Dépistage du VIH, test de grossesse, mais également surveillance des eaux, détection de contaminants agro-alimentaires : la biodétection est au coeur des problématiques de santé actuelles. Dans ce contexte, les biocapteurs plasmoniques connaissent depuis quelques années un essor particulièrement important : de plus en plus de sociétés, telles que HORIBA Scientific, proposent des prototypes commerciaux, destinés tant à des utilisateurs du domaine de la recherche que de l'industrie. Basée sur le phénomène de résonance des plasmons de surface (communément appelé SPR) la biodétection plasmonique repose sur l'extrême sensibilité d’une onde évanescente se propageant à l’interface entre un film d’or, la biopuce, et le milieu diélectrique couvrant, siège des interactions biomoléculaires étudiées. De manière plus concrète, toute adsorption de matériel biologique se produisant à cette interface entraîne une modification importante des propriétés optiques d’un faisceau de lumière réfléchi par la biopuce : le principe de transduction par SPR consiste alors à mesurer directement ces variations. A l'heure actuelle, différents modes d'interrogation, offrant des performances intéressantes, mais également des limitations propres à chaque configuration. Pour répondre aux exigences de précision et de dynamique de mesure posées par de nombreuses applications, un développement théorique et instrumental, présenté dans ce document, a été initié dans le but de proposer un nouveau un nouveau mode d'interrogation des biopuces plasmoniques : l'interrogation multi-spectrale. Les résultats obtenus par cette technique ont été exploités pour concevoir et réaliser une source multi-spectrale à base de LEDs, particulièrement avantageuse vis-à-vis des configurations existant à l'heure actuelle. La caractérisation du système développé dans le cadre du diagnostic génétique (mucoviscidose) et celui du cancer, ouvre la voie à une nouvelle génération de biocapteurs performants, compacts et de coût relativement raisonnable, présentant un potentiel industriel certain. / Biodetection is at the core of the current health concerns, as shown through the variety of applications to HIV screening, food contaminant analysis or water quality monitoring. In this field, plasmonic biosensing is a well-established label-free technique on the market: commercial systems from HORIBA Scientific are currently available for both research and industrial users.Based on the surface plasmon resonance (SPR) phenomenon, plasmonic biodetection uses the high sensitivity of an evanescent wave propagating along a metallic film (forming the biochip) and the surrounding dielectric medium interface. More specifically, the adsorption of biomolecules onto the metal surface induces a strong change in the optical properties of a light beam reflected by the biochip: the main principle of plasmonic transduction consists in measuring these physical changes. Several interrogation techniques have therefore been developed to access such optical information, but they fail in meeting the most demanding user requirements for precise, real-time, high-throughput measurement.Initiated by these issues, the instrumentation work presented in this document has led to the development of a novel SPR interrogation technique, referred to as multi-spectral interrogation. Moreover, the promising results obtained have been pushed forward to propose a multi-spectral illumination system based on LEDs, providing attractive performances compared to existing configurations. The biosensing potential of the developed system, demonstrated through applications to genetic diagnosis and cancer detection, opens the door to a new generation of compact, high-performance, low-cost SPR sensors.

Breeding for abiotic stress tolerance is one of main topics in plant breeding. Oilseed rape breeding programs were for a long time focused on morphological and physiological parameters. In this thesis few experiments focused on identification of genes involved in abiotic stress reaction were performed using RT-qPCR (quantitative reverse transcription PCR). Simultaneously SPR (surface plasmon resonance) method were used as modern optical method facilitating very low native protein concentration even in presence of other substances. This method facilitates quantification of concrete proteins by binding them to specific antigen and in oilseed rape research it was not used by now. ERD10 protein was identified by SPR as protein involved in cold stress reaction (or acclimation). The results show ERD10 accumulation in standard conditions affects dynamics of its accumulation change during cold stress. In case we are searching for genotypes great in acclimation ability even during short and warm autumn SPR method should be suitable method for fast, easy and relatively cheap screening of large number of genotypes in breeding collections. Also genes LTI78, RCI2A, NRP1 and two genes for hypothetical proteins were analysed. Their relative expression during cold stress was markedly increased too. Very little is known about these genes and proteins nowadays therefor it will be interesting topic of our oncoming experiment. Relative expression of genes picked according to MALDI-TOF/TOF analysis results was also tested in microspore embryo regenerants stressed by simulated drought. Genes for lactoylglutathione lyase I, phospholipase D 1 and peroxiredoxin antioxidase were tested. In tolerant cultivar was markedly decreased gene expression of peroxiredoxin antioxidase in standard conditions and early stress. These gene will be subject for next research as potential marker for more tolerant genotypes selection.

