Tesi sul tema "Tip-Enhanced and Surface-Enhanced Raman Spectoscopies"

Cette thèse a pour objectif le développement de la spectroscopie Raman exaltée de pointe (TERS) pour des applications en milieux liquides, et plus particulièrement pour l’étude de membranes lipidiques et de protéines amyloïdes qui sont impliquées dans des maladies neurodégénératives comme la maladie d’Alzheimer. La TERS s’affranchit de la limite de diffraction de la spectroscopie Raman conventionnelle en combinant la haute résolution spatiale de la microscopie à sonde locale et la spécificité chimique de la spectroscopie Raman exaltée de surface (SERS). En utilisant une pointe de microscope à sonde locale métallisée et effilée au niveau nanométrique, la TERS génère une exaltation localisée du signal Raman au sommet de la pointe. Ceci permet l’étude de biomolécules optiquement non-résonnantes à l’échelle nanométrique sans marquage moléculaire et de manière non-destructive. Les défis clés qui sont traités dans ce travail incluent la fabrication de pointes actives en TERS, l’optimisation d’un nouveau système TERS en réflexion totale interne (RTI) pour des utilisations en environnements liquides, et l’exploitation de données complexes obtenues par imagerie TERS hyperspectrale. Des protéines amyloïdes sous forme de fibrilles de protéine Tau ont été étudiées au moyen de notre instrument de RTI-TERS en prenant des fibrilles induites par de l’héparine comme référence pour évaluer la performance du système. Des études TERS de fibrilles Tau induites par de l’ARN ont donné un aperçu des mécanismes de formation sous-jacents des fibrilles amyloïdes. Par ailleurs, ces données ont été utilisées pour explorer le potentiel des méthodes chimiométriques, telles que l’Analyse en Composantes Principales (ACP) et l’Analyse en Cluster Hiérarchique (ACH), pour leur analyse fine. Ces méthodes ont été évaluées dans le contexte des méthodes plus traditionnelles de sélection de pics individuels. Cette thèse détaille aussi le développement d’un système RTI-TERS compatible avec le milieu liquide et son application à l’étude de bicouches lipidiques supportées en milieux aqueux. Cette avancée permet la caractérisation nanométrique de membranes lipidiques dans des milieux biologiquement pertinents et plus réalistes que l’air. Dans la perspective de futurs travaux examinant les interactions protéines-lipides, ces innovations sont cruciales pour comprendre la formation des fibrilles amyloïdes et leurs effets délétères sur les cellules neuronales. Au final, cette thèse a amélioré la TERS en tant qu’outil pour étudier les structures biomoléculaires à l’échelle nanométrique dans le contexte des maladies neurodégénératives, et le système RTI-TERS optimisé fournit une plateforme pour de futures recherches dans des applications biologiques et biomédicales
The aims of this thesis are the development of tip-enhanced Raman spectroscopy (TERS) for applications in liquid media, specifically for the study of lipid membranes and amyloid proteins which are implicated in neurodegenerative diseases like Alzheimer’s. TERS overcomes the diffraction limit of conventional Raman spectroscopy by combining the high spatial resolution of scanning probe microscopy with the chemical specificity of surface-enhanced Raman spectroscopy (SERS). By employing a metal-coated nano-tapered scanning probe microscopy probe tip, TERS generates a localised enhancement of the Raman signal at the tip apex. This enables the study of optically non-resonant biomolecules at the nanoscale in a label-free and non-destructive manner. The key challenges that are addressed in this work include the fabrication of TERS-active tips, the optimisation of our novel total-internal reflection (TIR)-TERS system for use in liquid environments, and the handling of the complex data obtained from hyperspectral TERS imaging. Amyloid proteins in the form of Tau fibrils were studied using this TIR-TERS setup with heparin-induced Tau fibrils being a benchmark for evaluating the performance of the system. TERS studies of RNA-induced Tau fibrils provided insight into the underlying formation mechanisms of amyloid fibrils. In addition, these data were used to explore the use of chemometric methods, such as Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA), for their fine analysis. These methods were evaluated in the context of more traditional peak-picking methods. This thesis also details the development of