Les processus d'altération des verres basaltiques constituant le pourtour vitrifié des laves en coussins influencent les grands cycles géochimiques terrestres puisqu'ils contribuent à l'évolution de la composition des océans et de la croûte océanique et sont des acteurs de la dynamique du climat. Au regard de la diversité des communautés bactériennes peuplant ces aquifères, il est admis que l'altération de ces roches est un processus biologiquement catalysé. Toutefois, peu d'études ont cherché à quantifier cette contribution bactérienne, au regard de la diversité et de la complexité des interactions possibles. Ce travail vise à expliciter et quantifier l'influence des ligands bactériens et notamment des sidérophores sur les processus d'altération, en décomposant les systèmes naturels en situations expérimentales simplifiées. Dans cette optique, trois verres basaltiques modèles, porteurs ou non de Fe(II)/Fe(III), ont été synthétisés. Des expériences de dissolution de ces verres, à pH neutre et à 25 C, ont été réalisées (1) en conditions abiotiques (solutions de sidérophores purs, milieu de culture stérile), (2) en présence de la souche Pseudomonas aeruginosa mais en confinant le verre dans des membranes de dialyse, (3) au contact direct de la souche. En parallèle de ces expériences, des analyses des solides ont été menées et un protocole d'analyse verticale de la composition d'un verre altéré par LA-ICP-MS a notamment été mis au point. L'analyse des solutions/milieux d'altération par ICP-OES a permis d'évaluer les cinétiques et la stoechiométrie de la dissolution. Les expériences en conditions abiotiques révèlent qu'en présence de sidérophores, l'extraction préférentielle du fer structural (respectivement de l'aluminium pour un verre sans fer) via des réactions de complexation en surface des verres, est le moteur de la dissolution du réseau vitreux. Réciproquement, dans les cultures bactériennes, la production de sidérophore par Pseudomonas aeruginosa est déclenchée lorsque (1) aucun contact direct verre/bactérie n'est possible, (2) le fer est absent de l'environnement cellulaire, (3) certains métaux toxiques ou pouvant nuire aux métabolismes bactériens (typiquement l'aluminium) sont néanmoins présents et (4) le fer n'est disponible que sous sa forme réduite. Outre l'accélération de l'hydrolyse du réseau vitreux en présence de sidérophores, l'influence positive de la constitution d'un biofilm en surface des verres sur la dissolution de ces derniers a également été observée. Ces résultats mettent en évidence la forte affinité de la souche pour les verres basaltiques ainsi que le rôle central du fer, oxydé ou réduit, dans les mécanismes dégagés / The alteration of ocean basalts partly controls the composition of seawater and of the oceanic crust which in turn influences the Earth's mantle geochemistry and may also have a significant impact on Earth's climate over a geological timescale. Regarding the existence of an extensive subsurface biosphere within the basaltic basement of the uppermost oceanic crust, the weathering of basaltic glass is now considered as a bacterial mediated process. However, the diversity and complexity of the involved mechanisms interfere with the quantification of the impact of microorganisms. This work was conducted to determine and quantify the influence of organic ligands produced by the cells, and notably siderophores on the alteration processes. Simplified experimental systems were designed to gradually mimic natural environments. Fe(III)-, Fe(II)-bearing, and Fe-free synthetic basaltic glasses were prepared and submitted to dissolution experiments at 25 C and near neutral pH conditions in (1) abiotic conditions (pure siderophore solutions and sterile bacterial medium), (2) in the presence of the bacterial strain Pseudomonas aeruginosa but isolated from the bacterial suspension by dialysis bags and (3) directly in contact with the strain. In parallel, solid analysis were conducted and LA-ICP-MS analysis protocols were notably developped. Dissolution kinetics and stoichiometry were determined by measuring elemental concentrations in solutions by ICP-OES. In abiotic conditions, the siderophore-promoted dissolution of the glass network appears to be driven by the