[pt] Neste trabalho é apresentado um estudo sobre nanopartículas metálicas, passando brevemente pela Teoria de Maxwell-Garnett do meio efetivo além de ter sido feita uma pequena pesquisa acerca dos trabalhos já existentes nessa área. É apresentada ainda uma proposta de um sensor à fibra óptica baseado no fenômeno da Ressonância Plasmonica de Superfície Localizada, que utiliza essas nanopartículas cujas propriedades ópticas são sensíveis às mudanças das características do meio onde estão imersas. Esse tipo de sensor é uma ferramenta simples e muito eficiente, além de ser de baixo custo financeiro. A descrição deste sensor mostra ainda três processos distintos de fabricação dessas nanopartículas, as configurações utilizadas para a aquisição dos dados experimentais, e a análise dos mesmos, incluindo a simulação computacional feita para o melhor entendimento dos resultados obtidos. / [en] In this work a study about metallic nanoparticles is presented. A brief revision of the Maxwell-Garnett Theory for the effective medium is made, in addition to a bibliographical research concerning the existing works on the topic. A proposal of a fiber optic sensor based in the Localized Surface Plasmon Resonance phenomena using these metallic nanoparticles, whose optic properties are sensible to changes in the medium in which they are immersed, is also made. This kind of sensor is a simple and efficient tool, in addition to also having low financial cost. Three distinct processes for fabrication of these nanoparticles are discussed. The setups used for the acquisition of the experimental data and the analysis of this data, including the computational simulation made to improve the understanding of the obtained results, are also discussed.

Neste trabalho estão apresentadas novas estratégias para funcionalizar superfícies de ouro baseadas na sustentação de bicamadas lipídicas em superfícies de sensores de imagem por Ressonância de Plasmons de Superfície (SPRi) e a construção de um biosensor para detecção de citocromo c. SPRi é uma técnica ótica de gravimetria em tempo real. Por meio de medidas de variações de índice de refração (n) próximas a uma interface, a adsorção e desorção de moléculas podem ser mensuradas. Inicialmente testamos várias estratégias para encontrar um suporte adequado que se ligasse na superfície de ouro e que oferecesse sustentação e estabilidade para a bicamada de fosfolipídeo biotinilado. Estudos de FT-IR e MEV mostraram que a quitosana facilita a formação de uma bicamada íntegra de fosfolipídeos, de tal modo, que a mesma alcança valores de espessura próximos àqueles previstos, ~ 34,5 Å. Além disso, mostramos que esse sistema apresenta vantagens perante outros modelos, tais como, (poli-lisina/fosfolipídeos) e (tiol hidrofóbico/fosfolipídeo). Utilizando-se o complexo químico biotina/estreptoavidina conseguimos imobilizar o anticorpo anti cit c na bicamada, mantendo-o afastado da superfície de ouro. A construção do biosensor foi acompanhada com experimentos de SPRi. O limite de detecção de citocromo c atingido foi de 10-11mol/L. Um sensor construído somente com BSA e anticorpo anti cit c apresentou sensibilidade semelhante. Esta sensibilidade é em torno de três ordens de grandeza superior aos experimentos de imunoblotting usualmente utilizados para detecção de cit c. A principal limitação deste biosensor, tal como de outros imunoensaios, está intimamente ligada às vantagens e desvantagens dos anticorpos como ferramentas analíticas. / In this work we developed new strategies to functionalize gold surfaces based on the support of lipid bilayers on the surfaces of surface plasmon resonance imaging sensors (SPRi) and the construction of a biosensor for detection of cytochrome c. SPRi is an optical gravimetric real time technique. Through measurements of changes in refractive index (n) in close proximity to an interface, the adsorption and desorption of molecules can be measured. Initially we tested several strategies for finding a suitable medium that would adsorb on the gold surface and also support and stabilize a biotinylated phospholipid bilayer. Studies of FT-IR and SEM showed that chitosan induces the formation of an intact phospholipid bilayer, so that it reaches thickness values close to those expected, ~ 34.5 Å. Furthermore, we showed that this system has advantages in relation to other models, such as (poli-lisine/phospholipids) and (thiol hydrophobic / phospholipid). Using the chemical complex biotin/streptavidin anti cyt c antibody could be immobilized in the bilayer, keeping it away from the gold surface. The construction of the biosensor was accompanied with SPRi experiments. The limit of detection of cytochrome c was achieved from 10-11mol / L. A sensor built only with BSA and anti cyt c showed similar sensitivity. This sensitivity is about three orders of magnitude higher than the immunoblotting experiments commonly used for detection of cyt c. The main limitation of this biosensor, like in other immunoassays, is linked to the advantages and disadvantages of antibodies as analytical tools.

