Дисертації з теми "Biosenseur redox"

La biologie des réactions redox est particulièrement difficile à étudier de par la compartimentation spatiale mais aussi cinétique des différents systèmes redox cellulaires. Les biosenseurs codés génétiquement, incluant la «redox-sensitive Yellow Fluorescent Protein» (rxYFP) sont une manière de contourner les limitations des méthodes conventionnelles de mesure du couple glutathion/glutathion disulfure (GSH/GSSG). Cette étude présente l’utilisation des biosenseurs rxYFP pour analyser les états redox dans des différents compartiments cellulaires, et leur dynamique en réponse au stress dans les cellules humaines. La rxYFP exprimée soit dans le cytosol, le noyau ou la matrice mitochondriale de cellules HeLa s’est révélée sensible aux changements de l’état redox intracellulaire provoqué par des traitements aussi bien réducteurs qu’oxydants. La rxYFP est capable de détecter des différences de l’état redox, entre les compartiments, mais aussi entre différentes lignées cellulaires. Les senseurs exprimés dans des kératinocytes humain de l’épiderme HEK001 ont réagi au stress induit par les UVA, de façon dose-dépendante, mais pas au stress induit par les UVB. De plus, ces senseurs ont pu détecter les changements redox induits par des faibles doses (30 µM) ainsi que par des doses modérées (100 µM) de peroxyde d’hydrogène (H2O2), de façon dynamique et spécifique des compartiments cellulaires. La rxYFP exprimé dans la matrice mitochondriale a montré une vitesse d’oxydation plus élevée que les senseurs rxYFP exprimés dans le cytosol ou le noyau, ce qui est attribuable à un pH local plus basique. De plus, la déplétion en GSH provoquée par un traitement au buthionine sulphoximine (BSO) a affecté spécifiquement le potentiel redox mitochondrial mais pas cytosolique ni nucléaire. Ces observations soutiennent l’idée que l’état redox du GSH mitochondrial est maintenu et régulé de façon indépendante par rapport à celui du cytosol ou du noyau. Nous avons également montré que dans les cellules humaines, les sondes rxYFP réagissent de façon prédominante avec le GSH/GSSG, puisque la déplétion en GSH ralentit la vitesse d’oxydation de la rxYFP en réponse à un traitement par H2O2. De plus, grâce à l’utilisation des sondes rxYFP et à l’analyse de l’état redox des antioxydants cellulaires, nous démontrons que l’oxydation des thiols se produit après l’activation des caspases au cours de l’apoptose induite par TRAIL. L’ensemble de nos données montrent la robustesse des senseurs rxYFP pour la mesure des changements d’état redox dans les cellules humaines. En complément d’autres senseurs redox ainsi que des méthodes conventionnelles de mesure des états redox, les senseurs rxYFP ciblés aux différents compartiments cellulaires sont un nouvel outil pour étudier l’homéostasie redox dans les cellules de mammifères, et permettent l’étude de l’état redox du glutathion et de la dynamique des changements redox avec une grande précision
The kinetic and spatial separation of redox systems renders redox biology studies a particularly challenging field. Genetically encoded biosensors including the glutathione-specific redox-sensitive yellow fluorescent protein (rxYFP) may provide an alternative way to overcome the limitations of conventional glutathione/glutathione disulfide (GSH/GSSG) redox measurements. This study describes the use of rxYFP sensors for investigating compartment-specific steady redox states and their dynamics in response to stress in human cells. RxYFP expressed either in the cytosol, nucleus or mitochondrial matrix of HeLa cells was responsive to the intracellular redox state changes induced by reducing as well as oxidizing agents. Compartment-targeted rxYFP sensors were able to detect different steady state redox conditions between the cytosol, nucleus and mitochondrial matrix as well as between the cell lines. These sensors expressed in human epidermal keratinocytes HEK001 responded to stress induced by UVA radiation in a dose-dependent manner but not to UVB radiation. Furthermore, rxYFP sensors were able to sense dynamic and compartment-specific redox changes caused by low dose (30 µM) and moderate dose (100 M) hydrogen peroxide (H2O2). Mitochondrial matrix-targeted rxYFP displayed a greater dynamics of oxidation in response to a H2O2 challenge than the cytosol- and nucleus-targeted sensors, largely due to a more alkaline local pH environment. Similarly, the depletion of glutathione induced by buthionine sulphoximine (BSO) affected selectively mitochondrial redox potential without inducing changes in cytosol and nucleus. Furthermore, using rxYFP probes and cellular antioxidants redox state analysis, we show that oxidation of thiols occurs after activation of caspases during TRAIL-induced apoptosis. These observations support the view that mitochondrial glutathione redox state is maintained and regulated independently from that of the cytosol and nucleus. We also showed that in human cells the rxYFP probes react predominantly with glutathione since the glutathione depletion slows down the dynamics of rxYFP oxidation in response to H2O2. Taken together, our data show the robustness of the rxYFP sensors to measure compartmental redox changes in human cells. Complementary to existing redox sensors and conventional redox measurements, compartment-targeted rxYFP sensors provide a novel tool for examining mammalian cell redox homeostasis, permitting high resolution readout of steady glutathione state and dynamics of redox changes