a liquid-compatible TIR-TERS system and its application to the study of supported lipid bilayers in aqueous media. This advancement enables the nanoscale investigation of lipid membranes in biologically relevant media, which is more representative compared to TERS in air. With the outlook of future works investigating protein-lipid interactions, these innovations are crucial for understanding amyloid fibril formation and their deleterious effects on neuronal cells. To conclude, this thesis enhances TERS as a tool for studying biomolecular structures in the context of neurodegenerative diseases at the nanoscale, and the optimised TIR-TERS system provides a platform for future research in biological and biomedical applications

L'analyse in situ d'interfaces électrochimiques à l'échelle nanométriques est un enjeu majeur pour la compréhension des mécanismes de transferts de charges et d'électrons dans les domaines du stockage d'énergie ou de l'électrocatalyse. Ce travail a permis le développement de la spectroscopie Raman exaltée de pointe (TERS) en milieu liquide et en conditions électrochimiques. Le TERS permet l'analyse de la structure de molécules ou de matériaux à l'échelle nanométrique du fait de l'exaltation localisée du champ électrique à l'extrémité d'une sonde de microscope à effet tunnel (STM) en or ou en argent. Un dispositif reposant sur l'illumination d'une pointe au travers d'un solvant organique a démontré la possibilité d'imager les inhomogénéités d'une monocouche auto-assemblée sur or. Une seconde approche reposant sur l'exaltation du signal Raman à l'apex d'une pointe de taille nanométrique utilisée comme microélectrode (spectroscopie Raman exaltée de surface de pointe, tip SERS) a permis de suivre la réduction d'une monocouche auto-assemblée et d'améliorer la compréhension de son mécanisme. Afin d'imager la surface d'une électrode polarisée, le couplage d'un STM utilisant une pointe TERS en conditions électrochimiques a montré une résolution latérale de moins de 8 nm pour sonder de variations locales de l'exaltation du champ électromagnétique induites par des singularités géométriques de surface. Par ailleurs, l'analyse TERS de couches organiques formées à partir de sels d'aryldiazoniums a permis de montrer des différences de structures selon type de greffage. Ce travail constitue donc une avancée majeure pour l'analyse locale de surfaces modifiées
The in situ investigation of electrochemical interfaces structures at the nanoscale is a key element in the understanding of charge and electron transfer mechanisms e.g. in the fields of energy storage or electrocatalysis. This thesis introduces the implementation of tip-enhanced Raman spectroscopy (TERS) in liquid and in electrochemical conditions enabling the nanoscale analysis of electrified solid/liquid interfaces through the strong and local electric field enhancement at gold or silver scanning tunneling microscopy (STM) probes. The ability of TERS to image inhomogeneities in the coverage density of a self-assembled monolayer (SAM) through a layer of organic solvent on gold was demonstrated. A TERS-inspired analytical tool was also developed, based on a TERS tip used simultaneously as a single-hot spot surface-enhanced Raman spectroscopy (SERS) platform and as a microelectrode (EC tip SERS). The reduction of an electroactive SAM could then be monitored by electrochemical and in situ SERS measurements. In situ electrochemical STM-TERS was also evidenced through the imaging of local variations of the electric field enhancement on peculiar sites of a gold electrode with a lateral resolution lower than 8 nm. Finally TERS also demonstrated to be efficient in investigating the structure of organic layers grafted either by electrochemical reduction or spontaneously. This work is therefore a major advance for the analysis of functionalized surfaces

