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京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19189号 / 工博第4066号 / 新制||工||1627(附属図書館) / 32181 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 北川 進, 教授 濵地 格, 教授 森 泰生 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

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

Bond rupture is an experimental methodology that is used to augment a conventional mass balance biosensor. A good point-of-care biosensor is fast, reliable, simple, cost-effective, and detects low concentrations of the target analyte. Biosensor development is a multidisciplinary field and bond rupture testing is of technical interest to many groups. The Bond rupture methodology endows a mass probe with the ability to discern bond strength. The recognition of specific bonds by mass loading is separated from erroneous non-specific binding by a probe of the force between the analyte and the transducer. Bond rupture is achieved by acoustic excitation of the point of attachment. The force is incremented gradually until rupture occurs. The advancement of bond rupture biosensors beyond the lab requires improved understanding of the mechanisms of bond rupture by base excitation, the transducers, and the supporting hardware. Bond rupture has traditionally been used in conjunction with the Quartz Crystal Microbalance (QCM). There exists, however, a variety of sensors and transducers to which the bond rupture methodology could be applied. The time, cost and experience required for comprehensive investigation of all avenues is prohibitive. To further the development of bond rupture characteristic experiments are designed and carried out on the QCM platform. Numerical simulations are constructed which model the current bond rupture approach. This work is limited to the simulation of bond rupture by base excitation. From the results of the experimental investigation a number of improvements to the bond rupture technique are proposed. Improvements are tested by simulation and the Surface Acoustic Wave (SAW) device is selected to advance the bond rupture craft. A prototype SAW bond rupture device is designed. The prototype device is manufactured and tested, confirming the principle of SAW bond rupture. Future work is required to progress the SAW bond rupture methodology before possible integration with other sensor systems. Because of this work, and the evaluation of the SAW bond rupture prototype, much is learned about the advancement of SAW device bond rupture.

Organic materials exhibiting second-order non-linear optical (NLO) properties are a key to the development of advanced electro-optic (EO) modulators used in fiber-optic communications system. This work addresses the fabrication and characterization of organic materials with NLO properties using a self-assembly approach by alternately dipping a charged substrate into positively and negatively charged polymers to build up layer-by-layer (LbL) films. The effect of solution pH on the formation of LbL films fabricated using the polycation poly(allylamine hydrochloride) (PAH) and the polyanion poly{1-[p-(3–-carboxy-4–-hydroxyphenylazo)benzenesulfonamido]-1,2-ethandiyl} (PCBS) was studied using a quartz crystal microbalance with dissipation (QCM-D) monitoring, ellipsometry, absorbance, and second harmonic generation (SHG) measurements. PCBS has an azo-benzene chromophore side group that, when sufficiently oriented, results in measurable SHG. Films of PAH/PCBS fabricated at neutral pH where both PAH and PCBS are highly charged led to thin bilayers, ~1 nm, with a 1:1 molar ratio of PCBS:PAH. This molar ratio was found to be important for long-range polar ordering of PCBS in these films. Increasing the rate of convection was found to reduce the time required for complete adsorption of the polyion. This can have a significant impact on fabrication of films with high bilayer numbers. A variation of the above technique, which involves adsorbing one of the constituents electrostatically and another covalently, was studied using PAH and a reactive dye, Procion Brown (PB), which has a significantly higher hyperpolarizability than PCBS. It was found that a high pH, ~10.5, was important for achieving covalent attachment of the PB to the underlying PAH films. This resulted in much higher SHG intensities compared to when PB was deposited pH at 8.5-9.5 where the attachment of PB was due to a combination of electrostatic and covalent interactions. QCM-D results for PAH/PB films revealed the presence of a high percentage of unreacted amine groups in the underlying PAH film. A rate constant value for PB attachment step to the underlying PAH was also calculated. To enhance the SHG intensity of these films, silver nanoprisms were synthesized and deposited onto films using physisorption. An enhancement in the SHG intensity was observed for both PAH/PCBS and PAH/PB films. / Ph. D.

