Surface plasmon resonance (SPR) biosensors represent one of the most advanced sensing technologies for real-time studies of biomolecular interactions. In this thesis, methods for functionalization of SPR substrates were optimized and studied via spectroscopic methods. Effects related to the SPR sensor microfluidic interface on the measured biomolecular interactions were analyzed, and furthermore, means to decrease mass-transport limitations were proposed. Several SPR-based assays regarding the detection of nucleic acids were developed, which allow for the detection of physiologically relevant concentrations of nucleic acids as well as point mutations in a nucleic acid sequence. Assays for the determination of the enzymatic activity of HIV integrase and ribonuclease H were developed. These assays can be employed for the design and synthesis of molecules that function either as antiviral drugs or as gene-regulating agents.

Ce travail de thèse propose d’étudier les propriétés photophysiques de systèmes moléculaires à la fois photochromes et fluorescents. Sous l’effet de la lumière, ces molécules peuvent subir une désexcitation radiative, non radiative ou une photo-isomérisation. Dans la première partie de ce travail, la compétition entre ces différentes voies a été étudiée en détail grâce à deux photochromes proches sur le plan structurel et présentant une émission de fluorescence de couleur différente pour chaque forme du photochrome. Les efficacités relatives et les mécanismes de désexcitation ont été étudiés à l’échelle macroscopique. Cette comparaison a été accompagnée d’une étude sous microscope pour laquelle des nano-bâtonnets d’or ont été intégrés au système photochrome-fluorescent de manière à étudier l’influence d’un champ plasmon de surface localisé sur les différents phénomènes de désexcitation à une échelle proche de celle de la nanoparticule individuelle. Une deuxième partie de ce travail consiste en la préparation et l’étude des propriétés photophysiques de nanoparticules organiques constituées de dyades fluorescentes et photochromes. La fluorescence de ce type de nano-objets peut être efficacement contrôlée par la lumière : la conversion de quelques unités photochromes peut permettre, par transfert d’énergie intermoléculaire, d’éteindre la totalité de la fluorescence d’une nanoparticule. L’observation de cet effet pour des nanoparticules individuelles a été mise en application pour le développement d’une méthode de microscopie optique super-résolution. / The main purpose of this PhD work is the study of fluorescent and photochromic molecular systems. Under light illumination, such molecules may undergo radiative and non-radiative deactivation or photoisomerization. In the first part of this work, this competition has been investigated for structurally related photochromic compounds with different fluorescence colors corresponding to both isomers. Efficiency and mechanisms of the deactivation pathways were unraveled at the macroscopic scale. This competition was further studied under microscope with gold nanorods included in the sample, in order to study the effect of localized surface plasmon resonance on the different deactivation processes.The second part of this work consists in the preparation and the photophysical study of organic nanoparticles composed of fluorescent-photochromic dyads. Fluorescence of such nano-objects can be efficiently driven by light : the switching of a few photochromic units is enough to turn off the entire fluorescence of a nanoparticle by intermolecular energy transfer. Observation of this effect at the level of individual nanoparticles allowed the developement of a super-resolution method for optical microscopy.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19959号 / 理博第4226号 / 新制||理||1607(附属図書館) / 33055 / 京都大学大学院理学研究科化学専攻 / (主査)教授 寺西 利治, 教授 倉田 博基, 教授 島川 祐一 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

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The colloidal self-assembly of plasmonic gold nanoparticles (AuNPs) is of interest to utilize the plasmonic coupling effects that arise between nanoparticles. The enhanced properties of anisotropic AuNPs make them particularly attractive in self-assemblies. Herein, a literature study into the different strategies used to obtain self-assemblies of AuNPs using molecular linkers is presented. The use of nanospheres (AuNS) and nanorods (AuNRs) were mainly reviewed. Thereafter, two different nanobipyramids (AuBPs) were investigated for use in self-assemblies. The concentration of cetyltrimethylammonium bromide (CTAB), which coats the AuNP surface, was manipulated to study the stability of the AuNPs. A stable, meta-stable and non-stable region were identified for the nanoparticles. At low CTAB levels, the AuNPs preferentially assemble end-to-end. The addition of L-cysteine to stable AuNP dispersion induced end-to-end assembly, showing promise as a molecular linker for AuBPs. The addition of excess CTAB stabilized the assemblies over time. The kinetic behaviour of the two AuBPs differed, suggesting the effect of the AuNP shape on the self-assembly kinetics. This study provides a starting point for the development of a robust self-assembly strategy for anisotropic AuNPs by using L-cysteine as a molecular linker. / Den kolloidala självsammansättningen av ytplasmoniska guld nanopartiklar (AuNPs) är av intresse för att utnyttja de plasmoniska kopplingseffekterna som uppstår mellan nanopartiklar. De fördelaktiga egenskaperna hos anisotropa AuNP gör dem särskilt intressanta för självsammansättningar. En litteraturstudie har gjorts på de olika strategier som används för att erhålla självsammansättningar av AuNPs med hjälp av molekylära länkar. Användningen av nanosfärer (AuNS) och nanostavar (AuNRs) i självsammansättningar undesöktes huvudsakligen. Därefter undersöktes två olika nanobipyramider (AuBPs) för användning i självsammansättningar. Koncentrationen av cetyltrimetylammonium bromid (CTAB), som täcker AuNP-ytan, manipulerades för att undersöka AuNPs stabilitet. En stabil, meta-stabil och instabil region identifierades för nanopartiklarna. Vid låga CTAB-nivåer sammansätts AuNPs ände-mot-ände. Tillsatsen av L-cystein till stabila AuNP dispersioner inducerade sammansättningar ände-mot-ände, vilket visar L-cysteins potential som en molekylär länk för AuBPs. Tillsatsen av en stor mängd CTAB stabiliserade självsammansättningarna för en längre tid. Det kinetiska beteendet hos de två AuBPs skilde sig, vilket tyder på effekten av AuNP-formen på den självsammansättningskinetiken. Denna studie erbjuder en startpunkt för utvecklingen av en robust självsammansättningstrategi för anisotropa AuNPs genom att använda L-cystein som en molekylär länk.

