Добірка наукової літератури з теми "Biosenseur redox"

Генетически кодируемые биосенсоры на основе флуоресцентных белков представляют собой инструмент исследования ряда биологических процессов в живых системах в режиме реального времени. За последние годы было создано целое семейство биосенсоров, позволяющих визуализировать в живых клетках изменения редокс-состояния пула глутатиона. Целью настоящей работы была разработка нового биосенсора для регистрации соотношения 2GSH/GSSG на основе красного флуоресцентного белка mKate2. Для этого методом направленного мутагенеза в структуру флуоресцентного белка вносили пару аминокислотных остатков цистеина, которые при окислении окружающего пула глутатиона формируют дисульфидную связь, что приводит к изменению спектральных характеристик. Полученный биосенсор был протестирован in vitro на выделенном препарате белка, в частности, были исследованы спектральные характеристики, рН-чувствительность белка, окислительно-восстановительный потенциал. Кроме того, биосенсор, названный roKate, был протестирован в культуре живых клеток млекопитающих. Он отличается высокой яркостью и повышенной стабильностью сигнала при изменениях рН в физиологическом диапазоне. От других представителей данного семейства биосенсоров roKate отличается необратимым изменением сигнала при окислении в клетках млекопитающих. Применение данного сенсора предпочтительно в экспериментах с наличием длительного промежутка времени между воздействием на биологическую систему и последующим анализом вызванного эффекта, например в условиях длительной пробоподготовки.

The development of genetically encoded redox biosensors has paved the way toward chemically specific, quantitative, dynamic, and compartment-specific redox measurements in cells and organisms. In particular, redox-sensitive green fluorescent proteins (roGFPs) have attracted major interest as tools to monitor biological redox changes in real time and in vivo. Most recently, the engineering of a redox relay that combines glutaredoxin (Grx) with roGFP2 as a translational fusion (Grx1-roGFP2) led to a biosensor for the glutathione redox potential ( EGSH). The expression of this probe in mitochondria is of particular interest as mitochondria are the major source of oxidants, and their redox status is closely connected to cell fate decisions. While Grx1-roGFP2 can be expressed in mammalian mitochondria, it fails to enter mitochondria in various nonmammalian model organisms. Here we report that inversion of domain order from Grx1-roGFP2 to roGFP2-Grx1 yields a biosensor with perfect mitochondrial targeting while fully maintaining its biosensor capabilities. The redesigned probe thus allows extending in vivo observations of mitochondrial redox homeostasis to important nonmammalian model organisms, particularly plants and insects.

Microbial activity has gained attention because of its impact on the environment and the quality of people’s lives. Most of today’s methods, which include genome sequencing and electrochemistry, are costly and difficult to manage. Our group proposed a method using the redox potential change to detect microbial activity, which is rooted in the concept that metabolic activity can change the redox potential of a microbial community. The redox potential change was captured by a biosensor consisting of porous boron nitride, ATP-DNA aptamer, and methylene blue as the fluorophore. This assembly can switch on or off when there is a redox potential change, and this change leads to a fluorescence change that can be examined using a multipurpose microplate reader. The results show that this biosensor can detect microbial community changes when its composition is changed or toxic metals are ingested.

It is known that redox reaction can take place among the solutions of potassium ferrocyanide (K4[Fe(CN)6]), glucose (C6H[Formula: see text]O6) and glucose oxidase (Glucose Oxidase, GOD). In this work, the method of electrochemical biosensor detection based on screen printed electrode was used to observe the redox reaction among these solutions. The relationship between redox reaction and parameters was studied by examining the effects of concentration and scanning speed of three solutions.