Poly(3,4-ethylendioxythiophen) (PEDOT) ist ein Kunststoff der zur Gruppe der intrinsisch leitfähigen Polymere (ILP) zählt. Aufgrund seiner chemischen und thermischen Stabilität findet er Verwendung in antistatischen Verkleidungen und als Elektrodenmaterial. PEDOT (und andere ILP) zeigen aufgrund ihrer Schaltbarkeit zwischen (reduzierten, ) neutralen und oxidierten Zuständen unterschiedliche Eigenschaften wie Leitfähigkeit, Farbe oder Viskoelastizität. Im Rahmen dieser Arbeit wurden die elektrochemischen und viskoelastischen Eigenschaften von PEDOT-Filmen untersucht. Dabei wurde die Quarzmikrowaage (QCM) in Verbindung mit potentiostatischen (Potentialsprung, PS) und potentiodynamischen (Cyclovoltammetrie, CV) elektrochemischen Methoden verwendet, so dass in situ elektrochemische und mechanische Eigenschaften der Filme zugänglich waren. Zur Bestimmung der viskoelastischen Eigenschaften wurde ein Auswertealgorithmus entwickelt, welcher auf ein mathematisches Modell zur Bestimmung des Schermoduls nach Efimov zurückgreift. Während der Herstellung wurden Parameter wie Lösungsmittel, Leitsalz, Vorpolarisations- und Abscheidungspotential variiert und die erhaltenen Filme bezüglich Schermodul und Morphologie charakterisiert. Es konnte gezeigt werden, dass die Elektrolytzusammensetzung einen entscheidenden Einfluss auf die viskoelastischen Eigenschaften der Filme besitzt, welche mit der Morphologie der Filme korrelieren. Des Weiteren wurden die Änderungen der viskoelastischen Eigenschaften dieser Filme untersucht, welche während dem elektronischen Schalten zwischen neutralem und oxidiertem Zustand aufgrund des Ionenaustausches erfolgen. CV- und PS-Experimente zeigten, dass die viskoelastischen Eigenschaften durch Konzentration und pH-Wert des Elektrolyten beeinflusst werden und in unterschiedlicher Weise auf die Potentialänderungen reagieren. Durch den Einbau von Magnetit-Partikeln in die Schichten konnten Hybridfilme erhalten werden, deren Eigenschaften durch das Anlegen eines äußeren Magnetfeldes beeinflusst werden können. Solche Filme zeigten in einem äußeren Magnetfeld (0,7T) höhere Schermodule und einen stark unterdrückten Ionenaustausch.

The Gram-negative bacterium Pseudomonas aeruginosa is a life-threatening nosocomial pathogen due to its generally low susceptibility toward antibiotics. Furthermore, many strains have acquired resistance mechanisms requiring new antimicrobials with novel mechanisms to enhance treatment options. Proline-rich antimicrobial peptides, such as the apidaecin analog Api137, are highly efficient against various Enterobacteriaceae infections in mice, but less active against P. aeruginosa in vitro. Here, we extended our recent work by optimizing lead peptides Api755 (gu-OIORPVYOPRPRPPHPRL-OH; gu = N,N,N′,N′-tetramethylguanidino, O = L-ornithine) and Api760 (gu-OWORPVYOPRPRPPHPRL-OH) by incorporation of Ile-Orn- and Trp-Orn-motifs, respectively. Api795 (gu-O(IO)2RPVYOPRPRPPHPRL-OH) and Api794 (gu-O(WO)3RPVYOPRPRPPHPRL-OH) were highly active against P. aeruginosa with minimal inhibitory concentrations of 8–16 and 8–32 μg/mL against Escherichia coli and Klebsiella pneumoniae. Assessed using a quartz crystal microbalance, these peptides inserted into a membrane layer and the surface activity increased gradually from Api137, over Api795, to Api794. This mode of action was confirmed by transmission electron microscopy indicating some membrane damage only at the high peptide concentrations. Api794 and Api795 were highly stable against serum proteases (half-life times >5 h) and non-hemolytic to human erythrocytes at peptide concentrations of 0.6 g/L. At this concentration, Api795 reduced the cell viability of HeLa cells only slightly, whereas the IC50 of Api794 was 0.23 ± 0.09 g/L. Confocal fluorescence microscopy revealed no colocalization of 5(6)-carboxyfluorescein-labeled Api794 or Api795 with the mitochondria, excluding interactions with the mitochondrial membrane. Interestingly, Api795 was localized in endosomes, whereas Api794 was present in endosomes and the cytosol. This was verified using flow cytometry showing a 50% higher uptake of Api794 in HeLa cells compared with Api795. The uptake was reduced for both peptides by 50 and 80%, respectively, after inhibiting endocytotic uptake with dynasore. In summary, Api794 and Api795 were highly active against P. aeruginosa in vitro. Both peptides passed across the bacterial membrane efficiently, most likely then disturbing the ribosome assembly, and resulting in further intracellular damage. Api795 with its IOIO-motif, which was particularly active and only slightly toxic in vitro, appears to represent a promising third generation lead compound for the development of novel antibiotics against P. aeruginosa.

This thesis mainly deals with the development of photochemical approaches to immobilize carbohydrates on surfaces for glycobiological studies. These approaches have been incorporated into a number of state-of-the-art nanobio-platforms, including carbohydrate microarrays, surface plasmon resonance (SPR), quartz crystal microbalance (QCM), atomic force microscopy (AFM), and glyconanomaterials. All the surfaces have displayed good binding capabilities and selectivities after functionalization with carbohydrates, and a range of important data have been obtained concerning surface characteristics and carbohydrate-protein interactions, based on the platforms established. Besides, a variety of non-carbohydrate and carbohydrate-based molecules have been synthesized, during which process the mutarotation of 1-glycosyl thiols and the stereocontrol in 1-S-glycosylation reactions have been thoroughly studied. / QC 20111004