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A new detection reagent that could possibly augment or replace antibodies research and diagnosis methods are aptamers. Aptamers are ssDNA, RNA or polypeptide constructs that function like active antibodies. Antibodies and aptamers both specifically bind to selected target molecules, and as such they enable the detection or targeting of the presence or absence of a specific antigen. In order to ensure that ssDNA aptamers perform similarly to antibodies, anti-VEGF-A polyclonal antibody and anti-VEGF-A ssDNA aptamer were evaluated against vascular endothelial growth factor A (VEGF-A) using Surface Plasmon Resonance (SPR). It was hypothesized that the anti-VEGF-A aptamer had the same, if not better, binding kinetics than the anti-VEGF-A polyclonal antibody, and as such offers an ideal replacement for use in in field, real-time testing assays. SPR revealed that both the polyclonal antibody and ssDNA aptamer bound the target antigen, VEGF-A. Additionally, from the SPR kinetic analysis, the anti-VEGF-A aptamer had KD values of 20-28 nM and the anti-VEGF-A antibody had KD values of 16-127 uM. The binding efficacy of the aptamer was several orders of magnitude better than that of the antibody. The aptamer was also stable in solution for a longer amount of time than the antibody, which denatured in solution after two weeks.

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Interactions between biomolecules and cellulose films at solid/liquid interfaces was studied by surface plasmon resonance spectroscopy (SPR), quartz crystal microbalance with dissipation monitoring (QCM-D) and in situ atomic force microscopy (AFM) measurements. This dissertation shows the porous character of nanocrystalline cellulose films as the key feature for enhanced adsorption of chemically modified polysaccharides and provides quantitative analysis of polymer supported phospholipid structures as a stable platform for studying membrane-related processes. Smooth cellulose I films were prepared by spincoating cellulose nanocrystal suspensions onto positively charged self-assembled monolayers on gold. The adsorption of pullulan cinnamate (PC) onto cellulose surfaces increased with increasing degree of cinnamate substitution. The interactions between PCs with higher degree of substitution (DS) and porous nanocrystalline cellulose (NC) films presumably generated looped multilayer PC structures that adsorbed more than twice as much onto NC films than onto regenerated cellulose (RC) films. PC chains not only covered the NC surface but also penetrated into the porous film. The porous features of NC film are responsible for the greater adsorption of polymer chains relative to tightly packed RC films. Adsorption of phospholipid vesicles onto RC and NC films was also studied. Aggregates of intact vesicle were observed on NC surfaces with high water content ~ 84 % by mass. Phospholipid patches with smooth features were found to assemble onto RC surfaces with a lower degree of hydration ~ 30 % by mass. Vesicle membrane breakage was triggered by a destabilizing agent, LysoPC. The great mass decrease, and changes in dissipation and degree of hydration for phospholipid structures after exposure to LysoPC corresponded to the transformation from vesicles to layered structures. Initial binding of LysoPC micelles to unruptured vesicles was clearly resolved in SPR, whereas the huge mass decrease associated with bound water hides the initial adsorption of LysoPC onto vesicles in QCM-D experiments. The intitial binding of LysoPC micelles onto vesicle membranes lasted for 200 seconds with a maximal increase of 14 % by mass prior to vesicle collapse. The role of cholesterol in phospholipid interactions with model cellulose surfaces was also considered. Supported vesicle layers over RC surfaces were observed for vesicle membranes containing ≥ 6.3 % by mole cholesterol, whereas phospholipid or phospholipid with lower cholesterol content formed disconnected lipid islands on RC surfaces. Meanwhile, intact vesicles were always observed on NC surfaces for phospholipid/cholesterol blends regardless of the cholesterol content. The intact vesicles on cellulose surfaces were attributed to the ability of cholesterol to accommodate vesicle deformation. These studies showed the impact of mesoscale structure of cellulose films on adsorbates. It sheds light on the role of the lignin-carbonhydrate-complex in plant cell wall structure and will inform the next generation of biomimetic nanocomposites. The designed polymer supported biomimetic membranes provide a perfect platform to develop intact and ruptured protoplast systems for the study of plant cell wall self-assembly. / Ph. D.