Made available in DSpace on 2017-07-20T12:40:18Z (GMT). No. of bitstreams: 1 Rosana M.pdf: 3183755 bytes, checksum: ceaa632c444d6daec2678d0f7fe428db (MD5) Previous issue date: 2016-02-23 / This thesis describes the use of self-assembled monolayers (SAM) for the development of biosensors based on horseradish peroxidase enzyme - HRP. In the first chapter, the enzyme was immobilized on self-assembled monolayers of thiolactic acid (Au-TLA) and mixed SAM composed of 11-mercaptoundecanoic acid (MUA) together with the TLA (SAMmix). The steps of construction and characterization of biosensors have been carried out by electrochemical techniques and morphological ones. The enzyme immobilization method on SAM which provided higher stability was by covalent bond. The Au/TLA/HRP biosensor was used for the determination of H2O2 by chronoamperometry, yielding a detection limit (DL) of 5.46 μmol L-1 and quantification (QL) of 18 μmol L-1. Despite the good results for the H2O2 detection presented by this biosensor, the device was stable for only 6 days. Therefore, in order to increase the stability of the biosensor, SAM containing both the mercaptoundecanoic acid molecule (MUA) and TLA was obtained, to the formation of SAMmix (Au/MUA:TLA). In this system, the electron transfer rate can be considerably affected because while the TLA enables an increase of SAMmix conductivity due to formation of “islands”, the MUA provides greater stability for HRP immobilization, although it partially blocks the surface. The biosensor Au-SAMmix-HRP prepared in the ratio 0.5: 1.0 MUA/TLA showed higher sensitivity compared to other modifications ratio, with an apparent Michaelis-Menten constant = 0.40 mmol L-1. This biosensor has been applied to the determination of hydroquinone (HQ) in the presence of a fixed amount of [H2O2] = 0.3 mmol L-1. By differential pulse voltammetry technique (DPV), the biosensor exhibited excellent electrocatalytic activity for HQ in the range 3-30 μmol L-1, with good sensitivity, DL = 1.26 μmol L-1 and QL = 4.23 μmol L -1. The SAMmix increased the stability of the biosensor for at least 15 days, which is more stable when compared to the biosensor Au/TLA/HRP. In the second chapter of this thesis, another strategy was used for immobilization of the HRP enzyme based on the formation of TLA monolayer on the top of gold nanoparticles (AuNPs) stabilized in the inorganic polymer, 3-n-propylpyridinium silsesquioxane chloride (SiPy+Cl-). The presence of AuNPs-SiPy+Cl- was confirmed by UV-Vis spectroscopy from the plasmon band at 521 nm. The AuNps showed good distribution with approximate size between 4-18 nm, evidenced by transmission electron microscopy (TEM) and by dynamic light scattering (DLS), with good stability (ζ = + 38.5 mV). The AuNPs were deposited on the glassy carbon electrode (GCE) and modified with the TLA for immobilization of HRP enzyme. The formation of this biosensor was confirmed by electrochemical impedance spectroscopy (EIS) and morphologically by field-effect scanning electron microscopy (SEM-FEG). The GCE/AuNPs/TLA/HRP biosensor was used for the detection of H2O2 with = 0.46 mmol L-1, which was similar to the Au-SAMmix-HRP biosensor. This biosensor has been applied to the determination of catechol (CT) in presence of [H2O2] = 0.03 mmol L-1. Using the DPV technique, the biosensor showed an excellent electrocatalytic activity for CT in the range 6-46 μmol L-1, with good sensitivity, DL = 0.852 μmol L-1 and QL = 2.84 μmol L -1. The stability of this device was approximately 25 days, being superior to other biosensors developed (TLA-HRP and SAMmix-HRP), which can be attributed to the three-dimensional