Surface- and tip-enhanced resonant Raman scattering (resonant SERS and TERS) by optical phonons in a monolayer of CdSe quantum dots (QDs) is demonstrated. The SERS enhancement was achieved by employing plasmonically active substrates consisting of gold arrays with varying nanocluster diameters prepared by electron-beam lithography. The magnitude of the SERS enhancement depends on the localized surface plasmon resonance (LSPR) energy, which is determined by the structural parameters. The LSPR positions as a function of nanocluster diameter were experimentally determined from spectroscopic micro-ellipsometry, and compared to numerical simulations showing good qualitative agreement. The monolayer of CdSe QDs was deposited by the Langmuir–Blodgett-based technique on the SERS substrates. By tuning the excitation energy close to the band gap of the CdSe QDs and to the LSPR energy, resonant SERS by longitudinal optical (LO) phonons of CdSe QDs was realized. A SERS enhancement factor of 2 × 10³ was achieved. This allowed the detection of higher order LO modes of CdSe QDs, evidencing the high crystalline quality of QDs. The dependence of LO phonon mode intensity on the size of Au nanoclusters reveals a resonant character, suggesting that the electromagnetic mechanism of the SERS enhancement is dominant. Finally, the resonant TERS spectrum from CdSe QDs was obtained using electrochemically etched gold tips providing an enhancement on the order of 10⁴. This is an important step towards the detection of the phonon spectrum from a single QD
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Nanomaterials have the remarkable characteristic of displaying physical properties different from their bulk counterparts. An additional degree of complexity and functionality arises when oxide nanoparticles interact with metallic nanostructures. In this context the Raman spectra due to plasmonic enhancement of iron oxide nanocrystals are here reported showing the activation of spin-waves. Iron oxide nanoparticles on gold and silver tips are found to display a band around 1584 cm−1 attributed to a spin-wave magnon mode. This magnon mode is not observed for nanoparticles deposited on silicon (111) or on glass substrates. Metal–nanoparticle interaction and the strongly localized electromagnetic field contribute to the appearance of this mode. The localized excitation that generates this mode is confirmed by tip-enhanced Raman spectroscopy (TERS). The appearance of the spin-waves only when the TERS tip is in close proximity to a nanocrystal edge suggests that the coupling of a localized plasmon with spin-waves arises due to broken symmetry at the nanoparticle border and the additional electric field confinement. Beyond phonon confinement effects previously reported in similar systems, this work offers significant insights on the plasmon-assisted generation and detection of spin-waves optically induced
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Depuis les années 2000, le développement de la spectroscopie Raman à exaltation de pointe (TERS) a permis l’accès de manière extrêmement localisée aux propriétés structurales et moléculaires à la surface de la matière et à des analyses physico-chimiques combinées. La technologie TERS associe les techniques de microscopie à sonde locale - ici le microscope à force atomique (AFM) - avec le champ proche optique. Elle bénéficie en particulier de la génération, à la surface métaux nobles, de plasmons de surface à l’origine d’exaltation d’ondes électromagnétiques pouvant être confinées dans un volume sub-longueur d'onde à l'extrémité des sondes AFM-TERS. Aujourd'hui le principal verrou technologique en TERS est la conception des sondes AFM en termes de reproductibilité à échelle nanométrique, et de fabrication en série. Ce travail de thèse effectué dans le cadre d’une thèse CIFRE (HORIBA Scientific) a eu pour but de concevoir un nouveau type de sonde AFM-TERS répondant aux exigences de performances et de fabrication actuelles. Pour atteindre cet objectif, une étude de simulation numérique a conduit à proposer une nanostructuration métallique de l’extrémité d’un levier AFM, afin de conduire à une exaltation électromagnétique optimisée. Un procédé de nano- et micro-fabrication a été développé au sein de la plateforme de micro et nano-fabrication de l'IEMN, combinant lithographie électronique et optique, évaporation métallique et gravure sur wafers silicium. Il permet la réalisation en série de sondes AFM dont chaque extrémité est composée d'une nano-antenne métallique de taille sub-longueur d'onde, composée d'un nanodisque supportant un nanocône. La méthode de fabrication proposée permet un contrôle des réponses plasmoniques en termes d’amplification du champ et d’accordabilité de la résonance, qui sont la clé des performances en spectroscopie Raman à exaltation de pointe. Une étude sur l’évaporation inclinée lors du procédé de nano-fabrication développé par lithographie électronique a également été réalisé dans le but de contrôler la forme des nanoparticules – de forme conique à cylindrique avec des parois poreuses -- isolées ou en réseaux denses. Les simulations numériques suggèrent que de tels objets peuvent être des candidats potentiels pour le TERS ou le SERS (spectroscopie Raman à exaltation de surface)
