[EN] The contamination of water resources with many industrial, agricultural and other chemicals is one of the key environmental problems that humanity is facing nowadays. Despite the fact that they are usually present at very low concentration, they possess a significant risk to aquatic and human life. To address this issue many national and international institutions set different regulations to monitor and control the water quality. Currently, the monitoring of compounds included in official watch lists is conducted by chromatographic and mass spectroscopic methods. These techniques are approved as "gold standards" for analytical quantitation of organic residues in water. Although they are sensitive and reproducible, cannot be used on-site. The need of sampling and centralized laboratory measurements makes not only the overall cost high but lowering the efficiency of the analysis. Therefore, there is an urgent need to develop suitable field methods to facilitate the in situ measurements at a low cost. Biosensors are therefore an alternative technology that can provide sensitive results in a fast and affordable way. This thesis has focused on the development of a biosensor based on immunoassays and compact disk technology, for the multiplex detection of priority water contaminants. As the methods based on the immunorecognition events are challenging in terms of the selectivity and sensitivity, the major part of the thesis was the selection of the immunoreagents, assay form and procedure. For the detection part, gold nanostructures were selected as sensitive tags for signal enhancement. Therefore, different nanoparticles were studied in order to select the optimal size in terms of the signal enhancement, sensitivity and antibody amount used. Also, the assays performances with signal enhancement and without any amplification were evaluated. The best immunoassay was selected for developing the multiplexed assay. Furthermore, an approach to improve the readout sensitivity of microimmunoassays based on used of gold nanoparticles as both capture and detection species was demonstrated. The method is based on the performance of the immunorecognition event in a homogeneous mode and detection part in the heterogeneous format. Finally, representative water samples were analysed to confirm the applicability of the multi-residue assay. The analytical properties have been established for each methodology and the obtained results have been validated by comparison with reference techniques. The investigations carried out in this work, have resulted in new insights in immunoassay technique that could be useful for screening applications. / [ES] La contaminación de aguas superficiales causada por plaguicidas y productos industriales es actualmente, uno de los grandes problemas medioambientales. Aunque estas sustancias están presentes a niveles muy bajos, tienen efectos perjudiciales en el medio en general y especialmente en humanos. Por este motivo, diferentes instituciones han regulado los niveles de contaminantes en áreas de controlar de la calidad de las aguas, creando listas prioritarias de sustancias peligrosas y tóxicas para el medio ambiente. Actualmente, la monitorización de los contaminantes incluidos en las listas oficiales se realiza mediante técnicas cromatográficas y espectrometría. Estos métodos analíticos están aprobados como técnicas de referencia para la determinación de residuos orgánicos presentes en aguas naturales. A pesar de ser técnicas fiables, reproducibles y sensibles, los métodos cromatográficos no están exentos de inconvenientes. Este tipo de metodologías requiere una instrumentación costosa y una laboriosa preparación de muestra, que hacen que el análisis sea en general complejo. Por ello, el desarrollo de métodos analíticos alternativos que faciliten hacer medidas in-situ a bajo coste y con gran capacidad de trabajo es de gran utilidad. En este sentido, las técnicas inmunoquímicas tienen un gran potencial analítico ya que son en general sensibles y selectivas, se pueden utilizar en el lugar de la toma de muestra y tienen capacidad multianalito. Esta tesis se ha centrado en el desarrollo de un sistema biosensor, basado en la tecnología de disco compacto, para la detección multianalito de diversos contaminantes prioritarios en aguas naturales. Las limitaciones más críticas para el desarrollo de un biosensor multianalito mediante métodos inmunoquímicos son los relacionados con su sensibilidad y selectividad. Por lo tanto, una parte importante de la tesis se ha centrado en la