Au cours des dix dernières années, le développement de biosenseurs redox fluorescents a permis de générer des outils permettant d'étudier les dynamiques in vivo de molécules comme les formes réduite et oxydée du glutathion ou le peroxyde d'hydrogène. La cystéine étant un métabolite clé du métabolisme du soufre, l'objectif de ce projet de thèse était de développer un biosenseur redox fluorescent spécifique de la cystéine en couplant une oxydoréductase à la roGFP2 (reduction-oxidation green fluorescent protein). Tout d'abord les activités de plusieurs isoformes de cystéine désulfurases (CD) et des protéines à domaine rhodanese (Rhd), catalysant respectivement la désulfuration de la cystéine et des réactions de trans-persulfuration ont été analysées in vitro afin de déterminer si elles pouvaient constituer de bons candidats pour cette activité oxydoréductase. Ces analyses ont mis en évidence qu'une protéine chimérique naturelle bactérienne possédant des domaines CD et Rhd oxyde efficacement la roGFP2, au travers de réactions de trans-persulfuration depuis la cystéine vers la roGFP2. Cette protéine candidate a ensuite été fusionnée à la roGFP2 pour générer le biosenseur CD-Rhd-roGFP2. In vitro, cette protéine est sensible à l'oxydation en présence de concentrations physiologiques en cystéine alors que l'oxydation par le thiosulfate, autre substrat potentiel du domaine Rhd, est négligeable. D'une part, les réactions de trans-persulfuration entre les domaines protéiques menant à l'oxydation de la roGFP2 ne sont pas ou très peu inhibées par les systèmes réducteurs physiologiques. Néanmoins, le système glutathion-glutarédoxine réduit spécifiquement la roGFP2. L'expression de ce biosenseur chez la bactérie Escherichia coli, a révélé une réponse dynamique en réponse à des ajouts exogènes de cystéine ou de cystine ouvrant la voie à des études similaires dans les organites d'autres organismes modèles eucaryotes
Over the last decade, the development of fluorescent redox biosensors has provided tools to study the in vivo dynamics of molecules such as the reduced and oxidized forms of glutathione or hydrogen peroxide. Cysteine being a key metabolite of sulfur metabolism, this PhD project aimed at developing a fluorescent redox biosensor specific for cysteine by coupling an oxidoreductase to roGFP2 (reduction-oxidation green fluorescent protein). First, the activities of several isoforms of cysteine desulfurases (CD) and rhodanese-domain containing proteins (Rhd), catalyzing cysteine desulfuration and trans-persufidation reactions, respectively, were analyzed in vitro in order to determine whether they could constitute good candidates for this oxidoreductase activity. These analyses revealed that a natural chimeric protein possessing both CD and Rhd domains efficiently oxidizes roGFP2, by catalyzing trans-persulfidation reactions from cysteine to roGFP2. This candidate protein was then fused to roGFP2 to generate the CD-Rhd-roGFP2 biosensor. In vitro, this protein is sensitive to oxidation in the presence of physiological concentrations of cysteine whereas oxidation by thiosulfate, another potential substrate of the Rhd domain, is negligible. In addition, the trans-persulfidation reactions between the protein domains leading to the oxidation of roGFP2 are not inhibited by physiological reducing systems. Nevertheless, the glutathione/glutaredoxin system specifically reduces roGFP2. The expression of this biosensor in the bacterium Escherichia coli revealed a dynamic response of the biosensor to exogenous addition of cysteine or cystine, paving the way for similar studies in organelles from other eukaryotic model organisms

This dissertation investigates a label-free sensing platform which can be used to detect DNA, enzyme or protein, based upon electrochemical detection which is suitable for implementation in microarray form. Two implementations are proposed based on mixed Ferrocene self-assembled monolayer (SAM) and the Azurin (metalloprotein) SAM. We have shown for the first time that electro-active SAM, functionalized with suitable receptors, can be employed for the detection of biomolecular interactions. Detection of streptavidin by biotin-functionalized Ferrocene SAM was successfully demonstrated. These results were made possible by the development of the fabrication protocols that optimize the SAM stability and reproducibility. Reliable samples, combined with theoretical modelling and modification of existing published model for electro-active SAM, has enabled us to experiment and analyse in depth various electrochemical detection techniques, based on changes in capacitance, voltammetric formal potential and current, Open Circuit Potential (OCP). It was found that AC voltammetry and OCP are the best measurement techniques. The use of OCP with an electro-active SAM had not been previously demonstrated and the theoretical basis for this technique was presented. Essential for this technique was the development of micro-electrodes to reduce parasitic capacitances that would reduce the available signal, enabling real-time detection of bio-molecular interaction. We also made possible to characterize the binding of a protein (streptavidin) to a biotin-functionalized Azurin SAM. Also a numerical analysis has been developed to analyse the effect of design parameters of the platform, such as the probe density and buffer concentration, which can greatly affect the assay sensitivity. This is achieved using 3D simulation with finite element method in COMSOL.