Since the start of the 2000s the evolution of tip-enhanced Raman spectroscopy (TERS) has enabled the simultaneous measurement of localized structural, molecular, and physicochemical properties. TERS technology combines scanning probe microscopy -- atomic force microscopy (AFM) -- with near field optical microscopy. The combined technique is referred to as AFM-TERS. The technique harnesses and exploits the generation of surface plasmons on metal surfaces. These plasmons lead to the generation of confined electromagnetic waves in a sub-wavelength volume at the very tip of the AFM-TERS probe. The main technological challenge today is the design and optimization of an AFM-TERS probe having nanometer-sized dimensions -- and the controlled, reproducible batch fabrication of such structures. The objective of the work presented in this PhD thesis was to design, fabricate, and characterize a new type of AFM probe capable of bettering the current state-of-the-art performances. The PhD was carried out in collaboration with HORIBA and funded partly by a French ‘CIFRE’ grant. In order to meet these objects, comprehensive numerical modelling led to the design of an optimized metal nanostructuring having maximum electromagnetic exaltation -- placed at the extremity of a silicon-based AFM cantilever. A new combined micro and nano fabrication process was developed to achieve this -- to be performed using the existing equipment found in the IEMN cleanroom. The process encompasses techniques such as masking using electron beam (ebeam) lithography and UV photolithography, thermal evaporation of metals and ‘lift-off’ techniques, and highly-controlled dry etching of small silicon mesas structures and deep etching for MEMS cantilever releasing. The process enables the batch-fabrication manufacture of AFM-TERS probes containing matter on the millimeter scale (the silicon probe support), the micrometer scale (the silicon cantilever), and the nanometer scale (the combined metallic disk and cone having sub-wavelength dimensions). This method allows nanostructuring on the optical/plasmonic behavior of TERS probes, the key factor which will lead to higher performance in TERS. Finally, a further study concerning the inclined evaporation of metallic nanostructures via an ebeam-derived lithographic shadow mask was performed in order to control the size and shape of the nanostructuring. The study proved this approach to be feasible. Furthermore, numerical modelling of such structures suggests that they are potential original candidates for both TERS and SERS (surface-enhanced Raman spectroscopy)

Ce travail de thèse s'articule autour des phénomènes d'exaltation de la diffusion Raman grâce aux propriétés optiques des métaux nobles (Or et Argent). Des expériences de Spectroscopie Raman Exaltée de Surface (SERS : Surface Enhanced Raman Spectroscopy) et de Spectroscopie Raman Exaltée par sonde locale (TERS : Tip Enhanced Raman Spectroscopy) ont permis l'exploration des ces phénomènes. Le premier volet de ce travail a consisté en la préparation de substrats « SERS-actifs » et en l'analyse de leurs pouvoir exaltant. Trois types de substrats ont été élaborés au laboratoire afin d'étudier les paramètres d'influence (structuration de la surface, longueur d'onde et polarisation de la lumière incidente, nature du métal, etc...). Le second volet du travail a été consacré à la mise en place d'un dispositif TERS. La conception des pointes métalliques a fait l'objet d'une attention particulière. De plus, un module a été élaboré afin d'associer un système de nano-positionnement de la pointe à un Raman confoncal commercial. Ce module a aussi été conçu pour permettre de focaliser le faisceau laser à l'extrémité de la nano-sonde métallique. La conception des outils ainsi que la compréhension des résultats expérimentaux sont corrélés à une analyse numérique. Les sources électromagnétiques (plasmons de surface et effet de pointe) de l'amplification de la diffusion Raman sont étudiées avec l'appui de simulations numériques par la méthode des éléments finis. Enfin les aspects chimiques des phénomènes d'exaltation sont abordés par DFT (Density Functiunal Theory).