Ce travail de thèse a été réalisé, dans le cadre de la convention CIFRE 1538/2010, au sein d'adixen Vacuum Products (aVP) à Annecy (France). Il a été en partie financé par le projet S.P.A.M. (Surface Physics for Advanced Manufacturing). Il s'agit d'un projet ITN financé par le programme Pierre et Marie Curie de la Communauté Européenne rassemblant des partenaires universitaires et industriels dont aVP. L'objectif de ce programme était de contribuer à l'étude et au développement de la lithographie et en particulier la lithographie à ultraviolet extrême (EUVL). Ce travail porte sur la problématique de la contamination moléculaire dans l'industrie des semi-conducteurs ainsi que les besoins de maitrise de contamination pour la photolithographie EUVL. Pour ce faire, des modèles mathématiques de sorption ont été recherchés, testés et validés à l'aide d'une microbalance à quartz (QCM). Cette technique, possédant une très haute sensibilité (au niveau du ng), permet d'étudier les phénomènes de sorption relatifs à tout matériau déposable sur un cristal de quartz mis au contact de différents gaz dont la pression partielle est maitrisée. Par conséquent, le protocole détaillé dans cette thèse peut être utilisé pour d'autres types d'expériences dans toute discipline nécessitant une telle précision. Le déroulement de notre plan d'expérience comprend deux types de matériaux naturellement différents : un polymère (PCBA) d'une part et deux substrats métalliques (SS AISI 304 et CuC1) d'autre part pour lesquels le transfert de masse n'intervient pas de la même manière. Les gaz d'étude ont été sélectionnés pour leur intérêt dans l'industrie des semi-conducteurs (vapeur d'eau, HF). Le résultat de l'interaction des gaz d'étude avec les substrats ciblés est suivi en direct par la QCM, ce qui permet non seulement de valider et/ou améliorer les modèles mathématiques déjà disponibles dans la bibliographie mais aussi de les ajuster aux données obtenues expérimentalement. Nous pouvons ainsi non seulement prévoir le comportement des contaminants à l'équilibre (isothermes) et à l'état transitoire mais aussi réaliser des estimations de sorption à des températures autres que celles retenues pour notre plan d'expérience / This thesis was carried out within the framework of the CIFRE 1538/2010 convention at adixen Vacuum Products (aVP) in Annecy (France). It is has been partly funded by the ITN project SPAM (Surface Physics for Advanced Manufacturing). SPAM is an ITN project funded by the Pierre and Marie Curie program of the European Community bringing together academic institutions and industrial partners including aVP. The objective of this program was to contribute to the study and development of lithography and extreme ultraviolet lithography (EUVL). This work deals with the issues caused by the airborne molecular contamination (AMC) in the semiconductor industry and their control needs in EUVL and the current photolithography. In order to tackle the problem, sorption mathematical models have been investigated and validated using a quartz crystal microbalance (QCM). This technique, which confers a high sensitivity (ng level), allows the study of the sorption phenomena related to any deposable material onto a quartz crystal in contact with different gases whose concentrations are accurately controlled. Consequently, the protocol detailed in this thesis may be used for other types of experiments in any discipline requiring such precision. The conduct of our experimental plan includes two types of naturally different materials: a polymer (PCBA) on the one hand and two metallic substrates (stainless steel AISI 304 and CuC1) on the other hand, for which the matter transfer does not occur in the same manner. Studied gases were selected for their interest in the semiconductor industry (water vapor, HF). The resulting interaction between the studied gases and the targeted substrates is continuously followed by the QCM, which allows not only to validate the mathematical models already proposed by the literature but also to fit the experimentally obtained data. This enables us not only to predict the behavior of the AMC at equilibrium (isotherms) and the transient state but also to provide sorption estimations at temperatures other than those specified in our experimental plan

Byl studován kontakt mezi koulí a deskou pod tangenciálním zatížením ve vodě. Jako zdroj střižných kmitů byl použit akustický rezonátor (křemíkové mikrováhy – QCM). Kontakt koule s povrchem resonátoru indukuje změnu resonanční frekvence a šířky pásma. Byla měřena změna frekvence f a změna šířky pásma v závislosti na amplitudě oscilací. S rostoucí amplitudou docházelo k poklesu f a růstem , což je chování typické pro parciální skluz. Díky aplikaci Cattaneo-Mindlinova modelu byl vypočítán kontaktní poloměr a třecí koeficient. Kontaktní poloměr při nízké amplitudě stoupal při zvětšujícím se normálovým zatížením. Tato závislost se dobře shodovala s JKR modelem. Třecí koeficient se nacházel v odpovídajícím rozsahu. Při zvyšování externí normálové síly, docházelo k nepatrnému snižování hodnoty třecího koeficientu. Toto chování je vysvětleno příspěvkem adhezivních sil k totální normálové síle. Výpočtem byly získány dva typy třecích koeficientů, první ze změny frekvence f a druhý ze změny šířky pásma . Tyto dvě hodnoty se spolu shodovaly z ± 20 % pro měření prováděná ve vodě, zatímco pro dvě měření prováděných na hydrofilním povrchu ve vzduchu se lišila. Tento nesoulad poukazuje na nedostatek Cattaneo Mindlinovy teorie a mohl by být vysvětlen přítomností kapilárních sil.