In this thesis, different aspects within the research field of protein spectro- and electro-chemistry on nanostructured materials are addressed. On the one hand, this work is related to the investigation of nanostructured transparent and conductive metal oxides as platform for the immobilization of electroactive enzymes. On the other hand the second part of this work is related to the immobilization of sulfite oxidase on gold nanoparticles modified electrode. Finally direct and mediated spectroelectrochemistry protein with high structure complexity such as the xanthine dehydrogenase from Rhodobacter capsulatus and its high homologues the mouse aldehyde oxidase homolog 1. Stable immobilization and reversible electrochemistry of cytochrome c in a transparent and conductive tin-doped and tin-rich indium oxide film with a well-defined mesoporosity is reported. The transparency and good conductivity, in combination with the large surface area of these materials, allow the incorporation of a high amount of electroactive biomolecules (between 250 and 2500 pmol cm-2) and their electrochemical and spectroscopic investigation. Both, the electrochemical behavior and the immobilization of proteins are influenced by the geometric parameters of the porous material, such as the structure and pore shape, the surface chemistry, as well as the protein size and charge. UV-Vis and resonance Raman spectroscopy, in combination with direct protein voltammetry, are employed for the characterization of cytochrome c immobilized in the mesoporous indium tin oxide and reveal no perturbation of the structural integrity of the redox protein. A long term protein immobilization is reached using these unmodified mesoporous indium oxide based materials, i.e. more than two weeks even at high ionic strength. The potential of this modified material as an amperometric biosensor for the detection of superoxide anions is demonstrated. A sensitivity of about 100 A M-1 m-2, in a linear measuring range of the superoxide concentration between 0.13 and 0.67 μM, is estimated. In addition an electrochemical switchable protein-based optical device is designed with the core part composed of cytochrome c immobilized on a mesoporous indium tin oxide film. A color developing redox sensitive dye is used as switchable component of the system. The cytochrome c-catalyzed oxidation of the dye by hydrogen peroxide is spectroscopically investigated. When the dye is co-immobilized with the protein, its redox state is easily controlled by application of an electrical potential at the supporting material. This enables to electrochemical reset the system to the initial state and repetitive signal generation. The case of negative charged proteins, which does not have a good interaction with the negative charged indium oxide based films, is also explored. The modification of an indium tin oxide film with a positive charged polymer and the employment of a antimony doped tin oxide film were investigated in this work in order to overcome the repulsion induced by similar charges of the protein and electrode. Human sulfite oxidase and its separated heme-containing domain are able to direct exchange electrons with the supporting material. A study of a new approach for sulfite biosensing, based on enhanced direct electron transfer of a human sulfite oxidase immobilized on a gold nanoparticles modified electrode is reported. The spherical gold nanoparticles were prepared via a novel method by reduction of HAuCl4 with branched poly(ethyleneimine) in an ionic liquid resulting in particles of about 10 nm in hydrodynamic diameter. These nanoparticles were covalently attached to a mercaptoundecanoic acid modified Au-electrode and act as platform where human sulfite oxidase is adsorbed. An enhanced interfacial electron transfer and electrocatalysis is therefore achieved. UV-Vis and resonance Raman spectroscopy, in combination with direct protein voltammetry, were employed for the characterization of the system and reveal no perturbation of the structural integrity of the redox protein. The proposed biosensor exhibited a quick steady-state current response, within 2 s and a linear detection range between 0.5 and 5.4 μM with high sensitivity (1.85 nA μM-1). The investigated system provides remarkable advantages, since it works at low applied potential and at very high ionic strength. Therefore these properties could make the proposed system useful in the development of bioelectronic devices and its application in real samples. Finally protein with high structure complexity such as the xanthine dehydrogenase from Rhodobacter capsulatus and the mouse aldehyde oxidase homolog 1 were spectroelectrochemically studied. It could be demonstrated that different cofactors present in the protein structure, like the FAD and the molybdenum cofactor, are able to directly exchange electrons with an electrode and are displayed as a single peak in a square wave voltammogram. Protein mutants bearing a serine substituted to the cysteines, bounding to the most exposed iron sulfur cluster additionally showed direct electron transfer which can be attributable to this cluster. On the other hand a mediated spectroelectrochemical titration of the protein bound FAD cofactor was performed in presence of transparent iron and cobalt complex mediators. The results showed the formation of the stable semiquinone and the fully reduced flavin. Two formal potentials for each single electron exchange step were then determined.
In dieser Arbeit werden verschiedenen Aspekte im Forschungsfeld der Protein-Spekro- und Elektro-Chemie an nanostrukturierte Materialien behandelt. Zum einen werden in dieser Arbeit nanostrukturierte, transparente und leitfähige Metalloxide als Basis für die Immobilisierung von elektroaktiven Enzym untersucht. Des Weiteren behandelt diese Arbeit die Immobilisierung von humaner Sulfitoxidase auf einer Gold-Nanopartikel-modifizierten Elektrode. Schließlich wird die direkte und die vermittelte Elektrochemie von Xanthindehydrogenase aus Rhodobacter capsulatus und Aldehydoxidase Homolog 1, aus Mause, vorgestellt. Im ersten Teil der Arbeit wird über die stabile Immobilisierung und reversible Elektrochemie von Cytochrom c in einem transparenten und leitfähigen Zinn-dotierten und Zinn-reichen Indiumoxid Film mit einer gut definierten Mesoporosität berichtet. Die Transparenz und gute Leitfähigkeit in Kombination mit der großen Oberfläche dieser Materialien erlauben die Inkorporation einer große Menge elektroaktiver Biomoleküle (zwischen 250 und 2500 pmol cm-2) und deren elektrochemische und spektroskopische Untersuchung. Das elektrochemische Verhalten und die Proteinimmobilisierung sind durch die geometrischen Parameter des porösen Materials, wie die Struktur und Porenform, die Oberflächenchemie, sowie die Größe und Ladung des Proteins beeinflusst. UV-Vis und Resonanz-Raman-Spektroskopie in Kombination mit direkter Protein-Voltammetrie werden für die Charakterisierung von Cytochrom c eingesetzt und zeigen keine Störung der strukturellen Integrität