selección de inmunoreactivos, formato y optimización de diferentes parámetros claves del ensayo. Una estrategia utilizada para aumentar la sensibilidad de los ensayos ha consistido en marcar la inmunoreacción con nanopartículas de oro. Para ello, se ha estudiado diferentes tipos (esféricas y cilíndricas) de distinto tamaño y se han comparado sus prestaciones analíticas (relación señal ruido, sensibilidad etc.) También, se han desarrollado inmunoensayos cuantitativos sin necesidad de amplificación de la señal. Por otro lado, se ha desarrollado una aproximación que hemos denominado "inmunocaptura" basada en el uso de nanopartículas de oro como especie de captura de analitos en disolución y que actúa como marcador de la inmunointeracción que tiene lugar en la fase sólida. Finalmente, se han analizado muestras de agua naturales dopadas con distintos niveles de los analitos objeto de estudio para evaluar la utilidad de las metodologías desarrolladas como herramienta de screening masivo en el área medioambiental. Los resultados obtenidos han sido comparados con los obtenidos mediante las técnicas de referencia. Las investigaciones realizadas han permitido desarrollar nuevos formatos de ensayo y conocimientos inmunoquímicos aplicados a la tecnología de disco compacto, aportando nuevas herramientas de screening que permiten la determinación simultánea de contaminantes en aguas naturales por debajo de las concentraciones establecidas en la normativa europea de calidad de agua. / [CA] La contaminació d'aigües superficials causada principalment per plaguicides i altres productes industrials és un dels grans problemes mediambientals actuals. Malgrat que aquestes substàncies estan presents en nivells molt baixos, tenen efectes perjudicials en humans i animals. Per aquest motiu, diferents institucions estatals han regulat els nivells de contaminants en àrees de control de la qualitat de l'aigua, creant llistes prioritàries de substàncies perilloses i tòxiques per al medi ambient. Actualment, la monitorització dels contaminants inclosos en les llistes oficials es realitza mitjançant tècniques cromatogràfiques i d'espectroscòpia de masses. Aquests mètodes analítics estan aprovats com a tècniques de referència per a la determinació de residus orgànics presents en aigües naturals. Malgrat ser tècniques fiables, reproduïbles i sensibles, els mètodes cromatogràfics no estan exempts d'inconvenients. Aquest tipus de metodologies requereix una instrumentació costosa i una laboriosa preparació de mostres que fan que l'anàlisi sigui, en general, complex. Per això, el desenvolupament de mètodes analítics alternatius que facilitin la possibilitat de fer mesures in-situ a baix cost i amb gran capacitat analítica és de gran utilitat. En aquest sentit, les tècniques inmunoquímiques tenen un gran potencial analític ja que són, en general, sensibles i selectives, es poden utilitzar en el lloc de presa de la mostra i tenen capacitat multianalit. Aquesta tesi s'ha centrat en el desenvolupament d'un sistema biosensor, basat en la tecnologia de disc compacte, per a la detecció multianalit de diversos contaminants prioritaris en aigües naturals. Les limitacions més crítiques per al desenvolupament d'un biosensor multianalit mitjançant mètodes inmunoquímics són sensibilitat i selectivitat. Per tant, una part important de la tesi es va centrar en la selecció de inmunoreactius, format i optimització de diferents paràmetres clau de l'assaig. La detecció es va dur a terme mitjançant l'ús de nanopartícules d'or com a marcadors de la inmunointeracció i amplificació de la senyal analítica. S'han estudiat diferents estructures d'or (esferes i cilindres) de diferents tamanys, i s'han comparat les seves prestacions analítiques (relació senyal-soroll, sensibilitat, etc.). També s'han desenvolupat immunoassaigs quantitatius sense necessitat d'amplificació del senyal. Per altra banda, s'ha desenvolupat una aproximació que hem denominat "inmunocaptura", basada en l'ús de nanopartícules d'or com a espècie de captura d'analits en dissolució i que actua com a marcador de la inmunointeracció que té lloc en la fase sòlida. Finalment, s'han analitzat mostres d'aigües naturals dopades amb diferents nivells dels analits objecte d'estudi per avaluar la utilitat de les metodologies desenvolupades com a eina de "screening" massiu en l'àrea mediambiental. Els resultats obtinguts han sigut avaluats per comparació amb els obtinguts mitjançant tècniques de referència. Les investigacions realitzades han permès desenvolupar nous formats d'assaig i coneixements inmunoquímics aplicats a la tecnologia de disc compacte, aportant noves eines de "screening" que permetin la determinació de contaminants en aigües naturals per sota dels límits de concentració establerts per les normes internacionals de la qualitat de l'aigua. / Dobosz, PD. (2017). Screening methodologies for the determination of water contaminant residues by compact disk technology [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/79548 / TESIS