immobilization of HRP molecules in this device. / Esta tese descreve a utilização das monocamadas auto-organizadas (SAM) para o desenvolvimento de biossensores enzimáticos a base da enzima horseradish peroxidase – HRP. No primeiro capítulo, diferentes biossensores foram desenvolvidos utilizando a SAM de ácido tioláctico (Au/TLA) e também a SAM mista composta por ácido 11-mercaptoundecanóico (MUA) juntamente com o TLA (SAMmista). As etapas de construção e caracterização dos biossensores foram realizadas pelas técnicas eletroquímicas e também morfológicas. O método de imobilização da enzima sobre a SAM que proporcionou maior estabilidade foi pela ligação covalente. O biossensor Au/TLA/HRP foi utilizado para a detecção do H2O2 por cronoamperometria, obtendo-se um limite de detecção (LD) de 5,46 μmol L-1 e de quantificação (LQ) de 18 μmol L-1. Apesar dos bons resultados na detecção do H2O2 apresentados por este biossensor, o dispositivo foi estável por apenas 6 dias. Portanto, no intuito de aumentar a estabilidade do biossensor utilizou a molécula de ácido mercaptoundecanóico (MUA) juntamente com TLA, para a formação da SAMmista (Au/MUA:TLA). Neste sistema, a velocidade de transferência eletrônica é sensivelmente afetada, pois enquanto o TLA possibilita o aumento da condutividade da SAMmista devido a formação de “ilhas”, o MUA confere maior estabilidade à monocamada para a imobilização da enzima HRP, apesar de bloquear parcialmente a superfície. O biossensor Au/SAMmista/HRP preparado na razão 0,5:1,0 MUA:TLA apresentou a melhor sensibilidade em comparação as outras modificações, com uma constante de Michaelis-Menten aparente, = 0,40 mmol L-1. Este biossensor foi aplicado na detecção da hidroquinona (HQ) na presença de uma quantidade fixa de [H2O2] = 0,3 mmol L-1. Pela técnica de voltametria de pulso diferencial (VPD), o biossensor exibiu uma excelente atividade eletrocatalítica para HQ na faixa de 3 a 30 μmol L-1, com boa sensibilidade, LD = 1,26 μmol L-1 e o LQ = 4,23 μmol L-1. A SAMmista aumentou a estabilidade do biossensor para 15 dias, sendo este mais estável quando comparado ao biossensor Au/TLA/HRP. No segundo capítulo da tese, outra estratégia para a imobilização da enzima HRP sobre o TLA foi realizada a partir de nanopartículas de ouro (AuNps) estabilizadas no cloreto de 3-n-propilpiridínio silsesquioxano (SiPy+Cl-). A presença das AuNps-SiPy+Cl- foi confirmada pela Espectroscopia UV-Vis a partir da banda plasmon em 521 nm, as quais apresentaram boa distribuição com tamanho aproximado entre 4 a 18 nm, constatado pela microscopia eletrônica de transmissão (MET) e por espalhamento dinâmico de luz (DLS), apresentando boa estabilidade (ζ = + 38,5 mV). As AuNps foram incorporadas sobre o eletrodo de carbono vítreo (ECV) e modificadas com o TLA para imobilização da enzima HRP. A formação deste biossensor foi comprovada pela espectroscopia de impedância eletroquímica (EIE) e morfologicamente pela microscopia de varredura de efeito de campo (MEV-FEG). O biossensor ECV/AuNps/TLA/HRP foi utilizado na detecção do H2O2 com = 0,43 mmol L-1, sendo próxima ao biossensor Au-SAMmista-HRP. Este biossensor foi aplicado na detecção do catecol (CT) na presença de [H2O2] = 0,03 mmol L-1. Pela técnica de VPD, o biossensor exibiu uma excelente atividade eletrocatalítica para CT na faixa de 6 a 46 μmol L-1, com boa sensibilidade, LD = 0,852 μmol L-1 e o LQ = 2,84 μmol L-1. A estabilidade deste dispositivo é de 25 dias, sendo superior aos outros biossensores desenvolvidos (TLA/HRP e SAMmista/HRP) devido a imobilização ocorrer tridimensionalmente aumentando a quantidade de moléculas da TLA e HRP no dispositivo.