des Redox-Proteins durch die Immobilisierung. Eine langfristige Immobilisierung des Proteins von mehr als zwei Wochen auch bei hoher Ionenstärke wurde unter Verwendung dieser unmodifizierten mesoporösen Indiumoxid-basierten Materialien erreicht. Das Potential dieses modifizierten Materials für die Verwendung in einem amperometrischen Biosensor zum Nachweis von Superoxid-Anionen wurde aufgezeigt. Es wurde eine Empfindlichkeit von etwa 100 A M-1 m-2, in einem linearen Messbereich der Superoxidkonzentration zwischen 0,13 und 0,67 µM, erreicht. Außerdem wurde ein elektrochemisch umschaltbares Protein-basiertes optisches Gerät konzipiert mit Cytochrom c und der mesoporösen Indiumzinnoxidschicht. Ein redox-sensitiver Farbstoff wurde als schaltbare Komponente des Systems verwendet. Die Cytochrom c Oxidation des Farbstoffs durch Wasserstoffperoxid wurde spektroskopisch untersucht. Der Redox-Zustand des Farbstoffs, co-immobilisiert mit dem Protein, ist leicht durch das Anlegen eines elektrischen Potentials an das Trägermaterial kontrollierbar. Dadurch wird die elektrochemische Zurücksetzung des Systems auf den Anfangszustand und eine repetitive Signalerzeugung ermöglicht. Für negativ geladene Proteine, die keine gute Interaktion mit dem negativ geladenen Indiumoxid-basierten Film zeigen wurden die Modifikation der Indiumzinnoxidschicht mit einem positiv geladenen Polymer sowie die Verwendung eines Antimon-dotierten Zinnoxid Films vorgeschlagen. Dadurch konnte die Abstoßung induziert durch die ähnliche Ladung des Proteins und der Elektrode überwunden werden. Es gelang für die humane Sulfit-Oxidase und die separate Häm-haltige Domäne der Austausch von Elektronen mit dem Trägermaterial. Im zweiten Teil der Arbeit wird über eine neue Methode für die Biosensorik von Sulfit berichtet, bei der direkte Elektronentransfer von humaner Sulfitoxidase immobilisierten auf einer mit Gold-Nanopartikeln modifizierten Elektrode verstärkt wurde. Die sphärischen Gold-Nanopartikeln, von etwa 10 nm im Durchmesser, wurden über eine neue Methode durch Reduktion von HAuCl4 mit verzweigtem Polyethylenimin in einer ionischen Flüssigkeit synthetisiert. Diese Nanopartikel wurden kovalent an eine mit Mercaptoundecansäure modifizierten Gold-Elektrode immobilisiert und dienen als Basis für die Adsorption von Sulfitoxidase adsorbiert wurde. Dadurch wurde ein schneller heterogener Elektronen-Transfer und verbesserte Elektrokatalyse erreicht. Für die Charakterisierung des verwendeten Systems eingesetzt wurden UV-Vis und Resonanz-Raman-Spektroskopie in Kombination mit direkter Protein-Voltammetrie. Es wurde keine Störung der strukturellen Integrität des Redox-Proteins beobachtet. Der vorgeschlagene Biosensor zeigte eine schnelle steady-state Stromantwort innerhalb von 2 s, eine lineare Detektion im Bereich zwischen 0,5 und 5,4 µM Sulfit mit einer hohen Empfindlichkeit (1,85 nA µM-1). Das untersuchte System bietet bemerkenswerte Vorteile da es ermöglicht bei niedriger angelegter Spannung und bei sehr hoher Ionenstärke zu arbeiten. Aufgrund dieser Eigenschaften hat das vorgeschlagene System großes Potential für die Entwicklung von bioelektronischen Geräten und der Anwendung in realen Proben. Schließlich werden im letzten Teil der Arbeit die komplexeren Enzymen Xanthindehydrogenase aus Rhodobacter capsulatus und Maus Aldehydoxidase Homolog 1 spektro- und elektrochemisch untersucht. Es konnte gezeigt werden, dass verschiedene Kofaktoren in der Proteinstruktur, wie FAD und der Molybdän Kofaktor direkt Elektronen mit einer Elektrode austauschen können, was durch einzelne Peaks im Square Wave Voltammogramm angezeigt wird. Es konnte eine zusätzliche redoxaktive Gruppe mit direktem Elektronen-Transfer nach Austausch eines Cysteins durch Serin am exponierten Eisen-Schwefel-Cluster gezeigt werden. Außerdem wurde eine vermittelte spektroelektrochemische Titration des FAD-Kofaktors in Anwesenheit von Mediatoren der Klasse der Eisen und Kobalt-Komplexe durchgeführt. Die Ergebnisse zeigen, dass FAD in R. capsulatus XDH zu einem stabilen Semichinone reduziert werden kann. Es gelang die formalen Potentiale für die zwei einzigen Elektrontransferprozesse zu bestimmen.

Along with an increasing understanding of the harmful effects on the environment of a wide range of pollutants has come the need for more sensitive, faster and less expensive detection methods of identification and quantitation. Many environmental pollutants occur in low levels and often in complex matrices thus analysis can be difficult, time consuming and costly. Because of the availability and easy cultivation of the microorganisms with potentially high specificity, there is considerable interest in the use of living microorganisms as the analytical component (the biocomponent) of sensors for pollutants. While a number of biosensors using bacteria have been developed, yeast has been comparatively rarely used as the biocomponent. Yeast are attractive because they are easy to culture and they are eukaryotes which means their biochemistry is in many respects closer to that of higher organisms. This thesis describes the development of whole cell bioassays that use yeast cells as a sensing element and redox mediators to probe the intracellular redox reactions to monitor the catabolic activity of the yeast resulting from the external substrate, steady-state voltammetry is utilised as the electrochemical detection technique. The isogenic differential enzyme analysis (IDEA) concept of Lincoln Ventures Limited, lead NERF funded research consortium uses bacteria that have been cultured using specific organic pollutants as the carbon source which are the biocomponent in sensors. The use of wild type yeast Arxula adeninivorans that has the ability to use a very wide variety of substrates as sources of carbon and nitrogen was used as an alternative to bacteria to validate the “IDEA” concept. Naphthalene and di-butyl phthalate were chosen as model target contaminant molecules. The performance, detection limits and the usefulness of yeast based biosensor applications for environmental analysis are discussed. This thesis also describes the development and optimisation of a simple, cost effective in vivo estrogens bioassay for the detection of estrogens using either genetically modified or a wild type yeast Saccharomyces cerevisiae. In this study, catabolic repression by glucose was exploited to achieve specificity to estrogens in complex environmental samples that eliminates the requirement for conventional sample preparation. This is the first time that the use of wild type yeast to quantify estrogens has been reported. The attractive features of the bioassay are its use of a non-GMO organism, its speed, its high specificity and sensitivity with a detection limit of 10-15 M. The similarity of binding affinities for major estrogens to those of human estrogens receptors makes this in vivo estrogen bioassay very useful for analytical/screening procedures. The electrochemical detection method also makes it easy to interface with a variety of electronic devices.