This thesis covers different topics in the field of synthetic organic chemistry combined with the field of surface science and glycobiology. First, the text presents a series of orthogonally protected oligosaccharides (tri-, penta-, and heptasaccharides) of varying length and structures, which are synthesized with the aim of developing novel heterobifunctional biocompatible cross-linkers. Successful conjugation with different chemical handles is also described and used to illustrate the potential implementation of defined carbohydrate based compounds have potential use in biosensing applications. The results of incubation experiments using living cells indicate that the linker is incorporated into cell surfaces and enriched in microdomains. Second, synthesis of various saccharide-terminated alkane thiols immobilized on gold surfaces is reported. The protein adsorption and antifouling characteristics of these surfaces were investigated using model proteins and the common fouling organisms, Ulva linza and Balanus amphitrite. Further, oligo(lactose)-based thiols (di-, tetra-, and hexasaccharides) were synthesized and immobilized on gold nanoparticles to investigate how well these rigid, rod-like oligosaccharides can stabilize such nanoparticles for future use in constructing hybrid nanoparticles. Finally, the thesis describes synthesis of a systematic series of oligo(ethylene) glycols possessing either hydrogen- or methyl-terminated groups. Investigation of the fundamental characteristics of self-assembled monolayers, will give important insights into the design of protein repellant surfaces.

The content of this thesis is based upon the interaction of light with metallic nanoparticles arranged in different array geometries. An incident electric field (light) can force the conduction electrons of a metallic nanoparticle to oscillate. At particular frequencies, in the optical regime for gold and silver particles, absorption and scattering of the light by the particle is enhanced, corresponding to the particle plasmon resonance. The spectral position and width of the particle plasmon resonance of an isolated single particle may be tuned by adjusting its size and shape, thus changing the surface charge distribution. Periodic arrays of particles offer additional control over the frequency and width of the resonance attributed to the re-radiating (scattering) property of plasmonic particles. By fabricating arrays with a pitch comparable to the wavelength of an isolated single particle plasmon resonance, a coherent interaction between particles may be produced, known as surface lattice resonances (SLRs). The electromagnetic coupling between in-plane particle plasmon modes for different particle array geometries is explored through experiment and theory. Firstly, SLRs in square, hexagonal and honeycomb arrays are investigated by normal-incidence extinction measurements and compared to a simple-coupled dipole model. Secondly, to verify the nature of the coupling between the scattered electric field associated with particle resonances, the incident electric field polarization-dependence of the extinction of rectangular arrays and chains is studied. Thirdly, the optical response of square arrays with a symmetric two-particle basis is investigated, particularly the retardation of the scattered electric field between particles in a pair. Fourthly, square arrays with an asymmetric two-particle basis are fabricated to explore the symmetric (dipole moments of both particles are parallel) and anti-symmetric (dipole moment of both particles anti-parallel) SLRs, excited by normal-incidence light.

No description available.