Le projet de ma thèse a consisté en la mise au point de biomatériaux capables d'agir en tant que capteurs moléculaires pour la construction de biocapteurs d'affinité tels que des immunocapteurs, aptacapteurs et capteurs d'ADN, basés sur la lecture électrochimique. Les biocapteurs électrochimiques deviennent une technique intéressante pour l'identification des biomolécules en raison de possibilités de miniaturisation, de faible coût et de la lecture directe des signaux électriques. Toutefois, le choix d'un transducteur, qui permet d'obtenir un signal électrochimique, est crucial dans la construction du biocapteur. Au cours de ma thèse, j'ai eu l'occasion de comparer l'efficacité de différents matériaux conductrices tels que les conducteurs polymères (polypyrrole), les nanotubes de carbone et des nanoparticules d'or. Pour obtenir une réponse électrochimique intense, j'ai associé ces plateformes avec un marqueur redox-ferrocène. Les biocapteurs ont été basés sur la détection directe, généralement avec un «signal off» (diminution de la réponse électrochimique lors de la détection). J'ai travaillé sur différents types de reconnaissance biologique comme anticorps/antigène, aptamer/protéine, sonde ADN/ADN cible. Ces biocapteurs sont particulièrement intéressants dans le domaine de la biologie et de la santé publique. Au début je me suis intéressée à la nouvelle protéine impliquée dans le virus de la grippe et démontrée son évolution dans le cycle viral avec l'objectif de développer de nouveaux médicaments pour cette maladie ainsi que de nouveaux outils de détection. J'ai construit ces biocapteurs basés sur polymère conductrice-polypyrrole associé avec le marqueur redox, ferrocène pour l'immobilisation des anticorps spécifique pour les protéines impliquées dans le virus de la grippe. De nouveaux biorécepteurs - aptamères et des techniques électrochimiques ont été ensuite développés pour concevoir un système sensible capable de détecter la protéine prion cellulaire au niveau pM dans les échantillons de plasma humaine. Les aptamères sont associés sur la plateforme composée de nanotubes de carbone, conjuguées avec des dendrimères poly(amidoamine) PAMAM. Les composites combinent les performances électriques de nanotubes mais permet simultanément l'attachement de nombreux biomolécules, en raison des nombreux groupes amines portant par des dendrimères. Puis j'étais aussi intéressé par la détection de l'ADN par le développement de biocapteurs à base de nanotubes de carbone pour deux maladies infectieuses telles que l'hépatite C avec des cibles d'ADN synthétiques et l'ADN de M. tuberculosis provenant d'échantillons PCR. Ces exemples ont été utilisés pour démontrer que le capteur d'ADN pourrait être généralisé à toutes les maladies infectieuses et utilisé dans le système «point of care». Des études précédentes ont consisté dans le dépôt de nanotubes de carbone sur la surface par adsorption et j'ai trouvé que c'était problématique en termes de reproductibilité. Alors, j'ai utilisé polypyrrole comme une matrice pour l'association des nanotubes de carbone. Cette méthode semble être la plus efficace et a permis de combiner les propriétés des nanotubes avec celles de polypyrrole conducteurs. Au cours de ma thèse, j'ai démontré que les capteurs électrochimiques d'affinité à base de polymères conducteurs et les nanomatériaux peuvent être appliqués dans différents domaines concernant les problèmes de santé. Ces biocapteurs sont prêts pour être intégrés dans les microsystèmes ainsi que dans les systèmes «point of care». / The project of my thesis consisted on the development of biomaterials that are able to act as molecular transducers for the construction of affinity biosensors such as immunosensors, aptasensors and DNA sensors, based on electrochemical reading. Electrochemical biosensors become an attractive technique for the identification of biomolecules due to miniaturization possibilities, low cost and direct lecture of electric signals. However the choice of a transducer, which allows obtaining electrochemical signal, is crucial in biosensor construction. During my thesis I had the opportunity to compare the efficacy of different conducting materials such as conducting polymers (polypyrrole), carbon nanotubes. To obtain an intense electrochemical response, I associated these platforms with a redox marker – ferrocene. The biosensors which I constructed were based on direct detection, usually with “signal off” (decrease in electrochemical response during detection). I worked on different types of biological recognition such as antibody/antigen, aptamer/protein, DNA probe/DNA target. These biosensors are especially attractive in the biological field and public health. First, I was interested in the new protein involved in Influenza virus and demonstrated its evolution in viral cycle with the objective to develop new drugs for this disease as well as new tools for detection. I constructed biosensors based on conducting polypyrrole which was studied extensively in our group. I used this polypyrrole matrix associated with redox marker, ferrocene for immobilization of antibody specific for protein involved in Influenza virus. By this approach I demonstrate that electrochemical biosensors can become effective tools in the daily laboratory work, especially useful for biologists who are often limited by commercially available methods. Then new bioreceptors - aptamers and electrochemical techniques have been developed to design a sensitive system able to detect cellular prion protein at pM level in plasma samples. Aptamers were associated on the platform composed of polypyrrole or carbon nanotubes conjugated with dendrimers poly(amidoamine) PAMAM. Composite combines the high electrical performance of transducers but simultaneously allows attachment of high number of biomolecules, due to numerous amine groups bearing by dendrimers. I was also interested in DNA detection and in the development of biosensors based on carbon nanotubes for two infectious diseases like hepatitis C with synthetic DNA targets and M. tuberculosis DNA from PCR samples. Such examples were used to demonstrate that DNA sensor could be generalised to all infectious diseases and used in point-of-care system. My previous studies consisted on the deposition of carbon nanotubes on the surface by adsorption and I found that it was problematic in terms of reproducibility, so important in biosensor construction. I used polypyrrole as a matrix for carbon nanotubes association. This method seems to be effective and allowed combination of nanotubes properties with those of conducting polypyrrole. During my thesis I demonstrated that electrochemical affinity sensors based on conducting polymers and nanomaterials can be applied in different fields concerning health problems. These biosensors are ready for integration in microsystems for application as analytical tools as well as in point-of-care systems.

