pH sensors with high sensitivity utilising coulometric transduction method : comparison of solid-contact ion-selective electrode and polyaniline-based ion-selective electrode

This thesis compares the analytical performance of solid-contact ion-selective electrodes (SCISEs) and polyaniline-based ion-selective electrodes for the determination of pH using a newly developed coulometric signal transduction readout method. For the SC-ISEs, the conducting polymer poly(styrene-sulfonate)-doped poly(3,4-ethylenedioxythiophene) (PEDOT(PSS)) served as an ion-to-electron transducer. The coulometric transduction readout method was able to amplify the analytical signal by increasing the thickness of the solid-contact layer. It also was shown that the thinner the ion-selective membrane, the shorter the response time, which could be associated with a more rapid ion transport between the ion-selective membrane and the solid-contact layer. For the polyaniline-based ISEs, the coulometric response was found to be not entirely proportional to pH. This could be explained by the cyclic voltammogram of polyaniline which contains several peaks, meaning that the redox capacitance of polyaniline depends on the potential, as does the coulometric response. Since the conducting polymer worked both as an ion-to-electron transducer and as a sensing membrane, the chronoamperometric response of the polyaniline-based pH sensor was directly affected by interfering ions. By integrating the resulting current against time, the coulometric signal readout has shown to be useful in reducing the noise level. Under optimised conditions, a 10 mC PEDOT(PSS) solid-contact pH sensor covered with a drop-casted ion-selective membrane was able to detect 0.18 % change in concentration; a 50-cycle polyaniline-based pH sensor could detect a 0.36% change in concentration using the novel coulometric transduction method

Etude de la catalyse enzymatique par couplage microscopie-électrochimie : application aux biopiles à combustible H2/O2

Les biopiles enzymatiques, qui utilisent des enzymes pour convertir l'énergie chimique en électricité, se présentent comme l'une des ressources énergétiques alternatives et propres les plus prometteuses. Cependant, l'immobilisation fonctionnelle de ces enzymes sur une électrode pour une catalyse efficace suscite encore de nombreux défis. Afin d’accéder à des informations résolues dans l'espace, il est nécessaire de coupler l'électrochimie à d’autres techniques de surface. Dans cette thèse, la microscopie de fluorescence confocale à balayage laser a été couplée à l'électrochimie pour la caractérisation de la catalyse électro-enzymatique. La principale réaction étudiée était la réaction de réduction de l'oxygène catalysée par la bilirubine oxydase de Myrothecium verrucaria. Cette réaction implique une consommation de protons couplée au transfert d'électrons. En utilisant une analyse in situ, les variations locales de pH qui se produisent à proximité de la bioélectrode pendant la catalyse enzymatique sont visualisées grâce à un fluorophore dont l’émission dépend du pH, la fluorescéine. L'activité de l'enzyme a d’abord été sondée par spectroscopie UV-vis et électrochimie. Nous avons ensuite montré que l'intensité de la fluorescence enregistrée est directement proportionnelle au courant catalytique. Les profils d'appauvrissement en protons à l’interface électrochimique dans des électrolytes tamponnés et non tamponnés ont été reconstruits, afin de déterminer l'influence de la force ionique sur l'environnement local des enzymes. Enfin, les enzymes ont été marquées avec des fluorophores, permettant de révéler les hétérogénéités locales de leur distribution interfaciale.Enzyme biofuel cells, which use enzymes to convert chemical energy into electricity, hold promise as one of the most promising alternative and clean energy resources. However, the immobilization of such enzymes on an electrode for efficient catalysis still raises many challenges. In order to access spatially resolved information, it is necessary to couple electrochemistry to other surface techniques. In this thesis, confocal laser scanning fluorescence microscopy was coupled with electrochemistry for the characterization of electro-enzymatic catalysis. The main reaction studied was the oxygen reduction reaction catalyzed by bilirubin oxidase from Myrothecium verrucaria. This reaction involves a consumption of protons coupled with electron transfer. Using in situ analysis, the local pH variations that occur near the bioelectrode during the enzymatic catalysis are visualized thanks to a fluorophore whose emission depends on the pH, fluorescein. The activity of the enzyme was first probed by UV-vis spectroscopy and electrochemistry. We then showed that the intensity of the fluorescence recorded is directly proportional to the catalytic current. Profiles of proton depletion at the electrochemical interface in buffered and unbuffered electrolytes were reconstructed to determine the influence of ionic strength on the local environment of enzymes. Finally, the enzymes were labeled with fluorophores, making it possible to reveal the local heterogeneities of their interfacial distribution