Nous avons développé un nano-fil conducteur, constitué uniquement de protéines et bio-inspiré des nano-fils bactériens conducteurs. Pour cela, une protéine chimère a été créée par l'association d'une protéine prion capable de s'auto-assembler en fibre et d'une métalloprotéine, une rubrédoxine, capable d'effectuer des transferts d'électrons. Comme montré par des techniques de microscopies et de spectroscopies (absorbance UV-visible et RPE), la protéine chimère est capable de former des fibres à la surface desquelles on retrouve les rubrédoxines. Les propriétés électroniques des nano-fils ont été caractérisées par des mesures courant-tension sur des échantillons secs et par électrochimie. Les mesures courant-tension ont montré que la conduction se faisait par plusieurs mécanismes. Les acides aminés aromatiques présents au centre du domaine prion semblent impliqués dans un des mécanismes de conduction. Les mesures électrochimiques ont quant à elles montré une conduction par sauts entre rubrédoxines. De plus, nous avons utilisé les nano-fils comme interface entre une enzyme, la laccase, et une électrode. Un courant électrocatalytique dû à la réduction de l'oxygène a été obtenu prouvant ainsi la capacité de nos nano-fils à agir comme médiateurs d'électrons. Les nano-fils conducteurs faits de protéines sont une structure intéressante pour comprendre le transport de charges dans les systèmes biologiques et sont également très prometteurs pour le développement de la bioélectronique et plus particulièrement de biocapteurs et de biopiles enzymatiques
The discovery of bacterial nanowires able to transport electrons on long range within biofilms and transfer them to electrodes is very promising for the development of bioelectronics and bio-electrochemical interfaces. However, their assembling process, their molecular composition and the electron transport mechanism are not fully understood yet. We took inspiration from bacterial nanowires to design conductive protein nanowires. We fused the sequence of a rubredoxin, an electron transfer iron-sulfur protein, to the sequence of HET-s(218-289), a prion domain that forms amyloid fibril by self-assembling under well-defined conditions. The resulting chimeric protein forms amyloid fibrils and displays redox proteins organized on the surface as shown by microscopy techniques and UV-Vis and EPR spectroscopy. Electron transfer mechanisms were studied in “dry state” current-voltage (I-V ) measurements and as hydrated film by electrochemistry. Dry state measurements allowed to evidence several conduction pathways with a possible role of aromatic residues in the conduction. Electrochemistry revealed electron transport by hopping between adjacent redox centers. This property allowed the use of our protein as mediator between a multicopper enzyme (laccase) and an electrode for electrocatalytic reduction of oxygen. These protein nanowires are interesting structures for the study of charge transport mechanisms in biological systems but are also very promising for the design of biosensors and enzymatic biofuel cells

Las etapas de transferencia electrónica o transferencia de carga involucradas en reacciones electroquímicas juegan un papel muy importante en un gran número de procesos biológicos y bioquímicos. Hoy en día, el interés de la comunidad científica se centra en explorar y entender exhaustivamente la naturaleza biológica y química de los fenómenos bioelectroquímicos que ocurren en los seres vivos, con el objeto de mimetizarlos en el laboratorio. Los procesos bioelectrocatalíticos presentan un amplio abanico de aplicaciones dirigidas al: (i) desarrollo de biorreactores electroquímicos para la mitigación de las emisiones de gases de efecto invernadero, la eliminación de contaminantes presentes en aguas residuales y urbanas, o la síntesis de productos con alto valor añadido para la industria, (ii) el desarrollo de biopilas y biobaterías, y (iii) el desarrollo de (bio)sensores electroquímicos con fines analíticos. Sin embargo, la implantación en el mercado de dispositivos basados en procesos biocatalíticos aún se enfrenta a varios desafíos, como son la robustez, la estabilidad a largo plazo, la reproducibilidad y la rentabilidad de producción en términos de materiales y fabricación de los dispositivos electroquímicos. La motivación de esta tesis doctoral es la de enfrentarse a algunos de los desafíos con los que se encuentra hoy en día la bioelectrocatálisis, para ello esta tesis doctoral se centra, principalmente en el estudio de nuevos materiales y mejora de rutas y estrategias bioelectrocatalíticas, con la finalidad de desarrollar dispositivos electroquímicos con aplicaciones analíticas y en la obtención de productos de valor añadido. En primer lugar esta tesis doctoral recoge el estudio y desarrollo de (bio)sensores electroquímicos para la determinación de lactato, L-cisteína, peróxido de hidrógeno y pH en medios biológicos complejos, y en segundo lugar estudia la bioelectrosíntesis de ácido fórmico a través de la reducción bioelectroquímica de dióxido de carbono.