Pour la première fois, en associant un microscope à effet tunnel (STM) et un microscope optique inversé,nous avons imagé les plasmons de surface excités électriquement sur un film d’or avec la pointe d’un STM.Par microscopie de fuite radiative, en observant l’image de l’interface air/or et celle du plan de Fourierassocié, nous avons distingué les plasmons propagatifs des plasmons localisés sous la pointe. Les plasmonspropagatifs sont caractérisés par une distance de propagation et une direction d’émission en accord aveccelles de plasmons propagatifs créés par excitation laser sur des films d’or de mêmes épaisseurs. Les fuitesradiatives des plasmons localisés s’étalent jusqu’à l’angle maximum d’observation. Plasmons propagatifs etlocalisés ont une large bande spectrale dans le visible. Si la pointe est plasmonique (en argent), lesplasmons localisés ont une composante supplémentaire due au couplage associé. Pour différents types depointe, nous avons déterminé les intensités relatives des plasmons localisés et propagatifs. Nous trouvonsque chaque mode plasmon (propagatif ou localisé) peut être préférentiellement sélectionné en modifiant lematériau de la pointe et sa forme. Une pointe en argent produit une intensité élevée de plasmons localisés,tandis qu’une pointe fine de tungstène (rayon de l’apex inférieur à 100 nm) produit essentiellement desplasmons propagatifs. Nous avons étudié la cohérence spatiale des plasmons propagatifs excités par la pointe du STM. Avec un film d’or opaque (épaisseur 200 nm) percé de paires de nanotrous nous avons réalisé une expérienceanalogue à celle des fentes d’Young. Des franges d’interférences sont observées. La mesure de leurvisibilité en fonction de la distance des nanotrous donne une longueur de cohérence des plasmons de 4.7±0.5 μm. Cette valeur, très proche de la valeur 3.7± 1.2 μm déduite de la largeur de la distribution spectraledes plasmons, indique que l’élargissement spectral des plasmons propagatifs est homogène.Nous avons aussi étudié la diffusion des plasmons propagatifs excités par la pointe du STM par desnanoparticules d’or déposées sur un film d’épaisseur 50 nm. Nous observons une diffusion élastique et unediffusion radiative. Des franges d’interférences sont observées dans la région d’émission lumineuseinterdite du plan de Fourier, dont la période est inversement proportionnelle à la distancepointe-nanoparticule d’or avec un facteur de proportionnalité égal à la longueur d’onde moyenne desplasmons. Il y a donc interférence entre la radiation des plasmons localisés et la radiation provenant de ladiffusion des plasmons propagatifs sur les nanoparticules d’or. Ceci indique que les plasmons localisés etpropagatifs excités électriquement par la pointe du STM sont différentes composantes du plasmon uniqueproduit par effet tunnel inélastique avec la pointe du STM. Ces résultats originaux sur les plasmons créés sur film d’or par un effet tunnel inélastique localisé à l’échelle atomique (i) élargissent la compréhension du processus et (ii) offrent des perspectives intéressantes pour une association de la nanoélectronique et de la nanophotonique. / For the first time, using a equipment combining a scanning tunneling microscope (STM) and an invertedoptical microscope, we excite and directly image STM-excited broadband propagating surface plasmons ona thin gold film. The STM-excited propagating surface plasmons have been imaged both in real space andFourier space by leakage radiation microscopy. Broadband localized surface plasmons due to the tip-goldfilm coupled plasmon resonance have also been detected. Quantitatively, we compare the intensities ofSTM-excited propagating and localized surface plasmons obtained with different STM tips. We find that the intensity of each plasmon mode can be selectively varied by changing the STM tip shape or material composition. A silver tip produces a high intensity of localized surface plasmons whereas a sharp (radius < 100 nm) tungsten tip produces mainly propagating surface plasmons. We have investigated the coherence of STM-excited propagating surface plasmons by performingexperiments on a 200 nm thick (opaque) gold film punctured by pairs of nanoholes. This work is analogousto Young’s double-slit experiment, and shows that STM-excited propagating surface plasmons have acoherence length of 4.7±0.5 μm. This coherent length is very close to the value 3.7±1.2 μm expected fromthe spectrum, which indicates that the spectrum broadening of STM-excited surface plasmons ishomogeneous. We have also studied the in-plane and radiative scattering of STM-excited propagating surface plasmons bygold nanoparticles deposited on a 50 nm thick gold film. In the Fourier space images, interference fringesare observed in the forbidden light region. This interference occurs between STM-excited localized surfaceplasmons (radiating at large angles from the tip position) and the radiative scattering by the goldnanoparticle of STM-excited propagating surface plasmons. This indicates that STM-excited localized andpropagating surface plasmons are different components of the same single plasmon produced by inelasticelectron tunneling with the STM tip. These results not only broaden the understanding about the excitation process of STM excited surface plasmons but also offer interesting perspectives for the connection between nanoelectronics andnanophotonics.