One of the main subjects of this thesis is to design a novel synthetic route to covalently functionalize carbon nanotubes with various molecules in a non-toxic way on both surface and suspension. Functionalized carbon nanotubes are of great interest in the field of molecular electronics, materials science and nano-bio applications because of their remarkable structural, chemical and physical properties. First, the single-walled carbon nanotubes are functionalized with gold nanoparticles by the route, which involves silanization and copper-free click chemistry (SPAAC). We characterize the functionalized nanotubes through XPS, IR and Raman spectroscopic techniques to identify the surface attachment of molecules after each step. We observe a drastic change in homogeneity and functionalization density of single-walled carbon nanotubes with gold nanoparticles concerning solvent through TEM. Employing the same route, biomolecules such as fluorescent dyes and single-stranded DNA molecules are integrated with SWNTs. Fluorescence lifetime analysis of AF647 functionalized SWNTs is reduced compared to free dye due to the fluorescence quenching phenomenon of carbon nanotubes. Functionalized SWNTs are characterized with FLIM, SEM, and Raman for better correlation at the same area of interest. Furthermore, the nanotubes are resolved at the nanoscale level through STORM imaging technique with a limited photon budget. Single-stranded DNA molecules of different lengths are used to investigate the fluorescence quenching as they are distance-dependent. DNA-PAINT is engaged in imaging the functionalized SWNTs with an unlimited photon budget, overcoming STORM's challenge. Lastly, the route is transferred to surface-grown CNTs through the CVD technique, in which both the gold nanoparticles and fluorescent dyes are grafted with nanotubes selectively. CVD is carried out on different substrates Si/SiO2, quartz substrate and quartz coverslip for substrate functionalization. Catalyst deposition plays a significant role in not only the CVD growth but also in the lifetime analysis of the substrate functionalized nanotubes. We observe similar fluorescence quenching of nanotubes in the substrate compared with nanotubes functionalized in suspension. Surface-grown nanotubes in the optically transparent substrate can be resolved through STORM at the nanoscale level. In conclusion, we demonstrate a synthetic design to functionalize SWNTs which provides the possibility to be versatile and non-toxic. Moreover, we show that the nanotubes can be functionalized through this route homogeneously and selectively on both surface and suspension. This work lays the foundation for tailoring SWNTs with not only a wide range of molecules and to study their functional characteristics but also to carry out functionalization on different substrates for various applications.

Tato práce se zabývá syntézou sférických zlatých nanočástic pro biomedicínské aplikace. Zlaté nanočástice byly syntetizovány ekologicky nezávadnými metodami, kterým je věnována i značná část rešerše. Cílem bylo nasyntetizovat stabilní koloidní zlaté nanočástice vhodné pro různé biomedicínké aplikace zejména, pro in vivo a in vitro zobrazovací metody, kterých přehled je take obsažen v teoretické části práce. Také byl proveden test cytotoxicity, jelikož částice mají být použitelné pro in vivo aplikace. Částice byly dale charakterizovány metodami SEM, DLS a UV-VIS.

The diploma thesis deals with synthesis of gold nanoparticles and their surface functionalization suitable for in vitro imaging. In this view there are requirements for optical properties such as stability, monodispersity and no presence of cytotocxicity. In order to fulfill these demands the synthesis by Turkevich method with surface modification by glutathione and polytethylenglycol was performed. Analytical methods such as DLS, SEM and Zeta potential measurement were utilized to characterize the physical and chemical properties of synthesized gold nanoparticles. Finally, MTT assay was performed to evaluate toxicity of gold nanoparticles using HEK 293 cell line.