Cellular functions such as DNA replication and protein translation are influenced by changes in the intracellular redox milieu. Exogenous (i.e., nutrients, deterioration of media components, xenobiotics) and endogenous factors (i.e., metabolism, growth) may alter the redox homeostasis of cells. Thus, monitoring redox changes in real time and in situ is deemed essential for optimizing the production of recombinant proteins. Recently, different redox-sensitive variants of green fluorescent proteins (e.g., rxYFP, roGFP2, and rxmRuby2) have been engineered and proved suitable to detect, in a non-invasive manner, perturbations in the pool of reduced and oxidized glutathione, the major low molecular mass thiol in mammals. In this study, we validate the use of cytosolic rxYFP on two cell lines widely used in biomanufacturing processes, namely, CHO-K1 cells expressing the human granulocyte macrophage colony-stimulating factor (hGM-CSF) and HEK-293. Flow cytometry was selected as the read-out technique for rxYFP signal given its high-throughput and statistical robustness. Growth kinetics and cellular metabolism (glucose consumption, lactate and ammonia production) of the redox reporter cells were comparable to those of the parental cell lines. The hGM-CSF production was not affected by the expression of the biosensor. The redox reporter cell lines showed a sensitive and reversible response to different redox stimuli (reducing and oxidant reagents). Under batch culture conditions, a significant and progressive oxidation of the biosensor occurred when CHO-K1-hGM-CSF cells entered the late-log phase. Medium replenishment restored, albeit partially, the intracellular redox homeostasis. Our study highlights the utility of genetically encoded redox biosensors to guide metabolic engineering or intervention strategies aimed at optimizing cell viability, growth, and productivity.

A novel, integrated experimental and modeling framework was applied to an inhibition-based bi-enzyme (IBE) electrochemical biosensor to detect acetylcholinesterase (AChE) inhibitors that may trigger neurological diseases. The biosensor was fabricated by co-immobilizing AChE and tyrosinase (Tyr) on the gold working electrode of a screen-printed electrode (SPE) array. The reaction chemistry included a redox-recycle amplification mechanism to improve the biosensor’s current output and sensitivity. A mechanistic mathematical model of the biosensor was used to simulate key diffusion and reaction steps, including diffusion of AChE’s reactant (phenylacetate) and inhibitor, the reaction kinetics of the two enzymes, and electrochemical reaction kinetics at the SPE’s working electrode. The model was validated by showing that it could reproduce a steady-state biosensor current as a function of the inhibitor (PMSF) concentration and unsteady-state dynamics of the biosensor current following the addition of a reactant (phenylacetate) and inhibitor phenylmethylsulfonylfluoride). The model’s utility for characterizing and optimizing biosensor performance was then demonstrated. It was used to calculate the sensitivity of the biosensor’s current output and the redox-recycle amplification factor as a function of experimental variables. It was used to calculate dimensionless Damkohler numbers and current-control coefficients that indicated the degree to which individual diffusion and reaction steps limited the biosensor’s output current. Finally, the model’s utility in designing IBE biosensors and operating conditions that achieve specific performance criteria was discussed.

Electrochemical impedance spectroscopy (EIS) is a sensitive analytical method for surface and bulk properties. Classical EIS and derived electrochemical capacitance spectroscopy (ECS) with a redox couple are label-free approaches for biosensor development, but doubts arise regarding interpretability when a redox couple is employed (redox EIS) due to interactions between electroactive probes and interfacial charges or forced potential. Here, we demonstrated redoxless ECS for directly determining surfactant adsorption on screen-printed carbon electrodes (SPCEs), validated through a simulation of equivalent circuits and the electrochemistry of electronic dummy cells. Redoxless ECS provides excellent capacitance plot loci for quantifying the interfacial permittivity of di-electric layers on electrode surfaces. Redoxless ECS was compared with redox EIS/ECS, revealing a favorable discrimination of interfacial capacitances under both low and high SDS coverage on SPCEs and demonstrating potential for probeless (reagentless) sensing. Furthermore, the proposed method was applied in an electrolyte without a redox couple and bare electrodes, obtaining a high performance for the adsorption of surfactants Tween-20, Triton-X100, sodium dodecyl sulfate, and tetrapropylammonium bromide. This approach offers a simple and straightforward means for a semi-quantitative evaluation of small molecule interactions with electrode surfaces. Our proposed approach may serve as a starting point for future probeless (reagentless) and label-free biosensors based on electrochemistry, eliminating disturbance with surface charge properties and minimizing forced potential bias by avoiding redox couples. An unambiguous and quantitative determination of physicochemical properties of biochemically recognizable layers will be relevant for biosensor development.

