Nous avons développé un module de detection de bactérie base sur le suivi électrochimique d’une PCR. Le système a été realisé en Copolymère d’Oléfin Cyclique (COC). Il s’agit ici de présenter une nouvelle approche pour concevoir des systèmes de despistage d’infection nosocomiale en quelques heures. INTRODUCTION Avec le développement des flux migratoires de population et l’apparition de résistance aux antibiotiques, les infections nosocomiales sont devenues un enjeu de santé majeur. Les besoins en systèmes de diagnostique accessibles financièrement augmentent considérablement. L’approche qui consiste à intégrer entièrement une chaîne d’analyse commence à avoir les faveurs des systèmes de santé et en particulier de l’Organisation Mondiale de la Santé (OMS). Une compagnie nord américaine Cepheid a par ailleurs déjà développé un système automatisé de détection de germes basé sur l’identification de l’ADN par PCR. Cependant le coût élevé des technologies ralentit sa démocratisation malgré un apport considérable pour les tests cliniques de routine. Ici nous nous proposons une stratégie d’analyse qui permet de maîtriser les drastiquement les coûts de consommable. Tout d’abord tout le système est réalisé en COC, un thermoplastique qui peut être façonné à très grande échelle pour quelques dizines de centimes l’unité. [1]. Ensuite le système de détection, implique un suivi électrochimique qui remplace avantageusement les plateformes optiques traditionnellement coûteuses. Si la première cible de ce système est le dépistage de maladie nosocomiale, il apparaît aussi qu’il est transposable à d’autres domaines où la détection basée sur une PCR peut apporter des solutions. THEORIE L’échantillon, un écouvillon nasal est prélevé sur le patient. Ensuite les bactéries sont lysées grâce à l’utilisation de détergent. Une fois les parois bactériennes détruites, l’ADN est extrait sur phase solide par l’intermédiaire de billes magnétiques de type « charge switch » assemblées en colonnes [2]. Après lavage l’ADN est élué, puis amplifié par PCR en temps réel. Durant cette phase l’amplification de l’ADN est suivie grâce à l’utilisation d’un intercalant redox. Ce principe de détection fait l’objet d’un brevet [3] et nous présentons ici sa première implémentation dans un système de type laboratoire sur puce. Le complexe redox qui sert d’intercalant est détecté par voltammétrie à onde carrée (en anglais : Square Wave Voltammetry SQWV). Ce complexe interagit avec l’ADN double brin par intercalation pendant la phase d’élongation, ainsi il n’est plus disponible pour l’échange d’électrons avec les électrodes de détection. En conséquence, on observe une chute du niveau de courant mesuré pendant la SQWV qui correspond à l’augmentation de la quantité d’ADN. PRATIQUE De noveaux protocoles de fabrication de systèmes en thermoplastique ont été développés permettant de réaliser des système en COC robustes et étanches. L’intégration des électrodes sérigraphiées a été réalisée avec succès. Le desgin de la puce a été choisi afin de permettre la parallélisation d’expérience en vue des tests de sensibilité de détection. Afin de réaliser les cycles de chauffage nécessaire à l’amplification par PCR un système dédié, comprenant un bloc thermique, un système de régulation et une partie logicielle à été développé. La preuve de concept de l’efficacité du système a été démontré sur de l’ADN modèle : le Litmus. Il est possible de distinguer différentes concentration en ADN de départ. / With the development of human mobility and antibiotics resistance, nosocomial infections have become a major health problem. The need for fast and efficient diagnosis systems, ,while affordable by the health care systems, is increasing. The “sample to result” integration allowed by microfludics is a strong asset in such developments. Cepheid developed an integrated system for germ genotyping based on real-time PCR, but due to expensive laser-induced fluorescence detection its cost still reduces its range of applications in routine clinics. Here, we propose a new strategy allowing further cost reduction. First, the whole analysis streamline is integrated in a single chip made of Cyclo Olefin Copolymer (COC) [1]. This material can be mass-processed by CD technology, to yield chips at a few cents a piece. Second, the detection involves an electrochemical method that alleviates need for optics. Methicillin Resistant Staphylococcus Aureus (MRSA) was chosen as