La manipulation de micro- et nano- particules en solution peut être réalisée grâce aux interactions de ces objets avec des champs électromagnétiques. La lumière ou bien encore les champs électriques continus (DC) ou alternatifs (AC) peuvent être utilisés. Dans le cas d’un champ électrique non uniforme appliqué entre deux électrodes séparées par quelques micromètres, des gradients de champs très intenses et localisés sont ainsi créés. Ces gradients de champ localisés au niveau des électrodes engendrent la création de mouvements de charges composant la solution (effets électro-hydrodynamique). Mais aussi des charges confinées au niveau des particules à l’interface liquide/solide. Les particules en suspension subissent alors une force attractive ou répulsive appelée diélectrophorèse. Cette force est décrite dans la littérature pour des particules isolantes de taille supérieure à 200 nm. Dans cette étude par détection optique (videomicroscopie par champ sombre ou spectroscopie en microfluidique) la gamme de taille de particules est élargie (40-150 nm) pour étudier leurs réponses diélectrophorètique. En effet la diélectrophorèse dépend de la taille des particules, de son environnement et des paramètres du champ appliqué (fréquence, amplitude, topologie) La diélectrophorèse est mise en compétition avec le mouvement Brownian pour des particules d’or d’aussi petites tailles. La réponse pour des nanoparticules d’or en solution alors connue, il est envisageable de faire varier les paramètres, comme l’environnement de la particule ou bien la complexité des systèmes étudiés. / The manipulation of micro- and nano- particles in solution can be achieved through the interactions of these objects with electromagnetic fields. Emitted light, continuous (DC) or alternating (AC) electric fields can be used. In the case of a non-uniform electric field applied between two electrodes separated by a few micrometers, very intense and localized field gradients are created. These field gradients localised close to the electrodes generates a motion of the mass solution (electro-hydrodynamic effects). The charges confined onto particles at the liquid/solid interface are also subjected to motion. Suspended particles undergo an attractive or repulsive force called dielectrophoresis.This force is described in the literature for insulating particles larger than 200 nm. In this study optical detection was used (dark field videomicroscopy or microfluidic spectroscopy) to expand the range of particle size (40-150 nm) and to study their dielectrophoretic responses. Indeed, the dielectrophoresis is dependent on the size of particles, their environment and the parameters of the applied electric field (frequency, amplitude, topology). The dielectrophoresis is in competition with the Brownian motion of these gold nanoparticles. By, knowing the dielectrophoretic response of these particles in solution, it is possible to vary parameters, such as the suspension composition of the particles or the complexity of the systems studied.

Correct diagnosis of disease is essential in the effort to save and improve lives. Point of care (POC) diagnostics are in-vitro tests that assist in patient diagnosis and can be used at the location of patient care. POC diagnostics are easy to use and provide near-instant readouts allowing medical providers and patients to make rapid decisions about treatment. Increased access to POC testing is especially beneficial to low-income and low resource areas that cannot afford expensive lab testing. The World Health Organization (WHO) has outlined at least 113 diseases for which POC diagnostics are needed. Because of this, developing effective, efficient, and economical methods for creating new POC tests is essential. Work in section one of this thesis describes strategies by which new POC bio-diagnostics can be created. The use of oxidized cellulose as a vector for antibody immobilization was explored in several cellulose-based materials to provide quick, economical tests while still obtaining effective limits of detection when used to detect the pregnancy hormone Human Chorionic Gonadotropin (HCG) in a proof of concept study. The majority of these tests could detect as low as 100 ng/mL of HCG well below the clinical level necessary for detection at 2400 ng/mL. The use of a hand-powered syringe-based POC named the fast flow immunoassay (FFI) was tested for its ability to increase observable signal in a sandwich immunoassay by passing the sample through the test filter multiple times. 10 passes through the filter resulted in a signal approximately 17x more intense than a 1-hour dot-blot sandwich immunoassay. Both oxidized cotton and FFI systems can be used to develop new POC assays quickly and economically. Future use of these POC systems could help expand the availability of diagnostic testing to disadvantaged areas. Gold-based drugs have been used and investigated as medications multiple times throughout history to treat various diseases such as Rheumatoid arthritis, parasitic infections, and cancer. In the last few decades, gold nanoparticles have been used as drug delivery agents and catalysts for various reactions. Recently catalytic gold nanocrystals have been characterized for their ability to treat neurodegenerative diseases. Although these results were promising, much is still unknown about their mechanism of action. Section two of this thesis investigates potential molecular pathways that gold nanocrystals could be affecting, specifically the IL-6/Jak/STAT3 inflammation pathway and the Nrf2 antioxidant pathway. The gold nanocrystals we tested did not affect these pathways at physiologically obtainable concentrations. Additional work was done to characterize protein interactome or protein corona of gold nanocrystals. Preliminary proteomic characterization of this protein corona in fetal bovine serum (FBS) identified 118 potential interactors and classified those based on function and structure. Future work will need to be done to follow up on these identifications and to determine what mechanistic implications they may have.