complexation and the preferential extraction of iron (respectively aluminium for no-Fe bearing glasses). Reciprocally, in biotic systems, the siderophore production is stimulated when (1) no direct interaction between the glass and bacteria is possible, (2) the system is Fe-defficient, (3) toxic metals (e.g. aluminium) are nevertheless present and (4) iron is only available under its reduced form. In addition to the promotion of hydrolysis rates of the silicate network by siderophores, biofilms forming at the glass surfaces were shown to have a positive impact on the dissolution kinetics. These results show the specific affinity of the strain for basaltic glasses and the central role of iron under its oxidized or reduced form in the dissolution mechanisms

[pt] Neste estudo, foram determinadas as concentrações de Cu, Fe, Zn, Cd, Pb e Ni no tecido muscular e fígado de três espécies de peixe (Mugil liza – tainha, iliófago; Micropogonias furnieri – corvina, carnívoro e Trichiurus lepturus – peixe espada, piscívoro), no tecido muscular de uma espécie de camarão (Farfantepenaeus brasiliensis – camarão rosa, onívoro) e também, no tecido muscular, vísceras e ovos de uma espécie de siri (Callinectes spp. – siri azul, onívoro) coletados na Baía da Ilha Grande. As concentrações dos elementos foram determinadas por espectrometria de massa com fonte de plasma indutivamente acoplado (ICP-MS). A metodologia analítica apresentou ótima exatidão e precisão quando comparada com amostras certificadas (DORM-3 e TORT-2). As concentrações de Cu, Zn, Cd, Ni e Pb apresentaram-se abaixo dos limites estabelecidos pela legislação brasileira e americana para a ingestão humana de pescado. As maiores concentrações de Cu, Fe, Zn, Cd, Ni e Pb foram observadas nos crustáceos. Nos peixes, as maiores concentrações dos elementos analisados foram observadas no fígado, e no siri, nas vísceras. O sexo dos indivíduos não influenciou no acúmulo dos elementos para os peixes, porém influenciou na acumulação de Cu, Fe, Zn, Cd e Ni no siri. Nos peixes, a maioria dos elementos não apresentou correlação com o comprimento dos indivíduos, com exceção do Pb, que apresentou correlação negativa na tainha e na corvina, e do cádmio, que apresentou correlação negativa na corvina. Nos crustáceos, alguns elementos também apresentaram correlação negativa com estes parâmetros, indicando que, tanto nos peixes quanto nos crustáceos, estes elementos não foram acumulados ao longo do crescimento. As concentrações encontradas foram diferentes nas estações seca e chuvosa. / [en] In this study the concentrations of Cu, Fe, Zn, Cd, Pb and Ni were determined in muscle tissue and liver of three fish species (Mugil liza – mullet, illiophagus; Micropogonias furnieri – croaker, carnivorous, and Trichiurus lepturus – cutlassfish, piscivore), in the muscle tissue of a shrimp species (Farfantepenaeus brasiliensis – pink shrimp, omnivore) and in muscle tissue, entrails and eggs of a soft crab (Callinectes spp. – siri azul, omnivore) sampled from the Ilha Grande Bay. The concentrations of the elements were determined by inductively coupled plasma mass spectrometry (ICP-MS). The methodology showed good accuracy and precision when tested with certified reference materials (DORM-3 and TORT-2). The concentrations of Cu, Zn, Cd, Ni and Pb were below the limits established by the Brazilian and American legislation for human ingestion of fish products. The highest concentrations of Cu, Fe, Zn, Pb, Cd and Ni were found in the crustacean samples. In fish, the highest concentrations were observed in the liver and, in the soft crab samples, in the entrails. Sex did not influence element accumulation in fish, but influenced the accumulation of Cu, Fe, Zn, Cd and Ni in the soft crab. In fish, most elements did not show any correlation to length and weight, with the exception of Pb, which showed negative correlations in mullet and croaker and Cd which showed negative correlations in croaker. In crustaceans, some elements also presented negative correlations to these parameters, indicating that both in fish and crustaceans these elements are not accumulated during organism growth. The concentrations of the elements analyzed were statistically different in the dry and rainy seasons.

