopy of the content and reactivity within each well. Then, a suspension of isolated mitochondria (yeast origin) was deposited on the ElecWell array and allowed to sediment within wells. We monitored by fluorescence the presence of individual mitochondria within wells (Fig. 1b) owing to their NADH content and variations [2]. Simultaneously, we monitored by cyclic voltammetry the variations of their oxygen consumption rate in response to specific activators and inhibitors of respiratory chain activity (ethanol, ADP, antimycine A). The resolution offered by the ElecWell platform is nearly a few thousands of mitochondria (Fig. 1c), corresponding to the mitochondrial content of a single cell. a) b) c) Figure 1. a) Integration of a platinum ring nanoelectrode into a SiO2-based microwell (radius: 3 µm) within the array (10 6 wells); b) Monitoring by fluorescence (NADH content) of isolated mitochondria deposited within wells. c) Detection by cyclic voltammetry (reduction current sampling) of the oxygen variations during mitochondrial activation (EtOH/ADP) and inhibition (Antimycin A)

Ring nanoelectrodes integrated into microwell arrays for the analysis of mitochondria isolated from leukemic cells

International audienceWe report here the fabrication and the electrochemical characterization of recessed disk microelectrodes (rDME) and ring nanoelectrodes (rRNE) integrated into microwell arrays. The technological process based on the reactive ion etching of a SiO2/Ti/Pt/Ti/SiO2 stack is optimized in order to realize functional electrochemical microdevices on glass substrate and so, enable the coupling of amperometric measurements with optical analysis. Multiphysic simulations and electrochemical characterizations are carried out to study and enhance the amperometric performance of recessed ring nanoelectrodes arrays (rRNEA) according to their geometry. Finally, all these results demonstrate that rRNEA are fitted for the detection of bio-electrochemical species at the microscale and consequently, for the analysis of the metabolic status of isolated mitochondria through the measurement of dissolved oxygen and hydrogen peroxide