Bortezomib is an inhibitor of proteasomes and an anti-cancer drug. Although bortezomib is considered a safe drug, as confirmed by cytotoxicity assays, recent reports highlighted the possibility of interaction between bortezomib and cellular components, with detrimental long-term effects. The evaluation of the interaction between bortezomib and dsDNA was investigated in bulk solution and using a dsDNA electrochemical biosensor. The binding of bortezomib to dsDNA involved its electroactive centers and led to small morphological modifications in the dsDNA double helix, which were electrochemically identified through changes in the guanine and adenine residue oxidation peaks and confirmed by electrophoretic and spectrophotometric measurements. The redox product of bortezomib amino group oxidation was electrochemically generated in situ on the surface of the dsDNA electrochemical biosensor. The redox product of bortezomib was shown to interact primarily with guanine residues, preventing their oxidation and leading to the formation of bortezomib–guanine adducts, which was confirmed by control experiments with polyhomonucleotides electrochemical biosensors and mass spectrometry. An interaction mechanism between dsDNA and bortezomib is proposed, and the formation of the bortezomib redox product–guanine adduct explained.

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

Iron oxide nanoparticles perform biological activity under physiological conditions. They exhibit enzyme-like properties that catalyze redox reactions mediated by natural enzymes of oxidoreductase and are classified into a typical of nanozymes that are defined as nanomaterials with enzyme-like activities. In addition, iron oxide nanoparticles widely exist in biological system, such as magnetosome and ferritin that not only regulate iron metabolism, but also regulate ROS homostasis. The enzyme-like properties of iron oxide nanoparticles render them with broad biomedical applications including immunoassay, biosensor, antimicrobial, anti-tumor, antioxidant. Taken together, iron oxide nanoparticles are bioactive materials and may perform particular biological function in life activity.

This chapter summarizes the developmental studies on environmental biosensors of enzymatic phosphate ion (Pi) biosensors for eutrophication and microbial biochemical oxygen demand (BOD) biosensors for organic pollution. In particular, an author focuses on the developmental studies that the author principally conducted, and describe the history and the insights into the future of these fields of environmental biosensors. In our developmental studies on the enzymatic Pi biosensors, we fabricated automatic instruments of a desktop-type and a submersible buoy-type, which was fabricated for remote biosensing of dam water. These instruments employed a luminol-chemiluminescence flow injection analysis (CL-FIA) system and enabled to have practical performances in precise Pi determination, operational stability, and accurate bioavailable Pi measurements. In the microbial BOD biosensor development, the author considered to apply the FIA concept enabling highly repeatable measurements to absorptiometric BOD measurements. Both precise temperature control and accurate time control to incubate measurement mixture of budding yeast cell suspension containing redox color indicator and sample enabled to obtain the highly repeatable results that led to highly sensitive BOD measurements. Looking back on our developmental studies, what the author was thinking at the time and the results obtained are described. Finally, the author discusses the developmental trends of these biosensor fields and new insights into the future perspectives.

This chapter summarizes the synthesis of transition metal-containing dendrimers and their biosensing and bioimaging applications as part of medical diagnostic procedures. This class of dendrimers has been utilized to investigate biological and pathological processes in human and animal systems. The three-dimensional architectures, solubility, lower toxicity and transition metal redox behaviour, as well as the optical and magnetic resonance properties, of this class of dendrimers make them excellent candidates for biosensing and bioimaging applications. These applications include using these dendrimers as contrast agents for various bioimaging techniques, such as magnetic resonance imaging (MRI), computed tomography (CT), and photoacoustic imaging (PA), with higher sensitivity and lower toxicity compared to existing small molecule contrasts. These metal-containing nanomaterials can also play a dual role as diagnostic agents in biosensor or bioimaging applications while exerting anticancer and antibacterial therapeutic effects. Metal-containing dendrimers may therefore be used in the field of imaging-guided therapeutic applications or theranostics (a combination of diagnostic techniques and therapeutic interventions). In addition to these transition metal-containing dendrimers, dendrimers containing the inner transition metal gadolinium are also described for their use as MRI contrasts.