[pt] Este trabalho teve como proposta o desenvolvimento de uma metodologia para determinação de Se e As em amostras de óleo cru, através da técnica de espectrometria de massas com plasma indutivamente acoplado e célula de reação dinâmica. A vantagem do método proposto está na etapa de preparação das amostras, na qual se emprega a diluição direta em solvente orgânico xileno, eliminando tratamentos prévios, como a digestão. Com isso, a análise fica mais fácil e rápida. Em seguida, foi feita a otimização da célula de reação, empregando o metano como gás de reação. Realizou-se um estudo univariado dos parâmetros de operação da célula, como a vazão do gás de reação e o parâmetro de rejeição q, a fim de reduzir as interferências sobre os isótopos 80Se e 75As. Padrões orgânicos de selênio e arsênio diluídos em xileno foram empregados para este estudo e observou-se uma eficiência de redução de interferentes no 80Se de 100%. No caso do 75As, observou-se que o uso da célula não se fez necessário, uma vez que a presença de cloro não foi detectada nas soluções orgânicas. Com os parâmetros da célula apropriados, as condições experimentais foram otimizadas a partir de um estudo univariado. As condições utilizadas foram potência de 1350 W, vazão de argônio de nebulização em 0,40 L min(-1) e argônio auxiliar de 0,7 L min(-1). A técnica de adição padrão foi empregada para calibração e os limites de detecção (LODs) instrumentais foram de 0,23 (80Se) e 0,018 (75As) ug kg(-1). A fim de avaliar a exatidão da metodologia aplicada, analisou-se o material certificado NIST 1634c e obtiveram-se boas recuperações, de 97,3% e 93,5%, respectivamente, para Se e As. Os intervalos de confiança mostraram que os resultados encontrados para o MRC estão dentro da faixa certificada para os dois elementos. A metodologia foi aplicada para análise de nove amostras de petróleo brasileiro. Utilizou-se a técnica de geração de hidretos como forma de comparação com o método proposto. As amostras foram decompostas em bloco de aquecimento e pré-reduzidas com HCl 6 mol L(-1). Curvas de calibração externa foram utilizadas na determinação dos isótopos 78Se, 82Se e 75As, resultando em LODs de aproximadamente 0,04 ug L (-1) para a determinação de Se total e 0,0022 ug L(-1) para o As. A exatidão foi avaliada com o NIST 1634c, obtendo boas recuperações de 99,7% (78Se); 99,2% (82Se) e 100,4% (75As). Um tratamento estatístico foi aplicado e mostrou que os resultados obtidos para selênio em todas as nove amostras de óleo foram concordantes pelas duas técnicas empregadas, a um nível de confiança de 95%. No entanto, somente quatro óleos tiveram resultados de arsênio concordantes pelas duas técnicas. Os outros óleos apresentaram concentrações inferiores para a metodologia de geração de hidretos em relação às concentrações encontradas pela técnica de introdução direta, possivelmente devido a perdas ocorridas durante as etapas de digestão e evaporação. Isto mostra a importância da metodologia desenvolvida para avaliações exatas das concentrações totais de Se e As em petróleos e derivados. / [en] A methodology was developed for the determination of Se and As in samples of crude oil using inductively coupled plasma mass spectrometry with a dynamic reaction cell. The proposed technique of sample preparation showed the advantage of direct dilution in organic solvent, xylene, eliminating previous treatments, such as digestion of samples, and resulting in an easy and fast analytical method. The cell reaction was optimized using methane as the reaction gas. An univariate study was performed with the operating parameters of the cell, the reaction gas flow rate and rejection parameter q, in order to reduce the interferences on isotopes 80Se and 75As. Organic standards for arsenic and selenium dissolved in xylene were used for this study and a 100% reduction efficiency was observed for 80Se. In the case of 75As, it was observed that the use of the cell was not necessary, since the presence of chlorine was not detected in the organic solutions. With the appropriate experimental conditions for the cell, the experimental conditions were optimized by an univariate study. The conditions used were 1350 W (RF power), 0.40 L min(-1) (Ar-nebulizer flow rate) and 0.7 L min(-1) (Ar-auxiliary flow rate). The standard addition technique was employed for calibration and the instrumental limits of detection (LOD) were 0.23 (80Se) and 0.018 (75As) mg kg(-1). In order to validate the methodology, the residual fuel oil certified reference material (CRM) NIST 1634c was analyzed and recoveries of 97% and 94% were obtained for Se and As, respectively. The confidence intervals showed that the results were within the CRM´s range for both elements. The methodology was applied to the analysis of nine samples of Brazilian crude oil. A hydride generation methodology was used for comparison with the proposed methodology. The samples were decomposed in a heated block and pre-reduced with HCl 6 mol L(-1). External calibration curves were used to determine 78Se, 82Se e 75As, resulting in LODs of approximately 0.04 ug L(-1) to determine total Se and 0.0022 ug L (-1) for the As. The accuracy was evaluated also with NIST 1634c, obtaining recoveries of 99.7% (78Se); 99.2% (82Se) e 100.4% (75As). The Snedecor statistical test showed that the results for selenium in all nine oil samples were in agreement by the two techniques at a 95% confidence level. However, only four oils had concordant results for arsenic by both techniques, the other oils showed lower concentrations for the hydride generation methodology in relation to concentrations found by the direct introduction technique. These results were attributed to losses during the digestion and evaporation steps, and this observation showed the importance of the purposed methodology for accurate assessments of total concentrations of Se and As in crude oil and refined products.