the initial primary target for validation. The technologies developed will further be applied to other strains of Bacteria or other fields such as agriculture, food testing, GMOs or security. The sample, a nasal swab, undergoes a chemical lysis, and DNA is extracted by selective capture on magnetic beads self-assembled into dense microcolumns arrays, as presented at MicroTAS 2009 [2]. DNA is then eluted, amplified by PCR in real time in the chip, while detected in situ by a high sensitivity electrochemical detection catalyzed by an intercalating redox compound. This proprietary technology [3] is being implemented here for the first time in a lab on chip. New protocols were developed to make a robust and leakage-free COC microfluidic chip with integrated electrodes. COC is a biocompatible and solvent resistant thermoplastic material. The COC microfluidic chip consists of a substrate with a hot-embossed microchannel and screen printed carbon and silver electrodes. The chip is sealed by solvent bonding [4]. This provides simple and cost effective fabrication, directly upscalable to mass production PCR Thermal control is done externally with a customized thermocycler. A redox compound is introduced together with DNA sample enabling electrochemical detection through Square Wave Voltammetry (SQWV). This compound intercalates in double-stranded DNA during the extension step, reducing its mobility and redox activity, so the peak current decreases during amplification of the target DNA. A sensitivity below 1ng/µL suitable for MRSA detection was achieved. The data were obtained by SQWV measurements. There is a clear correlation between the increase of DNA concentration and the decrease of the peak current for the redox compound: PCR amplification was also performed inside the chip, demonstrating the compatibility of this platform with thermal cycling and biomolecular reactions. Real-time electrochemical measurement during cycling was not possible yet due to experimental setup geometrical constraints, but work is in progress and results will be presented at the conference. Future experiments will focus on the integration of the bacteria lysis and DNA extraction steps on the same chip.

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.

Opto-electronic semiconductor technology continues to grow at an accelerated pace, as the industry seeks to perfect devices such as light emitting diodes for purposes of optical processing and communication. A strive for greater efficiency with shrinking device dimensions, continually pushes the technology from both a design and materials aspect. Nanosystems such a quantum dots, also face new material engineering challenges as they enter the realm of quantum mechanics, with each system and material having markedly different electronic properties. Traditionally, the semiconductor industry has focused on materials such Group II-VI and III-V compounds as the basis material for future opto-electronic needs. Unfortunately, these material systems can be expensive and have difficulties integrating into current Si-based technology. The industry is reluctant to leave silicon due in part to silicon's high quality oxide, and the enormous amount of research invested into silicon based circuit fabrication. Although recently materials such as GaN are starting to dominate the electro-optical industry since a Si-based substitute has not been found. The purpose of the dissertation was to examine several promising systems that could be easily integrated into current Si-based technology and also be produced using simple inexpensive fabrication techniques such ion implantation. The development of optically active nano-sized precipitates in silica to form the active layer of an opto-electronic device was achieved with ion implantation and thermal annealing. Three material systems were investigated. These systems consisted of carbon, silicon and metal silicide based nanocrystals. The physical morphology and electronic properties were monitored using a variety of material characterization techniques. Rutherford backscattering/channeling were used to monitor elemental concentrations, photoluminescence was used to monitor the opto-electronic properties and transmission electron microscopy was used to study the intricate morphology of individual precipitates. The electronic properties and the morphology were studied as a function of implant dose, anneal times and anneal temperatures.