Metal nanoparticles have been a primary focus in areas of catalysis and electrocatalysis applications as a result of their large surface area-to-volume ratios. While there is an increased interest in understanding the properties and behaviors of metal nanoparticles, they can become expensive over time. Recent research has incorporated the idea of using heteroatom-doped materials as a cheaper catalytic alternative to metal nanoparticles. In this study nitrogen-doping and phosphorous-doping techniques were applied to chemical vapor-deposited carbon ultramicroelectrodes in order to study the electrocatalytic properties toward the oxygen reduction reaction and the enhanced affinity for the deposition of gold nanoparticles onto the electrodes.

Studies of single, isolated nanoparticles provide better understanding of the structure-function relationship of nanoparticles since they avoid complications like interparticle distance and nanoparticle loading that are typically associated with collections of nanoparticles distributed on electrode supports. However, interpretation of results obtained from single nanoparticle immobilization studies can be difficult to interpret since the underlying nanoelectrode platform can contribute to the measured current, or the immobilization technique can adversely affect electron transfer. Here, we immobilized ligand-free gold nanoparticles on relatively electrocatalytically inert nitrogen-doped carbon ultramicroelectrodes that were prepared via a soft nitriding method. Sizes of the particles were estimated by a recently reported electrochemical method and were found to vary linearly with deposition time. The particles also exhibited electrocatalytic activity toward methanol oxidation. This immobilization strategy shows promise and may be translated to smaller nanoelectrodes in order to study electrocatalytic properties of single nanoparticles.

La thérapie génique est probablement une des approches la plus ambitieuse de l'histoire de l'humanité pour éliminer des maladies résistantes à tout autre traitement. Cependant, c'est une approche qui doit encore être développée afin d'obtenir un meilleur contrôle du processus de délivrance des médicaments et aussi de réduire les coûts. À cette fin, ce projet de thèse est axé sur l’optimisation et le contrôle de la délivrance d’oligonucléotides basée sur l'utilisation de nanobâtonnets d'or (Gold NanoRods, GNRs). De telles nanoparticules (40 nm de long et 10 nm de diamètre) sont internalisées par les cellules et grâce à leurs propriétés physiques extraordinaires permettent de délivrer les médicaments dans le cytoplasme de manière contrôlée. En effet, leur absorption très élevée dans le proche infrarouge du spectre électromagnétique permet de convertir l’énergie lumineuse en chaleur à l’intérieur et autour des nanobâtonnets, sans affecter la cellule. L’avantage d’une absorption dans l’infrarouge est qu’à cette longueur d’onde la lumière pénètre profondément dans les tissus humains (3 cm). Le contrôle de la température autour des nanoparticules permet la libération d'oligonucléotides par simple dénaturation du duplex à un instant donné.L’obtention de nanoparticules pouvant être considérées comme un « cargo » implique de remplir les conditions suivantes : stabilité de la forme colloïdale en milieu complexe, conservation des propriétés physiques et chimiques une fois administrées et possibilité d’immobiliser et de libérer le médicament de manière contrôlée.La première étape de mon projet a consisté à établir un protocole de synthèse de nanobâtonnets d’or afin d’obtenir une solution colloïdale mono-disperse et dont la bande d’absorption de plasmon longitudinal soit dans le proche infrarouge. L'étape suivante était d’optimiser un protocole de fonctionnalisation de la surface des GNRs. Le défi ici est associé à l'agrégation des GNRs lorsque le surfactant (CTAB) nécessaire au maintien des GNRs en solution est remplacé par des biomolécules (oligonucléotides). Cependant, après une étude systématique et détaillée, la déstabilisation de la couche protectrice de surfactant sur la surface métallique et l’ajout d’oligonucléotides ayant une fonction thiol à une des deux extrémités dans un rapport approprié ont permis une bio-fonctionnalisation efficace des nanobâtonnets. En conséquence, les nanoparticules fonctionnalisées, après redispersion dans la solution, ont les propriétés physico-chimiques nécessaires. En outre, l’immobilisation des oligonucléotides sur la surface des nanoparticules est spécifique (via la liaison thiol-Au) et permet leur transfert dans des solutions tamponnées ou dans des milieux complexes sans affecter leur stabilité. Après hybridation entre le simple brin immobilisé sur la surface des nanobâtonnets et le brin complémentaire, j’ai démontré que les oligonucléotides étaient stables et que le nombre de doubles brins qui se forment par hybridation peut être contrôlé. L’analyse des