L’objectif de ce travail de thèse a été de développer l’ingénierie des polymères liquides ioniques(PILs) de type imidazolium ainsi qu’une nouvelle famille de copolymères à blocs apparentés.Des PILs type imidazolium ont été utilisés en tant que polymères réactifs pour la catalyse organique etla modification chimique par post-polymérisation. Divers composés (homopolymères, copolymèresstatistiques de type styrénique, polymères réticulés) stables à l'air, portant divers contre-anions (bromures,hydrogénocarbonates, carboxylates), ont été spécialement conçus via des stratégies de synthèserelativement simples. La génération de carbènes N-hétérocycliques supportés sur polymères (poly(NHC)s)a permis de comparer les performances catalytiques de tous ces précurseurs à travers des réactions deréférence de la catalyse organique. Spécifiquement, les copolymères statistiques type styrénique peuventégalement être fonctionnalisés de manière stoechiométrique par post-polymérisation avec différentssubstrats électrophiles (e.g. CS2, isothiocyanate, métaux de transition).Une nouvelle famille de copolymère à blocs contenant un bloc poly(acétate de vinyle) et un bloc detype poly(bromure de N-vinyl-3-alkylimidazolium), a été synthétisé par CMRP. La capacité de cescomposés à s'auto-assembler en diverses mésostructures en masse comme en solution a ensuite étédémontrée. Des mesures de conductivité ionique ont montré l’influence de la préparation des échantillonset des conditions de mesures sur les valeurs obtenues. Le comportement en solution par la réactivité ioniquedu bloc PIL et la modification chimique du bloc hydrophobe poly(acétate de vinyle) en hydrophilepoly(alcool vinylique) ont permis la formation de différentes nanostructures micellaires.Mots clés : Polymères liquides ioniques, Copolymères à blocs, Imidazolium, catalyse organique,Modification post-polymérisation, Auto-assemblage, Conductivité ionique, nanoparticules d’or. / The aim of this PhD work is to expand the scope of engineered imidazolium-based poly(ionicliquid)s (PILs) and their related PIL-block copolymers (PIL BCPs).The use of the imidazolium-based PILs as true reactive polymers for organocatalysis and post-chemicalmodification is first described. Miscellaneous air-stable PIL derivatives featuring various counter-anions(e.g. bromides, hydrogen carbonates, carboxylates), including homopolymers, statistical copolymers ofstyrenic-type and crosslinked copolymer networks have been specifically designed by relatively simplesynthetic strategies. The generation of related polymer-supported N-heterocyclic carbenes, poly(NHC)s,enables comparing the catalytic performances in selected organocatalyzed reactions. Specific polystyrenebasedcoPILs can be also stoichiometrically derivatized by post-chemical modification using variouselectrophilic substrates (e.g. CS2, isothiocyanate, transition metals).A novel family of imidazolium-based PIL BCPs, namely poly(vinyl acetate)-b-poly(N-vinyl-3-alkylimidazolium bromide)s synthesized by CMRP, is then described. The ability of these compounds toself-assemble into various types of mesostructures in bulk or in solution has been demonstrated. Ionicconductivity measurements evidenced the influence of sample preparation and measurement conditions.The behavior in solution evidenced via the ionic responsiveness of the PIL block but also by post-chemicalmodification of the hydrophobic poly(vinyl acetate) block into hydrophilic poly(vinyl alcohol) theformation of various micelle-like nanostructures.Keywords: Poly(ionic liquid)s, Block copolymers, Imidazolium, Organocatalysis, Post-polymerizationmodification, Self-assembly, Ionic conductivity, Gold nanoparticles / El objetivo de esta tesis fue el desarrollo de polímeros de ingeniería iónicoslíquidos (pils) y tipo imidazolio una nueva familia de copolímeros de bloques relacionados.Lager tipo imidazolio fueron utilizados como reactivos para la catálisis orgánica ymodificación química de polímeros después de la polimerización. Varios compuestos(homopolímeros, copolímeros aleatorios de tipo estireno, polímeros reticulados) estable en elaire, contra de la realización diversos aniones (bromuros, bicarbonatos, carboxilatos), hansido especialmente diseñadas utilizando estrategias de síntesis relativamente simple. Se utilizóla generación de carbenos N-heterocíclicos soportado sobre polímeros (poli (NHC) s) paracomparar el rendimiento catalítico de estos precursores de referencia a través de reacciones decatálisis orgánica. Específicamente, los