Borides are implemented in a range of industrial applications due to their unique mechanical, electrical, thermal and catalytic properties. In particular, transition metal diborides are of special interest. In the recent years, borides of rhenium (Re), osmium (Os) and iridium (Ir) have been studied as for their ultra-hardness and superior stiffness. In this dissertation, a mechanochemical method is introduced to produce rhenium diboride (ReB2) powder, a novel hexagonal osmium diboride (h-OsB2), and iridium boride powders. Densification by Spark Plasma Sintering (SPS), thermal stability and mechanical properties of h-OsB2 were also studied. ReB2 was recently reported to exhibit high hardness and low compressibility, which both are strong functions of its stoichiometry, namely Re to B ratio. Most of the techniques used for ReB2 synthesis reported 1:2.5 Re to B ratio because of the loss of the B during high temperature synthesis. However, as a result of B excess, the amorphous boron, located along the grain boundaries of polycrystalline ReB2, would degrade the ReB2 properties. Therefore, techniques which could allow synthesizing the stoichiometric ReB2 preferably at room temperature are in high demand. ReB2 powder was synthesized at low temperature using mechanochemical route by milling elemental crystalline Re and amorphous B powders in the SPEX 8000 high energy ball mill for 80 hours. The formation of boron and perrhenic acids are also reported after ReB2 powder was exposed to the moist air environment for a twelve month period of time. Hexagonal osmium diboride (h-OsB2), a theoretically predicted high-pressure phase, has been synthesized for the first time by a mechanochemical method, i.e., high energy ball milling. X-ray diffraction (XRD) indicated the formation of h-OsB2 after 2.5 hours of milling, and the reaction reaches equilibrium after 18 hours of milling. The lattice parameters of the h-OsB2 are a=2.916Å and c=7.376 Å, with a P63/mmc space group. Transmission electron microscopy confirmed the appearance of the h-OsB2 phase. The thermal stability of h-OsB2 powder was studied by heating under argon up to 876 °C and cooling in vacuo down to -225 °C. The oxidation mechanism of h-OsB2 has also been proposed. The hexagonal phase partially converted to the orthorhombic phase (20 wt.%) after spark plasma sintering of h-OsB2 at 1500°C and 50MPa for 5 minutes. Hardness and Young*s modulus of the h-OsB2 were measured to be 31 ± 9 GPa and 574 ± 112 GPa, respectively by nanoindentation method. Prior to this research a number of compounds have been prepared in Ir-B system with lower than 2 boron stoichiometry, and no IrB2 phases have been synthesized experimentally. In this dissertation, three new iridium boride phases, ReB2-type IrB2, AlB2-type IrB2 and IrB have been synthesized with a similar mechanochemical method. The formation of these three phases has been confirmed by both X-ray diffraction (XRD) and transmission electron microscope (TEM) after 30 hours of ball milling and 48 hours of annealing. The IrB2 phases have hexagonal crystal structures and the new IrB phase has an orthorhombic crystal structure. The segregation of iridium from iridium borides* lattices has also been studied by high resolution TEM.

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Biogenic monoamines such as dopamine play an important role as major neurotransmitters. Simultaneous determination of the concentration changes is thus crucial to understand brain function. Additionally, quantification of pharmaceutically active compounds (PhACs) and their metabolites in biological fluids is an important issue for forensic tests, clinical toxicology and pharmaceutical analysis. We have developed two postcolumn luminescence detection methods coupled to a 2-dimensional-solid phase extraction (2D-SPE) system. The postcolumn reaction methods used in this study are the redox-dependent photoluminescence-following electron-transfer (PFET) and Fenton-based chemiluminescence techniques, for the determination of certain neurotransmitter and nonsteroidal anti-inflammatory drugs (NSAIDs). A stable [Os(tmphen)3]3+ (tmphen = 3,4,7,8-tetramethyl-1,10-phenanthroline) reagent was prepared in neutral aqueous solution by oxidation of [Os(tmphen)3]2+ with lead(IV) oxide. [Os(tmphen)3]2+ and [Os(tmphen)3]3+ are characterized by absorption spectroscopy. [Os(tmphen)3]3+ stability is compared with [Ru(tmphen)3]3+ in the same pH 7 environment. The properties of Os(III) and Ru(III) complexes were investigated for use as the oxidant in a PFET system. Studies of photophysical and electrochemical properties, the stability of the Os(III) and Ru(III) complexes, and analytical application in PFET detection of oxidizable analytes are presented. The spectroscopic properties of the complexes were not very advantageous, but careful control of the detection system and reaction