La possibilité de convertir l’activité catalytique d’une oxydoréductase en un courant électrique a permis le développement d’une grande diversité d’électrodes enzymatiques. Les anodes catalysant l’oxydation du glucose font partie des plus étudiées pour leurs applications dans la mesure de la glycémie ou dans des biopiles glucose/O2. Parmi les nombreuses stratégies disponibles, l’utilisation d’hydrogels à base de complexes d’osmium en guise de médiateurs rédox fournit d’excellents résultats, qui restent cependant limités en terme de densité de courant ou de sélectivité. Durant cette thèse, la glucose oxydase (GOx) a été déglycosylée. Les électrodes préparées avec la nouvelle enzyme délivraient des courants catalytiques plus élevés, ce qui laissait supposer initialement une diminution de la distance de saut d’électron entre la GOx et le médiateur rédox suite au retrait des oligosaccharides. Une étude avec des électrodes de différentes compositions suggère au contraire que la déglycosylation n’améliore pas le transfert électronique intrinsèque mais la structure globale de l’hydrogel. De fait, une enzyme plus petite et plus négativement chargée doit induire un volume d’hydrogel plus faible pour une même composition molaire. En second lieu, une réduction parasite de l’oxygène affectant ces anodes, non envisagée jusqu’à aujourd’hui, a été mise en évidence et étudiée. En effet, l’interférence de l’O2 n’est usuellement attribuée qu’à sa réactivité avec la GOx. La présente étude prouve que l’O2 se réduit aussi sur les complexes d’osmium si leur potentiel standard E°’ est inférieur à + 0,07 V vs. Ag/AgCl. La cinétique de cette réaction croît exponentiellement quand le E°’ du complexe diminue. En plus d’abaisser le courant d’oxydation et donc les performances de l’anode, la génération de peroxyde d’hydrogène pourrait aussi altérer sa stabilité. Ces résultats suggèrent que le choix d’un médiateur de E°’ donné doit aussi dépendre de l’amplitude de cette réduction
The possibility of converting the catalytic activity of oxidoreductase enzymes into electric current has led to the development of a high diversity of enzyme electrodes. Anodes catalysing glucose oxidation have been amongst the most studied, especially for their application in monitoring blood glucose or glucose/O2 biofuel cells. Although one of the numerous strategies available, the use of osmium-based hydrogels as redox mediators, has given excellent results, some limitations still remain such as rather low current densities, stability or selectivity Initially, the study focused on the deglycosylation of glucose oxidase (GOx). When most of the oligosaccharides around this glycoenzyme were removed, the ensuing increase in the electrode catalytic current seemed a priori to support the hypothesis of a decrease in the electron hopping distance between the enzyme redox centres and the redox mediator. However, a systematic study of electrode response for different compositions leads us to conclude that deglycosylation does not improve the intrinsic electron transfer but the whole hydrogel structure. This seems due to the smaller size and higher surface charge of the deglycosylated GOx inducing smaller hydrogel volumes than in the native-based GOx. The study then proceeded to examine the oxygen side reduction of commonly used osmium-based redox polymers. The interference of O2 on glucose oxidation current has generally been attributed to O2 reactivity with GOx. The present study shows that O2 reduction also occurs on osmium-based polymers if their formal potential E°’ is below + 0.07 V vs. Ag/AgCl. The kinetics of this reaction appears to increase exponentially when E°’ decreases. As well as reducing the oxidation current and, consequently, lowering anode performances, the generation of hydrogen peroxide could also modify electrode stability. These results suggest that the choice of redox mediator for a given E°'must also take into account the extent of O2 reduction

Biosensors are used for the detection of a range of analytes for applications in healthcare, food production, environmental monitoring and biodefence. However, many biosensing platforms are large, expensive, require skilled operators or necessitate the analyte to be labelled. Direct electrochemical detection methods present a particularly attractive platform due to the simplified instrumentation when compared to other techniques such as fluorescence-based biosensors. With modern integrated circuit capabilities electrochemical biosensors offer greater suitability for monolithic integration with any necessary signal processing circuitry. This thesis explores micro- and nanogap devices for both redox cycling and dielectric spectroscopy sensing mechanisms. By using two electrodes with interelectrode separation down to distances in the micro- and nanometre scale, several benefits can be realised. Firstly the close proximity of the two electrodes significantly reduces the interdiffusion time. This allows an electroactive species to be rapidly shuttled across the gap and switched between reduced and oxidised states. The result is feedback amplification of the amperometric response, increasing the signal. The second benefit is that the screening effect caused by electric double layers at the electrode–electrolyte interface is reduced due to the electric double layers occupying a larger fraction of the sensing volume. This significantly improves the sensor suitability for dielectric spectroscopy by increasing the potential drop across the biolayer. These two sensing mechanisms are demonstrated using a large area dual-plate microgap device for the detection of two different analytes. Utilising the first mode, detection of cysteine–cystine, an important redox couple involved in the signalling mechanism for the regulation of protein function, interaction and localisation is shown. The microgap device is then used for dielectric spectroscopy sensing of a mannose-specific uropathogenic Escherichia coli strain whilst also demonstrating the effect of ionic concentration on the capacitive response. The response of these devices is highly dependent on the interelectrode separation as well as the surface area of the electrodes. However, fabrication of large-area nanogap devices presents a significant challenge. This meant that careful optimisation and the development of novel techniques was necessary. This work reports the design, fabrication and characterisation of both a vertical and a horizontal coplanar large area nanogap device. The vertical nanogap device is fabricated using an inductively-coupled plasma reactive ion etching process to create a channel in a silicon substrate. A lower electrode is then optically patterned in the channel before anodically bonding a second identical electrode patterned on glass directly above. The horizontal nanogap device uses a different approach, utilising a state-of-the-art electron-beam lithography system to create a long serpentine nanogap with passivation to reduce fringing effects. The design allows the electron-beam lithography step to be substituted with nanoimprint lithography to reduce cost and improve throughput. Both of these devices have integrated microfluidic channels and provide a capacity for relatively high-throughput production.