copolímeros de tipo estireno también se puedenfuncionalizar sustratos por polimerización posterior estequiométricamente con diferenteselectrófilos (por ejemplo, metales CS2, isotiocianato, de transición).Una nueva familia de copolímero de bloque que contiene un poli (acetato de vinilo) y unbloque de poli (bromuro de N-vinil-3-alquilimidazolio) se sintetizó en CMRP. La capacidadde estos compuestos a auto-ensamblan en varias mesoestructuras como entonces se demostrósolución en masa. Mediciones de conductividad iónica han demostrado la influencia de lascondiciones de preparación y medición de la muestra en los valores obtenidos. Elcomportamiento en solución por el bloque PIL reactividad de iones, y la modificaciónquímica del bloque hidrófobo de poli (acetato de vinilo) hidrófilo poli (alcohol vinílico)permitió la formación de nanoestructuras diferentes micelares.Palabras clave: polímeros líquidos iónicos, copolímeros de bloque, imidazolio, catálisisorgánica, Cambiar post-polimerización, auto-ensamblaje, conductividad iónica, lasnanopartículas de oro.

<p>Chromatography is a powerful technique, essential in chemical analyses and preparative separation in industry and research. Many different kinds of chromatographic material are needed, due to the large variety of applications. Detailed methods of characterisation are needed to design new chromatographic materials and understand their properties. In this thesis, confocal Raman spectroscopy and surface enhanced Raman spectroscopy (SERS) have been applied to micrometer-size chromatographic beads, for which these techniques have not been used earlier. New methodology, optimized for use with the chromatographic beads, has been developed and evaluated. </p><p>Confocal spectroscopy has been used to determine distributions of functional groups within single chromatographic beads. This distribution is of great importance in determining the chromatographic properties, since the material is porous and the solute molecules can diffuse inside the beads. Most of the confocal experiments have been performed with Raman spectroscopy; fluorescence spectroscopy, using Nd³⁺ ions or dye-labelled proteins as fluorescence probes, has been used for comparison. </p><p>The concentration of adsorbed analytes is very low within the beads. SERS was therefore used to enhance the Raman signal. SERS-active surfaces were prepared by incorporating gold nano-particles into the interior of the bead. TEM measurements showed that the gold nano-particles could be observed throughout, and it was possible to record analyte spectra from different positions within the bead. Enhanced spectra could be obtained both for small test molecules and for larger bio-molecules, although the spectra for the smaller analytes were much more intense.</p>

Chromatography is a powerful technique, essential in chemical analyses and preparative separation in industry and research. Many different kinds of chromatographic material are needed, due to the large variety of applications. Detailed methods of characterisation are needed to design new chromatographic materials and understand their properties. In this thesis, confocal Raman spectroscopy and surface enhanced Raman spectroscopy (SERS) have been applied to micrometer-size chromatographic beads, for which these techniques have not been used earlier. New methodology, optimized for use with the chromatographic beads, has been developed and evaluated. Confocal spectroscopy has been used to determine distributions of functional groups within single chromatographic beads. This distribution is of great importance in determining the chromatographic properties, since the material is porous and the solute molecules can diffuse inside the beads. Most of the confocal experiments have been performed with Raman spectroscopy; fluorescence spectroscopy, using Nd3+ ions or dye-labelled proteins as fluorescence probes, has been used for comparison. The concentration of adsorbed analytes is very low within the beads. SERS was therefore used to enhance the Raman signal. SERS-active surfaces were prepared by incorporating gold nano-particles into the interior of the bead. TEM measurements showed that the gold nano-particles could be observed throughout, and it was possible to record analyte spectra from different positions within the bead. Enhanced spectra could be obtained both for small test molecules and for larger bio-molecules, although the spectra for the smaller analytes were much more intense.