conditions enabled sensitive detection of the analytes. The method was fully validated and the optimized system was capable of detecting dopamine and acetaminophen at about 30.2 µg L-1 and 33.5 µg L-1, respectively. The limit of detection (LOD) was 1.5 µg L-1 for acetaminophen and 4.3 µg L-1 for dopamine. The accuracy and precision were within bioanalytical method validation limits (90.9 to 101.5 % and RSD < 12.0 %, respectively). Typical analysis time was less than 15 minutes. Two Fenton-based flow-injection chemiluminescence (CL) methods were developed and validated for the determination of naproxen. Under the optimal experimental conditions the proposed methods exhibited advantages in a larger linear range from 2,760 ng mL-1 to 207,000 ng mL-1 for the first CL method and 41.4 ng mL-1 to 700.0 ng mL-1 for the second CL method. The LOD was 13.8 ng mL-1 for naproxen. The CL mechanisms for the system, H2O2-FeIIEDTA-naproxen was further studied by batch experiments, chemiluminescence spectroscopy, fluorometry, high pressure liquid chromatography (HPLC) and Fourier transform infrared spectroscopy (FTIR). The effects of various interferences commonly found in biological and wastewater systems on the chemiluminescence intensity were also investigated. We used these methods to determine NSAIDs in commercial pharmaceutical formulations. Another application of these method was for detecting NSAIDs in biological samples. A 2x-1-Dimensional Solid Phase Extraction (2x-1D SPE) method was developed for determination of acetaminophen and naproxen in urine. This method uses both the methanol concentration and the pH advantageously to preferentially isolate analytes of interest from complex sample matrix. These methods were fully validated and had sufficient sensitivity (limit of quantification: acetaminophen; 40.41 mg L-1 - 360.0 mg L-1 and naproxen; 23.03 mg L-1 - 214.8 mg L-1) for biological matrices and applications. / Chemistry

This thesis describes the study of a series of multimetallic supramolecules containing varied metals and ligands, synthesized by a building block method and characterized by mass spectrometry, electronic absorption spectroscopy, and electrochemistry. Incorporating different functional units into complex systems allowed these multimetallic supramolecules to perform various light activated tasks including DNA cleavage and hydrogen generation from water. The complex [({(bpy)₂Os(dpp)}₂Ru)₂(dpq)](PF₆)₁₂ and [{(bpy)₂M(dpp)}₂Ru(BL)PtCl₂](PF₆)₆ were synthesized (M = Os^II or Ru^II; BL = dpp or dpq; bpy = 2,2^'-bipyridine, dpp = 2,3-bis(2-pyridyl)pyrazine, dpq = 2,3-bis(2-pyridyl)quinoxaline). The building blocks displayed varied electrochemical properties upon complexation. The bridging ligands dpp and dpq display their reduction potentials shifted to less negative values when they changed from monochelating to bischelating. The electronic absorption spectra of the multimetallic systems displayed transitions of each contributing chromophore, with overlapping metal to ligand charge transfer (MLCT) transitions in visible region of spectrum. Spectroelectrochemistry revealed the nature of MLCTs and helped to identify fingerprint features of complex supramolecules. Photophysical measurements include emission spectroscopy with quantum yield measurements and emission lifetime measurements. Photophysical data provided detailed information to aid in developing an understanding of excited state properties of these complexes. Supported by the electrochemical data and spectroelectrochemistry, the hexametallic complex was suggested to have a HOMO localizing in the peripheral Os and a LUMO localizing in the central dpq, separating by a Ru energy barrier. This research systematically investigated photophysical properties of some building blocks and the mixed-metal, mixed-ligand supramolecules constructed by a variety of building blocks coupling light absorbing subunits to a reactive Pt metal center. Preliminary studies suggested [{(bpy)₂Ru(dpp)}₂Ru(dpq)PtCl₂](PF₆)₆ was a photocatalyst for H2 production from water in the presence of a sacrificial electron donor. The complex [{(bpy)₂Ru(dpp)}₂Ru(dpq)PtCl₂](PF₆)₆ had been studied for its catalytic ability in generating hydrogen and was found to have 34 product turnovers after 3 h photolysis. Photolysis and gel electrophoresis revealed that the tetrametallic complexes were able to bind to and then photocleave DNA through an oxygen mediated mechanism. The independence of ionic strength variation when [{(bpy)₂Ru(dpp)}₂Ru(dpp)PtCl₂](PF₆)₆ interacted with DNA, suggested the covalent interaction nature of the complex. These results suggest future work on understanding the excited state properties of supramolecular complexes is suggested. The designs of future photocatalysts for hydrogen production from water and anticancer photodynamic therapy drugs are also proposed. / Ph. D.