En complément des analyses de concentration et de spéciation, les analyses des isotopes stables du mercure par MC-ICP-MS sont à l’heure actuelle un outil majeur pour tracer les sources et les transformations de cet élément dans l’environnement. Cette thèse propose le développement de techniques de pré-concentrations couplées au MC-ICP-MS afin de réaliser des analyses isotopiques de mercure au niveau du ng.L-1 (Ultra-trace). La stratégie de pré-concentration en ligne va générer des signaux transitoires courts dont la gestion en MC-ICP-MS représente un réel défi. Afin d’y répondre, une méthode de traitement de données adaptée à ce type de signaux ainsi qu’une méthode de correction du phénomène de dérive isotopique ont été développées. Dans le but de déterminer des compositions isotopiques en mercure total à de faibles concentrations, le couplage d’une génération de vapeur froide, d’une double amalgamation sur or (CVG-DGA) et d’un MC-ICP-MS a été développé. Cette nouvelle technique donne des précisions externes de l’ordre de 0.20 à 0.30‰ (2SD) pour des concentrations en mercure de 5 ng.L-1 en solution. Cette thèse présente également une nouvelle méthode d’analyse isotopique par espèce chimique (CSIA) réalisée à l’aide du couplage d’une chromatographie en phase gazeuse équipée d’un injecteur PTV (GC-PTV) et d’un MC-ICP-MS. Celle-ci permet l’analyse isotopique de plusieurs espèces chimiques avec des précisions externes de l’ordre de 0.30 à 0.40‰ (2SD) pour des concentrations en mercure de 150 ng.L-1 dans des échantillons biologiques.Les développements analytiques de cette thèse ont permis de mesurer directement et de façon automatisée des compositions isotopiques de mercure à des niveaux d’ultra-trace (jusqu’à 5ng.L-1) tout en conservant des précisions compatibles avec les principales questions environnementales posées. Ceci va permettre d’analyser des compartiments environnementaux contenant de faibles quantités de mercure (eaux naturelles, planctons,…) et ouvre ainsi de nouvelles perspectives pour une meilleure compréhension du cycle bio-géochimique du mercure. / In addition to the quantitative and speciation analysis, the analysis of mercury stable isotopes by MC-ICP-MS are now a tool of choice to track sources and pathways of this element in the environment. This PhD thesis presents the development of hyphenation between pre-concentration techniques and MC-ICP-MS to measure isotopic composition of mercury at ng.L-1 levels (Ultra-trace). The on-line pre-concentration strategy will create short transient signals which represent a real challenge for MC-ICP-MS. In order to solve it, a data treatment strategy for this particular signals and a correction method for the isotopic drift were developed.The hyphenation between a cold vapor generation, a dual gold amalgamation (CVG-DGA) and a MC-ICP-MS was developed to determine total mercury isotopic composition. This new technique gives external precisions ranging from 0.20 to 0.30‰ (2SD) for Hg concentration in solution of 5 ng.L-1. This PhD thesis also reports a new method to perform mercury compound specific isotopic analysis (CSIA) using a gas chromatography fitted with a PTV injector (GC-PTV) coupled to the MC-ICP-MS. This allows the isotopic analysis of several species with external precisions ranging from 0.30 to 0.40‰ (2SD) for mercury concentration down to 150 ng.L-1 in biological samples.The analytical developments proposed in this PhD thesis allows to automatically and directly measure mercury isotopic compositions at ultra-trace levels (down to 5 ng.L-1) while keeping precision compatible with main environmental questions. This will allow to analyze environmental compartment containing very low amount of mercury (natural waters, planktons,…) and then open new perspectives for a better understanding of the bio-geochimical cycle of mercury.