Ce travail de thèse se focalise sur le couplage du microscope à force atomique haute–vitesse (HS-AFM) et de la spectroscopie Raman exaltée de surface (SERS) pour la détection des biomolécules. Nous avons élaboré un protocole de fabrication pour produire les substrats “SERS-actifs”. L’efficacité des substrats de nanoparticules cristalline d’or, d’argent ou bimétallique argent–or a été évaluée. Nous avons étudié l’impact des propriétés optiques et morphologiques des substrats sur l’intensité Raman en analysant des échantillons tests tels que la bipyridine éthylène et le bleu de méthylène. Nous nous sommes interessés à trois problematiques biologiques distinctes par analyses HS-AFM et SERS. Dans un premier cas, nous avons détecté la signature chimique de protéine cytochrome b5. Ce travail a été suivi par des études sur le changement de conformation de la protéine de choc thermique leuconostoc oenos Lo 18 en fonction de la concentration et du pH. La dernière application consiste en l’analyse des interactions membrane – virus. Afin de réaliser les analyses simultanées Raman/AFM, nous avons adapté notre protocole de fabrication pour couvrir la surface des pointes AFM commerciales par des nanoparticules d’or cristallines. Les études de diffusion Raman exaltée par effet de pointe (TERS) ont été effectuées sur les échantillons de disulfure de molybdène pour évaluer la qualité des pointes TERS. Pour finir, nous présentons une nouvelle configuration de couplage HS-AFM et spectroscopie Raman. Nous discutons des modifications et des défis rencontrés
This thesis focuses on the coupling of High–Speed Atomic Force Microscopy (HS-AFM) and Surface Enhanced Raman Spectroscopy (SERS) for biomolecule analysis. We have designed a fabrication protocol to manufacture “SERS-active” substrates. The efficacy of gold, silver and gold-silver bimetallic crystalline nanoparticle substrates were evaluated. We have investigated the impact of optical and morphological features of the substrates on Raman signal intensity by analyzing well-known samples such as bipyridine ethylene and methylene blue molecules. We took an interest in three distinct biological problematics with HS-AFM and SERS analyses. First, we have detected the chemical signature of cytochrome b5 protein. This study was followed by the investigation of conformational changes of small heat shock leuconostoc oenos Lo 18 protein in function of pH level and concentrations. The last application consists to the analyse a membrane and a virus interaction. In order to realize simultaneous Raman/AFM analysis, we have adapted our fabrication protocol to cover the surface of commercial AFM probes by crystalline gold nanoparticles. Tip – Enhanced Raman Spectroscopy (TERS) studies were performed on molybdenum disulfide to evaluate the quality of TERS probes. In the last part of this work, we have designed a new setup to combine Ando’s HS-AFM setup with Raman spectroscopy. We present the modifications that have been carried out and the challenges that we have encountered

Protein structures (cytochrome c) were visualized by TERS measurements on whole mitochondria referring to specific spectral features describing the electronic state of the heme moiety
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Protein structures (cytochrome c) were visualized by TERS measurements on whole mitochondria referring to specific spectral features describing the electronic state of the heme moiety.
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Raman spectroscopy is a powerful analytical method that allows deposited and/or immobilized cells to be evaluated without complex sample preparation or labeling. However, a main limitation of Raman spectroscopy in cell analysis is the extremely weak Raman intensity that results in low signal to noise ratios. Therefore, it is important to seize any opportunity that increases the intensity of the Raman signal and to understand whether and how the signal enhancement changes with respect to the substrate used. Our experimental results show clear differences in the spectroscopic response from cells on different surfaces. This result is partly due to the difference in spatial distribution of electric field at the substrate/cell interface as shown by numerical simulations. We found that the substrate also changes the spatial location of maximum field enhancement around the cells. Moreover, beyond conventional flat surfaces, we introduce an efficient nanostructured silver substrate that largely enhances the Raman signal intensity from a single yeast cell. This work contributes to the field of vibrational spectroscopy analysis by providing a fresh look at the significance of the substrate for Raman investigations in cell research.