Tesis por compendio / La presente tesis doctoral titulada “Complejos metálicos de transición para la detección cromo-fluorogénica de monóxido de carbono en aplicaciones medioambientales y biomédicas” se basa en la utilización de los principios de la Química de la Coordinación para el diseño y desarrollo de nuevos compuestos químicos capaces de detectar monóxido de carbono en aire mediante cambios de color y/o de fluorescencia. La primera familia de sondas colorimétricas que se presenta en el capítulo 3 está basada en unos complejos dinucleares de rodio hexacoordinados con ligandos trifenilfosfina y distintos ácidos carboxílicos. Primeramente, se expone el trabajo desarrollado con el complejo A de fórmula [Rh2(C6H4PPh2)2(O2CCH3)2]·(HO2CCH3)2 capaz de detectar selectivamente y con alta sensibilidad CO tanto en disolución como en aire. En presencia de CO se produce un cambio de color de morado a amarillo, debido a la coordinación del CO en las posiciones axiales del complejo. A continuación se amplia el trabajo con una colección de cinco complejos de rodio (II) B-F adsorbidos en gel de sílice y se estudia su uso como sondas para la detección de CO en aire mediante cambios de color visibles a simple vista. En tercer lugar, se procede a depositar los complejos en papel de celulosa para facilitar su aplicación en la detección práctica de CO. Se elige el complejo D [Rh2[(C6H4)P(C6H5)2]2(O2CCF3)2]· (CF3CO2H)2 como el más sensible a CO en este nuevo soporte y se inserta dentro de un sistema opto-electrónico capaz de cuantificar el CO presente en el aire; mediante la transducción del cambio de color del complejo en una señal eléctrica, y esta señal en un valor de concentración de CO determinado. Finalmente, teniendo en cuenta el papel del CO como agente terapéutico y aprovechando que la coordinación de CO en los complejos de rodio presentados es reversible; se seleccionan los dos complejos dicarbonílicos con cinéticas de liberación de CO en disolución más lentas A·(CO)2 y F·(CO)2 de fórmulas [Rh2[(C6H4)P(C6H5)2]2(O2CCH3)2]·(CO)2 y [Rh2[(m-CH3C6H3)P(m- CH3C6H4)2]2(O2CCH3)2]·(CO)2) como posibles moléculas liberadoras de CO (CO-RMs) a utilizar en estudios sobre inhibición de agentes indicadores de inflamación celular (ver capítulo 4). En la segunda parte de esta tesis doctoral se ha preparado una colección de vinil complejos de rutenio y de osmio G-K funcionalizados con grupos dadores de electrones (pireno, tolueno y benceno) y con un grupo aceptor de electrones (2,1,3-benzotiadiazol (BTD)). Estos complejos son coloreados, ya que presentan una banda de transferencia de carga. En presencia de CO se produce el desplazamiento del BTD con el consiguiente cambio de color (ver capítulo 5). En primer lugar, para posibilitar la detección fluorogénica de CO, se ha preparado el complejo G mediante el anclaje del fluoróforo pirenilvinilo como ligando dador de electrones del complejo de rutenio (II). En presencia de CO y mediante el desplazamiento del BTD se consigue un aumento de fluorescencia observable incluso a simple vista. Para concluir la segunda parte de la tesis se han sintetizado cuatro complejos más, H-K, dos de rutenio y dos de osmio; que han sido utilizados también para detectar CO en aire. Para la preparación de estas sondas también se ha utilizado gel de sílice como soporte. En presencia de CO se produce el desplazamiento del BTD (colorante) y los complejos metálicos cambian de color, permitiendo así la semicuantificación del CO en los rangos de concentración en que el CO es altamente tóxico incluso para exposiciones cortas. / Moragues Pons, ME. (2014). Transition metal complexes for chromo-fluorogenic detection of carbon monoxide in environmental and biomedical applications [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48453 / Premios Extraordinarios de tesis doctorales / Compendio