碩士
國立清華大學
工程與系統科學系
101
Sepsis is a serious infection disease usually caused by bacteria and posing immune system to attack body's own organs and tissues. Sepsis can be frightening because it can lead to serious complications that affect the functions of kidneys, lungs, brain, and hearing, and can even cause death. Traditionally, analysis of infectious bacteria is still based on culture-based protocols, which need days to obtain result. In addition, it can not be detected when the patient is in the initial stage with only several hundreds of bacteria in 1c.c whole blood. In order to push the detection limit, we use Mesoporous Silica Nanoparticles (MSNs). Porous Si-NPs give around 100 times binding surface area enhancement larger than solid Si-NPs can provide at the same size. The Porous Si-NPs give us over 2-3 order enhancement for Ox-Red signal. Mesoporous Silica Nanoparticles (MSNs) labeled with antibody were used as transducers to amplify the signal by increasing the signal to noise ratio, and reducing the response time. S. aureus containing samples have been tested by using anti- S. aureus magnetic beads(MBs-pSAb) as capture phase and sandwiching afterwards with MSNs modified antibodies(sSAb-MSNs)detected using Cyclic Voltammetry (CV).A detection limit of 10cells mL-1. And a linear range from 10 to 104 cells mL-1 of S. aureus was obtained. The results show that this biochip system has a great potential for single-bacterium detection.

The development of cobalt(II) phthalocyanine–cobalt(II) tetra(5-phenoxy-10,15,20-triphenylporphyrin), (CoPc–(CoTPP)[subscript 4]) pentamer as a novel redox mediator for amperometric enzyme electrode sensitive to glucose is described. A glassy carbon electrode (GCE) was first modified with the pentamer, then followed by the immobilization onto the GCE–CoPc–(CoTPP)[subscript 4] with glucose oxidase (GOx) through cross-linking with glutaraldehyde in the presence of bovine serum albumin (BSA) and Nafion® cation-exchange polymer. The proposed biosensor displayed good amperometric respose charateristics to glucose in pH 7.0 PBS solution; such as low overpotentials (+400 mV versus Ag|AgCl), very fast amperometric response time (~5 s), linear concentration range extended up to 11 mM, with 10 μM detection limit. The biosensor exhibited electrochemical Michaelis–Menten kinetics and showed an average apparent Michaelis–Menten constant (K′M) of 14.91 ± 0.46 mM over a storage period of 2 weeks.

The development of cobalt(II) phthalocyanine–cobalt(II) tetra(5-phenoxy-10,15,20-triphenylporphyrin), (CoPc–(CoTPP)[subscript 4]) pentamer as a novel redox mediator for amperometric enzyme electrode sensitive to glucose is described. A glassy carbon electrode (GCE) was first modified with the pentamer, then followed by the immobilization onto the GCE–CoPc–(CoTPP)[subscript 4] with glucose oxidase (GOx) through cross-linking with glutaraldehyde in the presence of bovine serum albumin (BSA) and Nafion® cation-exchange polymer. The proposed biosensor displayed good amperometric respose charateristics to glucose in pH 7.0 PBS solution; such as low overpotentials (+400 mV versus Ag|AgCl), very fast amperometric response time (~5 s), linear concentration range extended up to 11 mM, with 10 μM detection limit. The biosensor exhibited electrochemical Michaelis–Menten kinetics and showed an average apparent Michaelis–Menten constant (K′M) of 14.91 ± 0.46 mM over a storage period of 2 weeks.

Relatório de Estágio do Mestrado Integrado em Ciências Farmacêuticas apresentado à Faculdade de Farmácia
As espécies reativas de oxigénio são um grupo de moléculas derivadas do oxigénio, que são formadas por redução-oxidação (redox) ou por excitação eletrónica, e que têm diferentes distribuições temporais e espaciais e uma ampla gama de concentrações intracelulares e extracelulares. O peróxido de hidrogénio é umas destas espécies com mais interesse ao nível da sinalização redox celular sendo que as suas concentrações podem desencadear diferentes respostas, existindo níveis limiares que separam o estado patológico do estado fisiológico e por conseguinte, o estado de oxidative eustress do estado de oxidative distress. Assim, é importante compreender como é que esta molécula se comporta e encontra no corpo humano, por forma a realizar a sua deteção e monitorização. Atualmente, são aplicados vários métodos analíticos destacando-se os espectroscópicos (quimioluminescência e fluorescência) e os eletroanalítcos (sensores e biossensores). Os biossensores, embora ainda apresentem algumas desvantagens assumem-se como um dos maiores desafios na deteção e monitorização do peróxido de hidrogénio utilizando uma molécula de reconhecimento biológico ou mediadores de transferência de eletrão tais como os hexacianoferratos, conhecidos como “peroxidases artificiais”. Estes sensores são capazes de fornecer informações sobre a dinâmica de concentração em tempo real, que não é acessível com outros métodos. Torna-se, então, claro que o H2O2 desempenha funções fundamentais nometabolismo sendo a compreensão do seu papel biológico bastante relevante e os métodosde deteção e monitorização um enorme desafio. Assim, foram aplicadas diversas metodologias analíticas para compreensão da sua bioatividade no tecido cerebral, em células cancerígenas e outras células.