We have studied fundamental aspects of time-resolved two-photon excited fluorescence depolarisation. The thesis presents experimental as well as theoretical progress. We show that a multi-photon induced instrumental response function obtained from a suspension of gold nanoparticles is appropriate for the analysis of two-photon excited fluorescence decays obtained using time-correlated single photon counting detection. Theoretical expressions have been derived for the fluorescence anisotropy decay obtained upon two-photon excitation of various molecular systems in liquid solutions: a) an anisotropic rigid rotor that undergoes rotational diffusion in the presence of ultrafast unresolved restricted reorientations, e.g. librations. b) a molecular group covalently attached to a stationary macromolecule, and undergoing local reorientation in a uniaxial ordering potential. A new approach to the analysis of two-photon excited fluorescence depolarisation experiments was developed, which combines data obtained by using linearly and circularly polarised excitation light, in a global manner. In the analysis, knowledge about unresolved reorientations was obtained from one-photon excitation studies of the corresponding systems. By means of this procedure it has been possible to obtain quantitative information about the molecular two-photon absorption tensor for perylene and two of its derivatives. Thereby the symmetry of the final excited and intermediate vibronic states could be assigned. The analysis reveals that the two-photon transition studied with the 800 nm laser exhibits mixed character. An important finding from the experiments was that the two-photon absorption tensor appears to be solvent dependent. Furthermore, the thesis presents the first theoretical treatment of two-photon excited donor-donor energy migration in the presence of molecular reorientation and which applies the extended Förster theory. Explicit expressions for molecules that belong to the point groups D2h, D2 and C2v are given. Preliminary experiments are finally also reported on a two-photon excited donor-donor energy migration system consisting of a bisanthryl-bisteroid.

This study focuses on preparing surface enhanced fluorescence (SEF) substrates for use in the enhancement of the emission signal of rhodamine B and fluorescein dyes. Fluorescence spectroscopy has been widely utilized owing to its high sensitivity. SEF is a process where the interactions of fluorophores with the localized surface plasmons of metal nanoparticles results in fluorescence enhancement, increased photostability and rates of system radiative decay which leads to a decreased lifetime. One of the most important factors of SEF studies is to provide a uniform distance between fluorophore and metal nanoparticle in a controlled manner / otherwise, F&ouml / rster resonance energy transfer takes place from fluorophore to metal nanoparticle and emission intensity of fluorophore is quenched. The spherical gold and silver nanoparticles were prepared using the well known and straightforward chemical reduction method, in which sodium citrate acted both as a reducing agent and a stabilizer around the formed nanoparticles. Silver and gold were chosen because of their high plasmon field enhancement. Since plasmon field strongly depends on the shape and size of the nanoparticles, the prepared nanoparticles were characterized using absorption spectroscopy and field emission scanning electron microscopy (FE-SEM). Prior to deposition of silver or gold nanoparticles on glass slides, the slides were derivatized by immersing them into an aqueous solution of 3-Aminopropylethoxysilane (APTES). Following derivatization, silver or gold nanoparticles were deposited by immersing the slides into the colloid mixture. Metal nanoparticle coated slides were characterized using absorption spectroscopy and field emission scanning electron microscopy (FE-SEM). Surface enhanced Raman scattering (SERS) measurements were carried out to observe the plasmon efficiency of the deposited nanoparticles. The SERS measurements were repeated for the duration of two weeks in order to check the stability of the plasmon efficiency. In this study, different types of materials (silica, zinc oxide, gold, stearic acid.) were employed as spacers to observe their effects on fluorescence enhancement. Physical vapor deposition (PVD) and Langmuir-Blodgett (LB) film deposition techniques were used for the formation of the spacer within the substrate. Fluorescence enhancement of rhodamine B and fluorescein was observed on the prepared SEF substrates. Obtained enhancement factors indicate that SEF substrates have the potential for sensitivity improvements of fluorescence sensing in many fields.