[pt] Este trabalho foi desenvolvido a partir do projeto aprovado no Edital Meninas e Mulheres nas Ciências Exatas e da Terra, Engenharias e Computação, da Fundação Carlos Chagas de Apoio à Pesquisa do Estado do Rio de Janeiro (FAPERJ), que teve como objetivo promover o interesse de jovens mulheres para a pesquisa científica com projetos desenvolvidos em escolas públicas do estado do Rio de Janeiro. O cigarro e o anticoncepcional são temas comuns entre os jovens por isso, foram escolhidos como objeto de estudo deste trabalho. O cigarro apresenta substâncias tóxicas que causam doenças crônico-degenerativas. Por sua vez, os anticoncepcionais atuam nos hormônios que podem desregular os ciclos de elementos essenciais no organismo. Assim, o objetivo desse trabalho foi avaliar, empregando ferramentas estatísticas, a composição elementar de cabelos de pessoas fumantes e mulheres que tomam anticoncepcionais, comparadas à de pessoas que não usam essas substâncias. As amostras de cabelo foram coletadas de voluntários das 5 escolas e analisadas seguindo a metodologia estabelecida no Labspectro, Laboratório de Espectrometria Atômica, da PUC-Rio, para o mineralograma capilar, que consiste na determinação multielementar por espectrometria de massas com plasma indutivamente acoplado (ICP-MS). Utilizando o teste Kruskal-Wallis, foram identificadas concentrações significativamente menores (p menor que 0,05) de Na e Mg no grupo dos fumantes, e de Cu nos cabelos de mulheres que tomam anticoncepcionais. Na análise de correlação de Spearman, foram identificadas maiores correlações entre Cu/Pb e Cr/As, para o grupo de fumantes, enquanto correlações entre Co/Se/I para mulheres que tomam anticoncepcional. As análises de componentes principais confirmaram a análise de Spearman, mas não foi possível estabelecer separação dos grupos estudados. Dessa forma, o fumo e o uso de anticoncepcionais interferem nos resultados do mineralograma capilar, sendo necessários mais estudos para avaliar as implicações dessas diferenças à saúde. / [en] This work was developed from the project approved in the Call for Proposals for Girls and Women in the Exact and Earth Sciences, Engineering and Computing,from the Carlos Chagas Foundation for Research Support in the State of Rio de Janeiro (FAPERJ), which aimed to promote young women s interest in scientific research with projects developed in public schools in the state of Rio de Janeiro.Cigarettes and contraceptives are common themes among young people, who oftenend up using them prematurely and were therefore chosen as the subject of this study. Cigarettes contain toxic substances that cause chronic degenerative diseases due to nicotine addiction. In turn, contraceptives act on hormones that can disruptthe cycles of essential elements in the body. The aim of this study was therefore to evaluate, using statistical tools, the elemental composition of the hair of smokers and women who take contraceptives, compared to that of people who do not use these substances. The hair samples were collected from volunteers from the schools participating in the project and analyzed following the methodology established at Labspectro, the Atomic Spectrometry Laboratory at PUC-Rio, for the capillary mineralogram, which consists of multi-elemental determination by inductively coupled plasma mass spectrometry (ICP-MS). Using the Kruskal Wallis test, significantly lower concentrations (p less than 0.05) of Na and Mg were identified in the group of smokers, and of Cu in the hair of women taking contraceptives. In Spearman s correlation analysis, higher correlations were identified for some toxic elements, such as Cu/Pb and Cr/As, for the group of smokers, while correlations involving Co, Se and I were obtained for women taking contraceptives. Principal component analysis confirmed the results of Spearman s analysis, but it was not possible to establish a clear and distinct separation of the groups studied. Thus, it can be concluded that smoking and the use of contraceptives interfere with the results of the hair mineralogram, and further studies are needed to assess the health implications of these differences.

[pt] O cabelo humano pode ser utilizado para verificação do estado de saúde do indivíduo. Para tal, são quantificados os elementos essenciais e tóxicos por espectrometria de massa com plasma indutivamente acoplado (ICP-MS) em uma amostra de cabelo e os resultados comparados com valores de referência. Com essas informações, o médico pode avaliar se há algum tipo de desequilíbrio mineral ou intoxicação. O cachorro pode ser um bom bioindicador de exposição ambiental já que ele compartilha do mesmo ambiente que o ser humano, bebe a mesma água e, em alguns casos, come o mesmo alimento. O pelo canino tem quase a mesma composição que o cabelo humano, variando somente sua taxa de crescimento. Neste trabalho, desenvolveu-se um método para determinação dos elementos essenciais e tóxicos em pelo canino por ICP-MS. Para tanto, foram otimizados os procedimentos de lavagem do pelo, a massa mínima de amostra para garantir a homogeneidade, e o procedimento de decomposição. Verificou-se que as melhores condições foram: lavagem com água, massa de amostra entre 100 e 300 mg, e decomposição com HNO3 e H2O2. Os resultados foram comparados com os obtidos por espectrometria de emissão óptica com plasma indutivamente acoplado (ICP OES), técnica já bem estabelecida. O método mostrou-se versátil devido a sua capacidade analítica multielementar, com sensibilidade e acurácia adequadas (os valores de recuperação em relação aos valores certificados ficaram entre 80 e 120 por cento) para a amostra de referência. O trabalho apontou a necessidade de novos estudos para estabelecer valores de referência do mineralograma para cães, assim como avaliar a diferença entre raças. / [en] Human hair can be used to check the individual s health status. For this purpose, the essential and toxic elements are quantified in the inductively coupled plasma mass spectrometer (ICP-MS) and compared to a reference table to identify any possible mineral imbalance or intoxication. The dog can be a good bioindicator of environmental exposure since it shares the same environment as the human being, drink the same water and in some cases eat the same food. Canine fur has almost the same composition as human hair, varying only its growth. In this work, a method was developed to determine the essential and toxic elements in canine by ICP-MS. For that, the hair washing procedures were optimized, the minimal sample mass to ensure homogeneity, and the decomposition procedure. It was found that the best conditions were: washing with water, sample mass between 150 and 300 mg, and decomposition with HNO3 and H2O2. The results were compared with those obtained by optical emission spectrometry with inductively coupled plasma (ICP OES), a well-established technique. The method proved to be versatile due to its multi-element analytical capacity, with adequate sensitivity and accuracy (the recovery values in relation to the certified values were between 80 and 120 per cent) for the reference sample. The work indicated the necessity for further studies to established reference values of the mineralogram for dogs, as well evaluated the difference between races.