Reactive oxygen species are a group of molecules derived from oxygen, which are formed by reduction-oxidation (redox) or by electronic excitation, and which have different temporal and spatial distributions and a wide range of intracellular and extracellular concentrations. Hydrogen peroxide is one of this species interesting reactive oxygen species in terms of cellular redox signalling and its concentrations result in different responses. Different levels of hydrogen peroxide separate the pathological state from the physiological state and, the oxidative eustress state from the oxidative distress state. Thus, it is important to understandhow this molecule behaves and finds in the human body to carry out its detection and monitoring. Nowadays, several analytical methods are applied, highlighting spectroscopic(chemiluminescence and fluorescence) and electroanalytical (sensors and biosensors).Biosensors, although they still have some disadvantages, are one of the biggest in challenges in detection and monitoring of hydrogen peroxide using a biological recognition molecule or electron mediators such as hexacyanoferrate compounds known as an “artificial enzyme peroxidase”. This type of sensors can provide information regarding the real-time concentration dynamics of hydrogen peroxide, that are not accessible to other methods. It then becomes clear that hydrogen peroxide plays key roles in metabolism being the understanding of its biological role very relevant and the methods detection and monitoring a huge challenge. Thus, several analytical methodologies were applied to understand its bioactivity in brain tissue, cancer cells and other cells.

Mycobacterium tuberculosis (Mtb) is evolutionarily equipped to resist exogenous reactive oxygen species but shows vulnerability to an increase in endogenous ROS (eROS). Since eROS is an unavoidable consequence of aerobic metabolism, understanding how eROS levels are controlled is essential yet remains uncharacterized. By combining the Mrx1-roGFP2 redox biosensor with transposon mutagenesis, we identified 368 genes (redoxosome) responsible for maintaining non-toxic levels of eROS in Mtb. Integrating redoxosome with a global network of protein-protein interactions and transcriptional regulators revealed a hypothetical protein (rv0158) as a top node managing eROS and redox homeostasis in Mtb. RNA sequencing, seahorse XF flux measurements, and lipid analysis indicate that rv0158 is required to balance the deployment of fatty acid substrates between lipid anabolism and oxidation. Disruption of rv0158 perturbed redox balance in a carbon-source-specific manner, promoted killing in response to anti-TB drugs, reduced survival in macrophages, and lowered persistence in mice. We describe a novel pathogen response to moxifloxacin. Mtb, unlike Escherichia coli, decreases respiration in response to moxifloxacin. Nevertheless, cells were killed, as ROS increased due to NADH-dependent reductive stress. Moxifloxacin lethality was mitigated by supplementing bacterial cultures with a ROS scavenger (thiourea), and an iron chelator (bipyridyl), indicating ROS is part and not a consequence of death processes. Treatment with N-acetyl cysteine (NAC) accelerated respiration and ROS production, increased moxifloxacin lethality, and lowered the mutant prevention concentration. Thus, redox and bioenergetic imbalance contribute to the moxifloxacin-mediated killing of Mtb. These results provide a way to make fluoroquinolones more effective anti-tuberculosis agents. We have previously reported that Mtb H37Rv sets up a gradient of mycothiol redox potential: EMSH-oxidized (-240 mV) to EMSH-reduced (-320 mV) inside macrophages, where the EMSH -reduced Mtb subpopulation are significantly more tolerant to anti-TB drugs. Therefore, one of the keys to subverting drug-tolerance is to impede the emergence of EMSH -reduced subpopulation by inducing overwhelming oxidative stress. In this study, we exposed THP1-macrophages infected with Mtb H37Rv expressing Mrx1-roGFP2-biosensor, to a library of FDA-approved drugs (Enzo Life Sciences; BML-2842) and scored for the oxidative shift in the Mtb- EMSH at 24 hours post infection. Based on their activity to trigger oxidative stress inside the bacterium, non-cytotoxicity to host, and inhibition of bacterial growth inside macrophages, C5 molecule emerged as the top hit. Pre-treatment with C5 potentiated killing of Mtb by all tested antibiotics (isoniazid, rifampicin, and moxifloxacin) and reduced drug-tolerance.
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Parkinson’s disease is a neurodegenerative disorder characterized by a loss of dopaminergic neurons, where mitochondrial dysfunction and neuroinflammation are implicated in this process. However, the exact mechanisms of mitochondrial dysfunction, oxidative stress and neuroinflammation leading to the onset and development of Parkinson’s disease are not well understood. There is a lack of tools necessary to dissect these mechanisms, therefore we engineered genetically encoded fluorescent biosensors to monitor redox status and an inflammatory signal peptide with high spatiotemporal resolution. To measure intracellular redox dynamics, we developed red-shifted redox sensors and demonstrated their application in dual compartment imaging to study cross compartmental redox dynamics in live cells. To monitor extracellular inflammatory events, we developed a family of spectrally diverse genetically encoded fluorescent biosensors for the inflammatory mediator peptide, bradykinin. At the organismal level, we characterized the locomotor effects of mitochondrial toxicant-induced dopaminergic disruption in a zebrafish animal model and evaluated a behavioral assay as a method to screen for dopaminergic dysfunction. Pairing our intracellular redox sensors and our extracellular bradykinin sensors in a Parkinson’s disease animal model, such as a zebrafish toxicant-induced model will prove useful for dissecting the role of mitochondrial dysfunction and inflammation in Parkinson’s disease.