From Enzyme Stability to Enzymatic Bioelectrode Stabilization Processes

International audienceBioelectrocatalysis using redox enzymes appears as a sustainable way for biosensing, electricity production, or biosynthesis of fine products. Despite advances in the knowledge of parameters that drive the efficiency of enzymatic electrocatalysis, the weak stability of bioelectrodes prevents large scale development of bioelectrocatalysis. In this review, starting from the understanding of the parameters that drive protein instability, we will discuss the main strategies available to improve all enzyme stability, including use of chemicals, protein engineering and immobilization. Considering in a second step the additional requirements for use of redox enzymes, we will evaluate how far these general strategies can be applied to bioelectrocatalysis

Local pH Modulation during Electro-Enzymatic O2 Reduction: Characterization of the Influence of Ionic Strength by In Situ Fluorescence Microscopy

International audienceUnderstanding how environmental factors affect the bioelectrode efficiency and stability is of uttermost importance to develop high-performance bioelectrochemical devices. By coupling fluorescence confocal microscopy in situ to electrochemistry, this work focuses on the influence of the ionic strength on electro-enzymatic catalysis. In this context, the 4 e-/ 4 H + reduction of O2 into water by the bilirubin oxidase from Myrothecium verrucaria (MvBOD) is considered as a model. The effects of salt concentration on the enzyme activity and stability were probed by enzymatic assays performed in homogeneous catalysis conditions and monitored by UV-vis absorption spectroscopy. They were also investigated in heterogeneous catalysis conditions by electrochemical measurements with MvBOD immobilized at a graphite microelectrode. We demonstrate that the catalytic activity and stability of the enzyme both in solution and in the immobilized state at the bioelectrode were conserved with an electrolyte concentration of up to 0.5 M, both in a buffered and a non-buffered electrolyte. Relying on this, we used fluorescence confocal laser scanning microscopy coupled in situ to electrochemistry to explore the local pH of the electrolyte at the vicinity of the electrode surface at various ionic strengths and for several overpotentials. 3D proton depletion profiles generated by the interfacial electroenzymatic reaction were recorded in the presence of a pH sensitive fluorophore. These concentration profiles were shown to contract with increasing ionic strength, thus highlighting the need for a minimal electrolyte concentration to ensure availability of charged substrates at the electrode surface during electro-enzymatic experiments

In Situ Fluorescence Tomography Enables a 3D Mapping of Enzymatic O 2 Reduction at the Electrochemical Interface

International audienceGetting information about the fate of immobilized biomolecules and the evolution of their environment during turnover is a mandatory step towards bioelectrode optimization for effective use in biodevices. We demonstrate here the proof-ofprinciple characterization of the reactivity at an enzymatic electrode thanks to fluorescence confocal laser scanning microscopy (FCLSM) implemented in situ during the electrochemical experiment. The enzymatic O 2-reduction involves proton and electron transfers. Therefore, fluorescence variation of a pH-dependent fluorescent dye in the electrode vicinity enables the reaction visualization. Simultaneous collection of electrochemical and fluorescence signals gives valuable space-and time-resolved information. Once the technical challenges of such a coupling are overcome, in situ FCLSM affords a unique way to explore reactivity at the electrode surface and in the electrolyte volume. Unexpected features are observed, especially the pH evolution of the enzyme environment, which is also indicated by a characteristic concentration profile within the diffusion layer. This coupled approach gives also access to a cartography of the electrode surface response (i.e. heterogeneity), which cannot be obtained solely by an electrochemical mean