Water drops that nucleate and grow over an evaporating polymer solution exposed to a current of moist air remain noncoalescent and self-assemble into close packed arrays. The hexagonally close packed, nearly monodisperse drops, eventually evaporate away, leaving a polymer film, with ordered array of pores. Meanwhile, typical breath figures or dew that form when moist air contacts cold surfaces involve coalescence-assisted growth of highly polydisperse, disordered array of water drops. This dissertation provides the first quantitative attempt aimed at the elucidation of the mechanism of the breath figure templated assembly of the ordered arrays of pores in polymer films. The creation and evolution of a population of close packed drops occur in response to the heat and mass fluxes involved in water droplet condensation and solvent evaporation. The dynamics of drop nucleation, growth, noncoalescence and self-assembly are modeled by accounting for various transport and thermodynamic processes. The theoretical results for the rate and extent of evaporative cooling and growth are compared with experiments. Further, the dissertation describes a rich array of experimental observations about water droplet growth, noncoalescence, assembly and drying that have not been reported in the published literature so far. The theoretical framework developed in this study allows one to rationalize and predict the structure and size of pores formed in different polymer-solvent systems under given air flow conditions. While the ordered arrays of water drops present an example of dynamics, growth and assembly of spherical particles, the study on colloidal gold nanorods focuses on the behavior of rodlike particles. A comprehensive set of theoretical arguments based on the shape dependent hydrodynamics of rods were developed and used for centrifugation-assisted separation of rodlike particles from nanospheres that are typical byproducts of seed mediated growth of nanorods. Since the efficiency of shape separation is assessed using UV-Vis-NIR spectroscopy and transmission electron microscopy (TEM), the present dissertation elucidates the shape dependent parameters that affect the optical response and phase behavior of colloidal gold nanorods. The drying of a drop of colloidal gold nanorods on glass slides creates coffee ring like deposits near the contact line, which is preceded by the formation of a liquid crystalline phase. The assemblies of rods on TEM grids are shown to be the result of equilibrium and non-equilibrium processes, and the ordered phases are compared with two dimensional liquid crystals. The methodology of pattern characterization developed in this dissertation is then used to analyze the structure of the exocuticle of iridescent beetle Chrysina gloriosa. The patterns were characterized using Voronoi analysis and the effect of curvature on the fractions on hexagonal order of tiles was determined. Further, these patterns were found to be analogous to the focal conic domains formed spontaneously on the free surface of a cholesteric liquid crystal. In summary, the dissertation provides the crucial understanding required for the widespread use of breath figure templated assembly as a method for manufacturing porous films, that requires only a drop of polymer solution (dilute) and a whiff of breath! Further, the dissertation establishes the physical basis and methodology for separating and characterizing colloidal gold nanorods. The dissertation also suggests the basis for the formation and structure of tiles that decorate the exoskeleton of an iridescent beetle Chrysina gloriosa.

Cancer is one of the leading causes of death worldwide, and affects roughly 1.5 million new people in the United States every year. One of the leading tools in the detection and treatment of cancer is radiation. Tumors can be detected and identified using CT or PET scans, and can then be treated with external beam radiotherapy or brachytherapy. By taking advantage of the physical properties of gold and the biological properties of nanoparticles, gold nanoparticles (GNPs) can be used to improve both cancer radiotherapy and imaging. By infusing a tumor with GNPs, either using passive extravasation of nanoparticles by the tumor vasculature or active targeting of an antibody-conjugated nanoparticle to a specific tumor marker, the higher photon cross-section of gold will cause more radiation dose to be deposited in the tumor during photon-based radiotherapy. In principle, this would allow escalation of dose to the tumor while not increasing the dose to normal healthy tissue. Additionally, if a tumor infused with GNPs was irradiated by an external kilo-voltage source, the fluorescence emitted by the gold atoms would allow one to localize and quantify the GNP concentration. This work has two main aims: to quantify the GNP-mediated dose enhancement during GNRT on a nanometer scale, and to develop a refined imaging modality capable of quantifying GNP location and concentration within a small-animal-sized object. In order to quantify the GNP-mediated dose enhancement on a nanometer scale, a computational model was developed. This model combines both large-scale and small-scale calculations in order to accurately determine the heterogeneous dose distribution of GNPs. The secondary electron spectra were calculated using condensed history Monte Carlo, which is able to accurately take into account changes in beam quality throughout the tumor and calculate the average energy spectrum of the secondary charged particles created. Then, the dose distributions of these electron spectra were calculated on a nanometer scale using event-by-event Monte Carlo. The second aim is to develop an imaging system capable of reconstructing a tomographic image of GNP location and concentration in a small animal-sized object by capturing gold fluorescence photons emitted during irradiation of the object by an external beam. This would not only allow for localization of GNPs during gold nanoparticle-aided radiation therapy (GNRT), but also facilitate the use of GNPs as imaging agents for drug-delivery or other similar studies. The purpose of this study is to develop a cone-beam implementation of XFCT that meets realistic constrains on image resolution, detection limit, scan time, and dose. A Monte Carlo model of this imaging geometry was developed and used to test the methods of data acquisition and image reconstruction. The results of this study were then used to drive the production of a functioning benchtop, polychromatic cone-beam XFCT system.