Study and development of electrochemical biosensors for the detection of pollutants in aqueous medium

Les biocapteurs sont des moyens d'analyse en plein essor à la fois rapides, sélectifs et peu coûteux applicables à des domaines extrêmement variés (environnement, santé, agroalimentaire,…). Dans ce type d'outil, un élément sensible de nature biologique (anticorps, enzyme, microorganisme, ADN…) doté d'un pouvoir de reconnaissance pour un analyte ou un groupe d'analytes est associé à un transducteur pouvant être de type électrochimique, optique ou thermique. Dans ce travail, nous nous sommes intéressés au développement de différents biocapteurs, en se basant à l'immobilisation d'enzymes sur des microélectrodes en vue de la détection électrochimique d'analytes d'intérêt dans le domaine environnemental. Nous avons montré les potentialités d'application de deux biocapteurs conductimétrique et impédimétrique à base de lipase Candida Rugosa pour la détection directe et rapide des pesticides organophosphorés. Nous avons cherché à mieux comprendre le fonctionnement des biocapteurs et optimisé le procédé d'immobilisation des enzymes ainsi que différents paramètres de mesure afin de maximiser les performances analytiques des outils développés. Nous avons également élaboré un biocapteur impédimètrique pour une détermination très sensible de la phospholipase A2 en utilisant les nanoparticules d'or afin de renforcer la conductivité électrique. Enfin, nous avons développé un biocapteur de tyrosinase pour la détection du Bisphénol A, en se basant sur la modification électrochimique des électrodes de diamant dopé qui possèdent des propriétés électriques remarquables et uniques, avec l'ajout de nanotube de carbone multi-feuillet, ce qui permet d'améliorer des performances analytiques du biocapteur, en particulier sa limite de détection et sa stabilité. Les biocapteurs à base de lipase et de tyrosinase ont été appliqués avec succès à la quantification des composés organophosphorés et du bisphénol A respectivement dans plusieurs échantillons d'eaux réellesA biosensor is an analytical device, used for the detection of an analyte, that combines a biological component so-called a receptor (e.g. enzyme, antibody, DNA, microorganism) to a physical transducer (e.g. electrochemical, optical or thermal). In last decade, biosensors are quite promising tools since they are rapid, selective and cost effective, with an increasing interest for their application in various fields (e.g environment, food, health). In this work, we developed different biosensors based on immobilized enzymes onto microelectrodes in view of electrochemical detection. First, we demonstrated the potentialities of conductometric and impedimeteric biosensors based on lipase from Candida Rugosa for direct and rapid detection of organophosphate pesticides. In order to enhance the analytical performances of the developed devices, we tried to optimize enzyme immobilization as well as several operational parameters. We also elaborated an impedimetric biosensor for a sensitive determination of phospholipase A2 activity. Finally, a tyrosinase biosensor was developed for ultra-sensitive detection of Bisphenol A, based on the unique proprieties of boron doped diamond electrodes and taking advantage the use of multi walled carbon naotubes to improve the stability and detection limit of biosensor. We also demonstrated the applicability of both biosensors based on lipase and tyrosinase for the detection of organophosphate pesticides and bisphenol A in river wate

Immobilization of Candida Rugosa Lipase on Aluminosilicate Incorporated in a Polymeric Membrane for the Elaboration of an Impedimetric Biosensor

This work describes the development of a new biosensor for the detection of an organophosphate insecticide (Diazinon) in an aqueous medium. The method is based up on the immobilization of Candida Rugosa Lipase enzyme by adsorption on Aluminosilicate in order to incorporate it into a polymeric membrane. The electrical properties of the polymeric membrane have been characterized by electrochemical impedance spectroscopy (EIS). The linear range of the biosensor is from 10-8 M to 10-6 M with a detection limit of 10-8 M

Impedimetric Biosensor for the Determination of Phospholipase A 2 Activity in Snake Venom

International audienceAn impedimetric biosensor to determine the activity of phospholipase A2 has been developed for the first time. This biosensor is based on the immobilization of the enzyme phospholipase A2 (PLA2) from snake venom on the surface of a gold electrode. The enzymatic reaction takes place in the presence of the L-α -phosphatidylcholine (lecithin) substrate, in 10 mM pH 7.4 phosphate buffer. The impedance of the enzymatically modified electrode/ electrolyte interface increased as the concentration of lecithin increases. A detection limit of 10−11 M and a linear range of 10−10 M to 10−5 M were obtained. At a fixed concentration of lecithin (10−3 mM), when the PLA2 enzyme with a certain level of activity is injected, the impedance of the enzymatically modified electrode/ electrolyte interface changed in the inverse direction, due to the competitive effect of the injected PLA2 enzyme. A linear range of 10 to 400 ng/L (corresponding to 16 to 640 U/L) was obtained for commercial naja mossambica mossambica snake venom. The activity of PLA2 from naja cobra snake venom (Origin Thailand) was determined to be 75% of that of naja mossambica mossambica snake venom