Hlavním předmětem této diplomové práce jsou elektromagnetické simulace pomocí metody konečných prvků (FEM) k vyšetření vlivu grafenu na hrotem zesílenou Ramanovu spektroskopii (TERS) a povrchem zesílenou infračervenou absorpční spektroskopii (SEIRA) a k prozkoumání citlivosti sondy skenovacího optického mikroskopu blízkého pole (SNOM) ke složkám elektromagnetického pole v závislosti na parametrech sondy (průměru apertury v pokovení). Nejprve je proveden výpočet TERS systému složeného ze stříbrného hrotu nacházejícího se nad zlatým substrátem s tenkou vrstvou molekul, jehož účelem je porozumění principů TERS. Poté je na molekuly přidána grafenová vrstva, aby se prozkoumal její vliv ve viditelné (TERS) a infračervené (SEIRA) oblasti spektra. Druhá část práce se zabývá výpočty energiového toku SNOM hrotem složeným z pokoveného skleněného vlákna interagujícím s blízkým polem povrchových plasmonových polaritonů. Zde uvažujeme zlatou vrstvu se čtyřmi štěrbinami uspořádanými do čtverce na skleněném substrátu sloužícími jako zdroj stojatého vlnění povrchových plasmonů s prostorově oddělenými maximy složek elektrického pole orientovanými rovnoběžně či kolmo na vzorek. Ve výpočtech hrotem zesílené spektroskopie zjišťujeme, že grafen přispívá pouze malým dílem k zesílení pole ve viditelné oblasti spektra, ovšem v infračervené oblasti má grafen vliv pro záření s energií menší než dvojnásobek Fermiho energie grafenu, pro kterou je hodnota zesílení pole větší než v případě výpočtu bez grafenu. Avšak pro velmi vysoké vlnové délky zesílení pole v přítomnosti grafenu klesá pod (konstantní) hodnotu pro případ bez grafenu. Při studiu citlivosti SNOM hrotu k jednotlivým složkám pole shledáváme, že pro hrot se zlatým pokovením je energiový tok skleněným jádrem hrotu kombinací příspěvků energie prošlé aperturou a periodické výměny energie mezi povrchovým plasmonem šířícím se po vnějším okraji pokovení a mody propagujícími se v jádře. Dále zjišťujeme, že hroty s malou aperturou (či bez apertury) jsou více citlivé na složku elektrického pole orientovanou kolmo ke vzorku (rovnoběžně s osou hrotu), zatímco hroty s velkou aperturou sbírají spíše signál ze složky rovnoběžné s povrchem vzorku. V případě hrotu s hliníkovým pokovením jsou hroty citlivější ke složce pole rovnoběžné s povrchem, což je způsobeno slabším průnikem pole skrze pokovení.

Die Raman-Spektroskopie ist eine der nützlichsten optischen Methoden zur Untersuchung der Phononen organischer und anorganischer Materialien. Mit der fortschreitenden Miniaturisierung von elektronischen Bauelementen und der damit einhergehenden Verkleinerung der Strukturen von der Mikrometer- zur Nanometerskala nehmen das Streuvolumen und somit auch das Raman-Signal drastisch ab. Daher werden neue Herangehensweisen benötigt um sie mit optischer Spektroskopie zu untersuchen. Ein häufig genutzter Ansatz um die Signalintensität zu erhöhen ist die Verwendung von Resonanz-Raman-Streuung, das heißt dass die Anregungsenergie an die Energie eines optischen Überganges in der Struktur angepasst wird. In dieser Arbeit wurden InAs/Al(Ga)As-basierte Multilagen mit einer Periodizität unterhalb des Beugungslimits mittels Resonanz-Mikro-Raman-Spektroskopie und Raster-Kraft-Mikroskopie (AFM) den jeweiligen Schichten zugeordnet. Ein effizienterer Weg um die Raman-Sensitivität zu erhöhen ist die Verwendung der oberflächenverstärkten Raman-Streuung (SERS). Sie beruht hauptsächlich auf der Verstärkung der elektromagnetischen Strahlung aufgrund von lokalisierten Oberflächenplasmonenresonanzen in Metallnanostrukturen. Beide oben genannten Signalverstärkungsmethoden wurden in dieser Arbeit zur oberflächenverstärkten Resonanz-Raman-Streuung kombiniert um geringe Mengen organischer und anorganischer Materialien (ultradünne Cobalt-Phthalocyanin-Schichten (CoPc), CuS und CdSe Nanokristalle) zu untersuchen. Damit wurden in beiden Fällen Verstärkungsfaktoren in der Größenordnung 103 bis 104 erreicht, wobei bewiesen werden konnte, dass der dominante Verstärkungsmechanismus die elektromagnetische Verstärkung ist. Spitzenverstärkte Raman-Spektroskopie (TERS) ist ein Spezialfall von SERS, bei dem das Auflösungsvermögen von Licht unterschritten werden kann, was zu einer drastischen Verbesserung der lateralen Auflösung gegenüber der konventionellen Mikro-Raman-Spektroskopie führt. Dies konnte mit Hilfe einer Spitze erreicht werden, die als einzelner plasmonischer Streuer wirkt. Dabei wird die Spitze in einer kontrollierten Weise gegenüber der Probe bewegt. Die Anwendung von TERS erforderte zunächst die Entwicklung und Optimierung eines AFM-basierten TERS-Aufbaus und TERS-aktiver Spitzen, welche Gegenstand dieser Arbeit war. TERS-Bilder mit Auflösungen unter 15 nm konnten auf einer Testprobe mit kohlenstoffhaltigen Verbindungen realisiert werden. Die TERS-Verstärkung und ihre Abhängigkeit vom Substratmaterial, der Substratmorphologie sowie der AFM-Betriebsart wurden anhand der CoPc-Schichten, die auf nanostrukturierten Goldsubstraten abgeschieden wurden, evaluiert. Weiterhin konnte gezeigt werden, dass die hohe örtliche Auflösung der TERS-Verstärkung die selektive Detektion des Signals weniger CdSe-Nanokristalle möglich macht.