Spårelementkoncentrationen i polerprov av kvarts från Bergby-Hamrångeområdet har analyserats genom LA-ICP-MS. Spårelementkoncentrationen i kvarts från enkla pegmatiter och LCT-pegmatiter har analyserats och registrerat koncentrationer för 23 olika isotoper. Koncentrationerna för Al27, Ti49, Li7, Rb85 och B11 hos 84 provpunkter har plottats i tvåaxliga-diagram. Tertiära diagram av Al/10-Li-Ti och Ti-Al/10-Rb*50 har sammanställts och i dessa har fält för LCT-pegmatiter markerats ut baserat på data från Müller m.fl. (2021). I rapporten beskrivs LA-ICP-MS översiktligt för läsare utan tidigare erfarenhet. Syftet med undersökningen var att besvara, 1) Om kvarts i polerproverna från BergbyHamrånge kan klassas som ultraren och 2) Att diskutera spårelementkoncentrationerna och undersöka alternativa användningsområden för kvarts som inte är ultraren. / The trace element concentration in pole samples of quartz from the Bergby-Hamrånge area has been analyzed by LA-ICP-MS. The trace element concentration in quartz from simple pegmatites and LCT-pegmatites has been analyzed and recorded concentrations for 23 different isotopes. Concentrations of Al27, Ti49, Li7, Rb85 and B11 at 84 sample points have been plotted in biaxial graphs. Tertiary diagrams of Al/10-Li-Ti and Ti-Al/10-Rb*50 have been compiled and in these, fields for LCT-pegmatites have been highlighted based on data from Müller et al. (2021). The report demands no previous knowledge of LA-ICP-MS to be understood. The purpose of the investigation was to answer, 1) If quartz samples from Bergby-Hamrånge can be classified as ultrapure quartz and 2) To discuss the trace element concentrations and investigate alternative uses for quartz that is not ultrapure.

La caractérisation de nanomatériaux dans des matrices alimentaires et animales suscite un intérêt scientifique important afin d’évaluer les risques potentiels de l’exposition liés à l’utilisation grandissante des nanomatériaux par plusieurs industries, y compris un certain nombre d’applications agroalimentaires. Un facteur limitant à l’étude et la réglementation des nanomatériaux dans des matrices complexes telle que la nourriture est l’absence de méthodes standardisées pour l’extraction et l’analyse de nanoparticules, tout en évitant l’altération de certaines caractéristiques physicochimiques des nanoparticules. Les travaux présentés dans ce mémoire abordent l’optimisation de plusieurs approches de préparation d’échantillon (hydrolyse enzymatique et alcaline) pour l’extraction de nanoparticules d’Ag préalablement équilibrées dans une matrice de boeuf haché mi-maigre. Les nanoparticules extraites ont été analysées par spectrométrie de masse à plasma à couplage inductif en mode particule unique (SP-ICP-MS) permettant la mesure de leur taille et concentration, mais aussi de la concentration en métal dissous, le tout à de très faibles concentrations (de l’ordre du ng/L). La validation de l’analyse par SP-ICP-MS a été réalisée par évaluation de la répétabilité, de la détermination des limites de détection et par une investigation de l’influence du traitement de données sur l’interprétation des résultats. Les pertes de nanoparticules lors de la préparation des échantillons ont été minimisées par l’identification et l’optimisation de paramètres clés tels que la composition du médium d’extraction, l’utilisation d’ultrasons et de la manipulation de l’échantillon après dégradation de la matrice. Les meilleurs recouvrements ont été obtenus par hydrolyse alcaline de la matrice en utilisant de l’hydroxyde de tetramethylammonium (TMAH), mais les échantillons obtenus étaient moins stables et plus susceptibles aux altérations des propriétés physicochimiques des nanoparticules que pour la dégradation par hydrolyse enzymatique utilisant lipase et pancréatine de porc. / The regulation and characterization of nanomaterials in foods and animal matrices are of great interest due to the potential risks associated with their exposure and the increasing number of instances where they are used within the food industry. One factor limiting the scientifically rigorous regulation of nanoparticles in foods is the lack of standardized procedures for the extraction of nanoparticles (NP) from complex matrices, without alteration of their physico-chemical properties. To this end, two sample preparation approaches (enzymatic- and alkaline-based hydrolyses) were tested and optimized in order to extract 40 nm Ag NP, following their equilibration with a fatty ground beef matrix. Extracted NP were characterized using single particle inductively coupled plasma mass spectrometry (SP-ICP-MS), allowing the determination of NP size and concentrations and also dissolved metal concentrations at trace levels. Validation of the SP-ICP-MS analysis was achieved by an evaluation of the repeatability and accuracy and by a determination of the various detection limits. Finally, we also looked into the influence of data treatment on interpretation of the results. NP losses during the sample preparation were minimized by identifying and optimizing key parameters such as the composition of the extraction media, usage of ultrasonication or the handling of the sample after separation from the undigested matter, among other points. The alkaline approach using TMAH (tetramethylammonium hydroxide) was found to have the highest recoveries, however processed samples were found to be less stable and more prone to alteration of the Ag NP physicochemical characteristics than samples processed using an enzymatic digestion based upon pork pancreatin and lipase.