Human practices have resulted in great damage to the environment. Carbon-depletion, water and air pollution, as well as global warming are examples of the environmental footprints caused by several industries and their related applications. Detergency (cleaning) is widely practiced operation in household, industry and institutional sectors and thus consumes significant amounts of water, energy and chemicals and, therefore, contributes appreciably to the environmental destruction. This process is still not fully understood, on a molecular level, and not acceptably optimised. Therefore, this study is a contribution toward a better fundamental understanding and optimisation of protein stain removal from interfaces, which may ultimately result in the development of environmentally friendly and sustainable cleaning products and technologies. In this research, the cleaning of rubisco, a grassy protein stain, from different surfaces (hydrophobic, hydrophilic, and dyed) using different cleaning agent formulations was investigated. These studies encompassed experimental work and fundamental analysis in terms of mathematical modelling. The results revealed a consistent correlation between enzyme adsorption kinetics and stain cleanability. Higher adsorption and desorption absolute rates ( a k and d k ) resulted in higher enzyme mobility and thus higher stain cleanability regardless of the enzyme adsorbed amount. Surface chemistry underlying the stain has indirectly influenced stain cleanability through the alteration of enzyme adsorption kinetics. Such fundamental findings may aid in screening enzyme candidates for detergent formulations and may also assist in designing easily cleanable surfaces. Another fundamental finding is the cooperative cleaning mechanism of surfactant and enzyme of rubisco stain from different interfaces. The break down of intermolecular physical bonds between interfacial rubisco molecules by surfactants and the break down of the intramolecular covalent bonds by enzyme resulted in a higher protein displacement from interfaces. The overall protein removal by the two different actions of surfactant and enzyme showed a superiority of a biosurfactant-enzyme formulation. This finding may have significant implications on developing sustainable detergents that have superior cleaning performance and no or minimal environmental hazard. Overall, the findings reported in this Ph.D. thesis may form a basis for further comprehensive scientific research, which may ultimately provide detergent market with more efficient and optimum cleaning products and technologies.

<p>The aims of this study are to first immobilize positively charged gold nanorods to negatively charged cell culture surfaces. Second, to use polyelectrolytes for controlling the distance between gold nanorods and fluorophores. This is used to optimally determine the distance, of which maximum fluorescence enhancement is achieved, between gold nanorods and fluorophores. In order to approach these aims, we use UV/VIS absorption spectroscopy, fluorescence spectroscopy, atomic force microscopy, and ellipsometry. The results show that we could control the immobilization of gold nanorods on plastic microwell plates and create reproducible polyelectrolyte layers, in order to control the distance between the gold nanorods and fluorophores. In addition, the localized surface plasmon resonance wavelength red shifted as the PELs increased. In conclusion, we found that the maximum fluorescence enhancement of the fluorophores (Cy7) is about 2.3 times at a fluorophores-nanoparticles separation of approximately 9-12 nm. This work contributes some research information towards the design of optical biochip platforms based on plasmon-enhanced fluorescence.</p>

This thesis concerns the development of surface ligation techniques for the preparation of carbohydrate biosensors. Several methodologies were developed based on efficient photochemical insertion reactions which quickly functionalize polymeric materials, with either carbohydrates or functional groups such as alkynes or alkenes. The alkyne/alkene surfaces were then treated with carbohydrate azides or thiols and reacted under chemoselective Cu-catalyzed azide-alkyne cycloaddition (CuAAC) or photo-radical thiol-ene/yne click chemistry, thus creating a range of carbohydrate biosensor surfaces under ambient conditions. The methodologies were evaluated by quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) flow through instrumentations with recurring injections of a range of lectins, allowing for real-time analysis of the surface interactions. The developed methods were proven robust and versatile, and the generated carbohydrate biosensors showed high specificities and good capacities for lectin binding.  The methods were then used to investigate how varying the glycan linker length and/or a sulfur-linkage affect the subsequent protein binding. The survey was further explored by investigating the impact of sulfur in glycosidic linkages on protein binding, through competition assays with various O/S-linked disaccharides in solution interactions with lectins. / QC 20120309