Surface- Enhanced Raman Scattering (SERS) and Tip-Enhanced Raman Scattering (TERS) exploit the enhancement of the local electromagnetic field induced by optical nanoantennas and nano-tips to largely amplify the Raman scattering of molecules located in the near-field of these structures, the so called hot-spots. With SERS or TERS, Raman scattering amplifications up to ten orders of magnitude can be obtained. SERS sensors based on nanoparticles of different materials and shapes have been developed for applications in the detection of pollutants, biomarkers, explosives, food pathogens, etc. Large area, high reproducibility, chemical stability, even flexibility, combined to high enhancement factors are key factors that are driving the research efforts in this field. TERS, in addition to SERS, permits to place and spatially control the position of the hot spot at the tip apex, allowing for imaging with nanoscale resolution or, under proper conditions, atomic scale resolution. Several TERS commercial setups are now available, coupling compact Raman spectrometers with with scanning probe microscopy (SPM) platforms, like atomic force microscopy (AFM), scanning tunneling microscopy (STM) or Shear-Force Microscopy (ShFM). These techniques can offer new original approaches in many fields, such as analytical chemistry, biology and biotechnology, forensic science and cultural heritage. One of the most appealing application field for SERS and TERS is biomolecular spectroscopy. Biomolecular sensors have become fundamental in biomedical research and the human health improvement. One of the crucial applications is in early stage diagnosis of diseases, through the detection, identification and quantification of some specific proteins, generally called biomarkers, that are present in very low quantities in body fluids (sub-nanomolar range). Immunoassays, such as enzyme-linked immunosorbent assay (ELISA), protein arrays, Western blots, immune-histochemistry and immune-fluorescence are well assessed probes The sensitivity is generally in the µM range, although new strategies have been proposed to achieve sub-femtomolar sensitivity. Moreover, these techniques are “indirect” methods, characterized by long operation times (several hours) and, especially at ultralow concentrations, can yield a large number of false positive detection events. Surface- and Tip- Enhanced Raman spectroscopies can tailor molecular sensitivity down to the atto-molar range, and to the single-molecule level in some particular configurations. Different concepts of SERS-based biosensors have been demonstrated so far. Raman dye-labeled sensors exploit SERS-active labels (NPs coated with high Raman cross-section dyes and functionalized with antibodies against the target molecule) to spot proteins, permitting their indirect detection (the signal of the dye is monitored) also in-vivo. Direct, label-free SERS sensors, in which the vibrational fingerprint of the target molecule is used for detection, are, however, desirable. This is due to operational rapidity, simplicity and richness of information embedded in the vibrational spectrum (e.g. on the functional state of a protein). Recently much effort has been addressed to SERS detection of biomolecules in liquid environment, i.e. their natural habitat. New tools for the manipulation of plasmonic nanoparticles in liquid environments like proteins buffered solutions, or in cells cultures are required for this task. The use of optical forces exerted by tightly focused laser beams on micro and nanostructures, is among the most promising emergent strategies for manipulation, control and SERS detection of molecular and biomolecular compounds in liquid. Optical forces can be used to attract or push micro and nano-objects from the focus of a lens (typically a microscope objective) in a contactless way. In particular, the use of the radiation pressure, has no restriction on excitation wavelength and permits to push nanoparticles along the beam direction, permitting to induce and control the formation of SERS-active aggregates in liquid. This latter point is yet largely unexplored among the scientific community. A crucial point for an efficient biosensor is the selective interaction of its active surface with biomolecules. In this framework the employment of bioreceptors increases the affinity of the sensor with the target molecule. Antibodies have been used in many SERS detection schemes for the functionalization of metal nanoparticles, but usually a Raman-active dye molecule is used as label for protein detection, due to the large dimensions of antibodies that prevent biomolecules to feel the plasmonic field enhancement. A very interesting alternative to antibodies is represented by DNA aptamers, whose nucleobases sequence can be properly designed for the specific binding with target biomolecules. Aptamers are more efficient and smaller with respect to antibodies, allowing