A microconductometric biosensor based on lipase extracted from Candida rugosa for direct and rapid detection of organophosphate pesticides

International audienceOrganophosphate pesticides (OPs) have been intensively used as insecticides in agriculture; after entering the aquatic environment, they may affect a wide range of organisms. A conductometric enzymatic biosensor based on lipase extracted from Candida rugosa (CRL) has therefore been developed for the direct and rapid quantitative detection of organophosphate pesticides: diazinon, methyl parathion and methyl paraoxon in water. The biosensor signal and response time were obtained under optimum conditions, the enzyme being immobilised in the presence of gold nanoparticles. Under these conditions, the enzymatic biosensor was able to measure concentrations as low as 60 mu g/L of diazinon, 26 mu g/L of methyl parathion and 25 mu g/L of methyl paraoxon very rapidly (response time: 3min). Moreover, this CRL biosensor was not sensitive to interferences such as carbamates. It presented good storage stability for 21days when kept at 4 degrees C and it was successfully applied to real samples

Highly sensitive electrochemical biosensor for bisphenol A detection based on a diazonium-functionalized boron-doped diamond electrode modified with a multi-walled carbon nanotube-tyrosinase hybrid film (vol 74, pg 830, 2015)

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Highly sensitive electrochemical biosensor for bisphenol A detection based on a diazonium-functionalized boron-doped diamond electrode modified with a multi-walled carbon nanotube-tyrosinase hybrid film (vol 74, pg 830, 2015)

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Robust Electrografting on Self-Organized 3D Graphene Electrodes

International audienceA self-organized three-dimensional (3D) graphene electrode processed by pulsed laser deposition with thermal annealing is reported. This substrate shows great performance in electron transfer kinetics regarding ferrocene redox probes in solution. A robust electrografting strategy for covalently attaching a redox probe onto these graphene electrodes is also reported. The modification protocol consists of a combination of diazonium salt electrografting and click chemistry by means of Cu I-catalyzed alkyne–azide cycloaddition. Our modification strategy applied to 3D graphene electrodes was analyzed by means of atomic force microscopy, scanning electron microscopy, Raman spectroscopy, cyclic voltammetry, and X-ray photoelectron spectroscopy (XPS). For XPS chemical surface analysis, special attention was paid to the distribution and chemical state of iron and nitrogen in order to highlight the functionalization of the graphene-based substrate by electrochemically grafting a ferrocene derivative. Dense grafting was observed, offering 4.9×10 –10 mol cm –2 surface coverage and showing a stable signal over 22 days. The electrografting was performed in the form of multilayers, which offers higher ferrocene loading than a dense monolayer on a flat surface. This work opens highly promising perspectives for the development of self-organized 3D graphene electrodes with various sensing functionalities

Development of new graphene-substrate materials through laser technology for electrochemical sensing applications

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Structure, electrochemical properties and functionalization of amorphous CN films deposited by femtosecond pulsed laser ablation

International audienceAmorphous carbon nitride (a-C:N) material has attracted much attention in research and development Recently, it has become a more promising electrode material than conventional carbon based electrodes in electrochemical and biosensor applications. Nitrogen containing amorphous carbon (a-C:N) thin films have been synthesized by femtosecond pulsed laser deposition (fs-PLD) coupled with plasma assistance through Direct Current (DC) bias power supply. During the deposition process, various nitrogen pressures (0 to 10 Pa) and DC bias (0 to -350 V) were used in order to explore a wide range of nitrogen content into the films. The structure and chemical composition of the films have been studied by using Raman spectroscopy, electron energy-loss spectroscopy (EELS) and high-resolution transmission electron microscopy (HRTEM). Increasing the nitrogen pressure or adding a DC bias induced an increase of the N content, up to 21 at%. Nitrogen content increase induces a higher sp(2) character of the film. However DC bias has been found to increase the film structural disorder, which was detrimental to the electrochemical properties. Indeed the electrochemical measurements, investigated by cyclic voltammetry (CV), demonstrated that a-C:N film with moderate nitrogen content (10 at.%) exhibited the best behavior, in terms of reversibility and electron transfer kinetics. Electrochemical grafting from diazonium salts was successfully achieved on this film, with a surface coverage of covalently bonded molecules close to the dense packed monolayer of ferrocene molecules. Such a film may be a promising electrode material in electrochemical detection of electroactive pollutants on bare film, and of biopathogen molecules after surface grafting of the specific affinity receptor

DC nitrogen plasma assisted Femtosecond Pulsed Laser Deposition of Carbon-Nitrogen alloy for environmental analytical Microsystems: thin films characterization and in situ plume analysis

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