In this study, a novel and simple electrochemical glucose biosensor based on a silicon nanowire array (SNA) electrode was proposed. Metal-assisted etching (MAE) method using an AgNO3 and HF mixing solution as the etchant was employed to grow the silicon nanowire array (SNA) electrode. A thin gold shell is then sputtered over each silicon nanowire. Potassium ferricyanide, glucose oxidase (GOx), and a Nafion thin film were then sequentially coated onto the fabricated SNA for glucose detection. The processing time of the MAE and sputtering as well as the GOx concentration were optimized in terms of the redox peak currents of the SNA electrode. Compared with the corresponding plane gold electrode, the effective sensing area of the synthesized SNA electrode was measured to be 6.12 folds. Actual glucose detections demonstrated that the proposed SNA array electrode could operate in a linear range of 0.55 mM-11.02 mM and a very high sensitivity of 346 μA mM−1 cm−2. The proposed SNA electrode based glucose biosensor possesses advantages of simple fabrication process, low cost, and high sensitivity. It is feasible for future clinical applications.

The original objectives of this research project were to: (1) develop immunoassays, photometric sensors, and electrochemical sensors for real-time measurement of progesterone and estradiol in milk, (2) develop biosensors for measurement of caseins in milk, and (3) integrate and adapt these sensor technologies to create an automated electronic sensing system for operation in dairy parlors during milking. The overall direction of research was not changed, although the work was expanded to include other milk components such as urea and lactose. A second generation biosensor for on-line measurement of bovine progesterone was designed and tested. Anti-progesterone antibody was coated on small disks of nitrocellulose membrane, which were inserted in the reaction chamber prior to testing, and a real-time assay was developed. The biosensor was designed using micropumps and valves under computer control, and assayed fluid volumes on the order of 1 ml. An automated sampler was designed to draw a test volume of milk from the long milk tube using a 4-way pinch valve. The system could execute a measurement cycle in about 10 min. Progesterone could be measured at concentrations low enough to distinguish luteal-phase from follicular-phase cows. The potential of the sensor to detect actual ovulatory events was compared with standard methods of estrus detection, including human observation and an activity monitor. The biosensor correctly identified all ovulatory events during its testperiod, but the variability at low progesterone concentrations triggered some false positives. Direct on-line measurement and intelligent interpretation of reproductive hormone profiles offers the potential for substantial improvement in reproductive management. A simple potentiometric method for measurement of milk protein was developed and tested. The method was based on the fact that proteins bind iodine. When proteins are added to a solution of the redox couple iodine/iodide (I-I2), the concentration of free iodine is changed and, as a consequence, the potential between two electrodes immersed in the solution is changed. The method worked well with analytical casein solutions and accurately measured concentrations of analytical caseins added to fresh milk. When tested with actual milk samples, the correlation between the sensor readings and the reference lab results (of both total proteins and casein content) was inferior to that of analytical casein. A number of different technologies were explored for the analysis of milk urea, and a manometric technique was selected for the final design. In the new sensor, urea in the sample was hydrolyzed to ammonium and carbonate by the enzyme urease, and subsequent shaking of the sample with citric acid in a sealed cell allowed urea to be estimated as a change in partial pressure of carbon dioxide. The pressure change in the cell was measured with a miniature piezoresistive pressure sensor, and effects of background dissolved gases and vapor pressures were corrected for by repeating the measurement of pressure developed in the sample without the addition of urease. Results were accurate in the physiological range of milk, the assay was faster than the typical milking period, and no toxic reagents were required. A sampling device was designed and built to passively draw milk from the long milk tube in the parlor. An electrochemical sensor for lactose was developed starting with a three-cascaded-enzyme sensor, evolving into two enzymes and CO2[Fe (CN)6] as a mediator, and then into a microflow injection system using poly-osmium modified screen-printed electrodes. The sensor was designed to serve multiple milking positions, using a manifold valve, a sampling valve, and two pumps. Disposable screen-printed electrodes with enzymatic membranes were used. The sensor was optimized for electrode coating components, flow rate, pH, and sample size, and the results correlated well (r2= 0.967) with known lactose concentrations.