propriétés des nanomatériaux a constitué la seconde étape clé de mon travail, car elle revêt une importance cruciale pour la délivrance contrôlée de médicaments. J'ai décidé d'appliquer des méthodes uniquement optiques couvrant l'absorption des nanobâtonnets et l'analyse de la fluorescence des oligonucléotides marqués et des images TEM.Au cours du projet, il a donc été possible d’établir une nouvelle approche de fonctionnalisation et de créer un protocole de caractérisation efficace, axé sur les oligonucléotides. / Gene therapy is probably one of the most ambitious approaches in human history that aims to eliminate diseases, often those completely resistant to other treatments. However, this approach requires further development in order to obtain better control over the process of drug delivery and reduce costs. For this purpose, this project has focused on delivery of oligonucleotides using gold nanorods (GNRs). Such nanoparticles, (40 mm in length and 10 nm in diameter) can be internalized by cells and their extraordinary physical properties allow the delivery of drugs to the cytoplasm of cells in a controlled manner. Indeed, their strong absorption in the near-infrared part of the electromagnetic spectrum allows conversion of the energy of light into heat around the nanorods without affecting the cells. The advantage of absorption in the infrared is that at this wavelength the light can penetrate human tissues (3 cm). Control of the temperature around the nanoparticles allows the release of oligonucleotides by simple denaturation of the duplex at a given time.Obtaining nanoparticles that can be considered as a "cargo ship" implies fulfilling the following conditions: stability of the colloidal form in a complex medium, preservation of the physical and chemical properties once administered and the ability to immobilize and release the drug in a controlled manner.The first step of my project was to establish a nanorods synthesis protocol in order to obtain a monodisperse colloidal solution whose longitudinal absorption band is in the near infrared. The next step was to optimize the functionalisation protocol of the surface of the GNRs. The challenge here is associated with the aggregation of GNRs when the surfactant (CTAB) needed to maintain the GNRs in solution is replaced by biomolecules (oligonucleotides). However, after a systematic and detailed study, the destabilisation of the surfactant protective layer on the metal surface and the addition of oligonucleotides having a thiol function at one of the two extremities in a suitable ratio allowed an efficient bio-functionalisation of the nanoparticles. As a consequence, the functionalised nanoparticles, after redispersion in solution, possess the necessary physicochemical properties. In addition, the immobilisation of oligonucleotides on the surface of the nanoparticles is specific (via the thiol-Au bond) and allows their transfer into buffered solutions or in complex media without affecting their stability. After hybridisation between the single strand immobilized on the surface of the nanorods and the complementary strand, I demonstrated that the oligonucleotides were stable and that the number of double strands that are formed by hybridization can be controlled. The analysis of the properties of nanomaterials was the next important part of the work, as it is of crucial importance for the controlled delivery of drugs. I decided to apply only optical methods covering nanorods absorption and fluorescence analysis of labeled oligonucleotides and TEM images.In summary, during the project it was possible to establish a new functionalization approach and create a protocol for efficient characterization, focused on oligonucleotides. We expect that these observations will aid further research in the field of gene delivery based on gold nanoparticles.

This dissertation thesis is focus on using toxicity tests with testing organism - terrestrial isopod Porcellio scaber. This organism is use for toxicity assessment of selected anorganic compunds which could contaminate terrestrial ecosystem. This organism was selected for several reasons - sophisticated test methodology; well known biology of these animals and their ecological relevance. Besides classical endpoints such as mortality, change in mass of test specimens and the effect of the presence and concentration of contaminants onto food consumption; we monitored changes in the morphometric characteristics at the level of tissues and cells, the cytotoxicity and possibly bioaccumulation in various parts of the body of the organism. Selected test substance in this thesis were nanoparticles and inorganic salts. Wherein for chosen gold nanoparticles was carried out a complete study presents several levels of organization of living matter (organism, tissue, cell). For selected salts compound, this study was carried out on the behavior of organisms and classical endpoints.