In dieser Arbeit wurde ein Verfahren zur rückführbaren Proteinquantifizierung unter Verwendung von Schwefelisotopenverdünnung mit induktiv gekoppelter Plasmamassenspektrometrie (ID-ICP-MS) entwickelt. Die Methode dient zur zuverlässigen Quantifizierung laborinterner Proteinstandards. Sie eignet sich nur zur Analyse reiner Proteine, deren Stöchiometrie bekannt ist, da die Proteinkonzentration aus dem Schwefelgehalt bestimmt wird. Nicht proteingebundener Schwefel wird durch Membranfiltration von der Proteinfraktion abgetrennt und mit ID-ICP-MS quantifiziert. Der Gesamtschwefelgehalt in der Probe wird ebenfalls mittels ID-ICP-MS quantifiziert und um die Menge an ungebundenem Schwefel korrigiert. Die Optimierung der Probenvorbereitung zeigte, dass ein Aufschluss die Unsicherheit des Ergebnisses verbessert. Für die Methodenentwicklung wurden ein zertifiziertes Rinderserumalbumin-Referenzmaterial (BSA), sowie ein kommerzielles Avidin verwendet. Die Bestimmung der Proteinmassenfraktionen und Unsicherheitsbudgets erfolgte nach Korrektur für den ungebundenen Schwefel (0,4 % für BSA, 30 % für Avidin). Die Methode wurde auf das Tau-Protein angewendet, das ein Biomarker für die sogenannten „Tauopathien“ ist – eine Gruppe neurodegenerativer Krankheiten, die die Alzheimer-Krankheit und die frontotemporale Demenz einschließen. Durch Verwendung eines isotopenangereicherten Spikes, der an das SI-System angebunden ist, wurde die metrologische Rückführbarkeit für den Massenanteil des Tau-Proteins erreicht. Dieser Tau-Standard wurde zur absoluten Quantifizierung toxischer transgener Tau-Spezies in der löslichen Hirnfraktion transgener Mäuse verwendet. Die entwickelte Methode ist für die rückführbare Quantifizierung von Proteinen zur Verwendung als Standards in der biologischen und medizinischen Forschung anwendbar. Sie zeigte eine ähnliche Präzision wie etablierte Verfahren, erforderte jedoch weniger Probenvorbereitung und keine spezies-spezifischen Standards. / In this work, a method for traceable protein quantification using sulphur isotope dilution inductively coupled plasma mass spectrometry (ID-ICP-MS) was developed. The method is intended for the reliable quantification of in-house protein standards. It is only suited for pure protein formulations for proteins of known stoichiometry because the protein concentration is determined from the sulphur content. Non-protein bound sulphur is separated from the protein fraction by membrane filtration and is quantified by ID-ICP-MS. The total sulphur content in the sample is as well quantified by ID-ICP-MS and corrected for the amount of non-protein bound sulphur. Optimisation of the sample preparation showed that digestion improves the uncertainty of the result. For method development, a certified reference material bovine serum albumin (BSA) and a commercial avidin were used. The protein mass fractions with full uncertainty budgets were determined after correction for unbound sulphur. The developed procedure was applied to the tau protein, which is a biomarker fort he so-called „tauopathies“ – a group of neurodegenerative diseases including Alzheimer’s disease and Frontotemporal dementia. By employing an isotopically enriched spike, which is linked to the SI system, full metrological traceability was achieved for the tau mass fraction. The mass fraction of (0.328 ± 0.036) g kg-1 for tau was determined by ID-ICP-MS and confirmed by amino acid analysis. This tau standard was used for the absolute quantification of toxic transgenic tau species in the soluble brain fraction of transgenic mice. The developed method is applicable for the traceable quantification of proteins for use as standards in biological and medical research. The precision of the method was similar to established absolute protein quantification procedures while requiring less sample preparation and no species-specific standards.

Durch die Untersuchungen der vorliegenden Arbeit ist es erfolgreich gelungen die beiden gram-positiven Mikroorganismen Lysinibacillus sphaericus JG-A12 und Lysinibacillus sphaericus JG-B53 unter geregelten und idealen Kultivierungsbedingungen im Bioreaktor in hinreichenden Biomasseausbeuten zu kultivieren. Aus der Biomasse beider Stämme ist es anschließend gelungen, die primären Zellwandkomponenten bestehend aus Membranlipiden, Peptidoglykan mit sekundären Zellwandpolymeren und S-Layer-Proteinen in reiner Form und in guten Ausbeuten zu extrahieren. Diese Zellwandkomponenten wurden dann unter Verwendung von biochemischer und strukturanalytischer Methoden charakterisiert. Dabei ist es erstmals gelungen, die Membranlipide beider genutzter Mikroorganismen in Bezug auf deren Zusammensetzungen der enthalten hydrophoben Fettsäuren und der hydrophilen phosphathaltigen Kopfgruppen zu charakterisieren. Durch die vergleichend durchgeführten Metallbindungsversuche im Batch-Verfahren konnten Bindungspräferenzen intakter Zellen von Lysinibacillus sphaericus JG-A12 und Lysinibacillus sphaericus JG-B53 und deren isolierten Zellwandkomponenten mit den Metallen As, Au, Cd, Eu, Pb, Pd, Pt bzw. U untersucht werden. Dabei konnten sowohl in den Untersuchungen intakter Zellen und der primären Zellwandbestandteile deutlich höhere Metallsorptionsraten und Metallentfernungseffizienzen für Lysinibacillus sphaericus JG-B53 festgestellt werden als dies bei Lysinibacillus sphaericus JG-A12 nachzuweisen war. Dies macht diesen Stamm für potentielle technische Anwendungen als metallselektives biosorptives Material weitaus interessanter. Die Untersuchungen der Einzelkomponenten in Suspension lieferten jedoch nur begrenzt Informationen zur Interaktion der Metalle mit den Schichten wie sie unter natürlichen Bedingungen in der Zelle vorkommen. Daher wurden unter Verwendung der QCM-D erstmals die primären Zellwandkomponenten beider Mikroorganismen (S-Layer und Peptidoglykan) sowie von Referenzlipiden an Grenzflächen erfolgreich im nanoskaligen Bereich abgeschieden und online verfolgt. Dadurch war es möglich vereinfachte Einzelschichtsysteme der gram-positiven bakteriellen Zellwand nachzubilden. In den Untersuchungen konnten stabile Schichten generiert werden, welche vergleichbar zu dem Schichtsystem vitaler Zellen sind. Zusätzlich konnte bei den Abscheidungen der S-Layer-Proteine SlfB und Slp1 der positive Effekt von Polyelektrolytmodifizierungen auf das Rekristallisationsverhalten, die Schichtstabilität und den Bedeckungsgrad auf der technischen Oberfläche aufgezeigt werden. Zur Untersuchung der Metallinteraktion zellulärer Einzelschichtsysteme wurden in dieser Arbeit exemplarisch nach den erfolgreichen Untersuchungen zur Rekristallisation, die S-Layer-Proteine als erste Interaktionsschicht des Gesamtzellsystems mit der QCM-D untersucht. Diese stabilen und intakten Schichten konnten analog zu den Schichtuntersuchungen der reinen biologischen Komponenten und nach den QCM-D Metallinteraktionsstudien mit den S-Layer Strukturen mittels der Rasterkraftmikroskopie (AFM) untersucht und bildgebend dargestellt werden. In weiteren spektroskopischen Untersuchungen (TRLFS) der Zellwandkomponenten konnten die lumineszierenden Eigenschaften von Europium ausgenutzt werden, um das Metallbindungsverhalten der einzelnen Komponenten als auch des Gesamtsystems der Zellen beider Mikroorganismen zu bestimmen. Somit konnte Europium als spektroskopische Sonde eingesetzt werden um Rückschlüsse die Biomolekül-Metallwechselwirkungen zu ermöglichen. Dabei konnten vor allem mit den beiden oberflächennahen Zellschichten Lösung teilweise sehr starke Metall-Biomolekül-Wechselwirkungen beobachtet werden.