for a direct characterization of the vibrational fingerprint of the biomolecules selectively linked to DNA strands. In addition chemical manipulation of aptamers is simpler, enabling the possibility to include a free thiol group at the end of DNA sequence for increasing the affinity with gold surfaces of SERS biosensors. Therefore the combination of the high sensibility and specificity within a label free approach, make SERS biosensors very appealing tools that can overcome the limitations of the conventional immunoassays in the way towards early diagnosis of cancer diseases. The work presented in this Thesis was focused, on one side, on the study of the basic electromagnetic processes in SERS, including polarization- issues, targeted at optimizing the excitation geometry of a nanosensor and, on the other side, on the development of new strategies for high sensitivity and specific detection of biomolecules in dry and liquid conditions by means of Surface- and Tip- enhanced Raman spectroscopy. In the initial part the attention has been focused on polarization issues related to the twofold electromagnetic enhancement mechanism in Surface enhanced Raman Scattering (SERS). We have discussed how the re-radiation effect can strongly alter the polarization state of the SERS radiation, influencing the SERS depolarization ratio. We have developed a model to relate the SERS depolarization to the molecular depolarization ratio, an intriguing physical quantity that provides information on the molecular orientation. Furthermore, we have studied, both theoretically and experimentally, the polarization properties of SERS from near-field coupled nanowires excited with circularly polarized light. From a practical point of view, this configuration turns out to be very attractive since the signal intensity becomes insensitive to the exact orientation of the sample, making sensors more robust to optical misalignments. Afterwards, driven by the necessity to develop new SERS sensors featuring large area, low cost, highly reproducibility and field enhancement, we have characterized SERS enhancement of novel nanostructures, namely Au nanocrescents evaporated over monolayers of polymeric nanospheres and asymmetric Au nanoclusters grown on flexible PDMS substrates. In both cases we have found a signal amplification up to four orders of magnitude. We have shown the possibility to employ Au nanocrescents in the detection of haemoglobin, reaching a detection limit of 100 pM. Au/PDMS plasmonic substrates were employed for first measurements on mitochondria, suggesting the possibility to detect via SERS the properties of cytochrome c molecules contained in these organelle. Subsequently we faced the problems related to the in-liquid SERS detection of biomolecules. We have exploited a strategy (LIQUISOR) based on optical forces to push and form SERS-active aggregates of gold nanorods (NR) mixed with proteins. Working on Bovine Serum Albumin (BSA), we have reached a limit of detection of 50nM, we have studied the growth kinetics of the optically induced aggregate, its morphology by SEM images and demonstrated that LIQUISOR can provide quantitative information on the protein concentration. A model has been developed that describes the process of nanorod/protein binding and size increase of the bio-nanorod composite, supported by dynamic light scattering measurements. To assess the efficiency and versatility of the LIQUISOR methodology we have applied it to the detection of Lysozyme (Lys), Hemoglobin (Hgb) and Catalase (Cat). Detection at physiological pH was demonstrated in all cases, reaching sensitivities of few ng/mL (picomolar) and high SERS gains (seven orders of magnitude for Hgb). Finally, we carried out first proof of principle experiments of of high sensitive and selective LIQUISOR detection of MnSOD (4.5 nM) and Ochratoxin A (1 µM), exploiting aptamers to functionalize the gold NRs and add specificity to the LIQUISOR methodology. In the last part of this thesis the attention was moved to Tip enhanced Raman spectroscopy. Firstly we have developed a fast and efficient double-step electrochemical etching for the fabrication of nanotips featuring radius of curvature lower than 35 nm starting from Au wires of 125 µm diameter. Homemade tips were used to obtain TERS spectra of Rhodamine 6G (R6G), Methylene Blue, Crystal violet (CV) and Alizarin. TERS enhancement factors EF higher than four orders of magnitude were obtained. TERS imaging was demonstrated on a molecular film of R6G and CV adsorbed on a flat gold substrate to discriminate aggregates of molecules with a spatial resolution of 3 nm. TERS was finally applied in the biomolecular field to high sensitivity spectroscopy of HypF-N oligomers, analogues of amyloid oligomers, the constitutive elements of amyloid fibrils, responsible for several neurodegenerative diseases. Our first results highlight the potentiality of TERS effect for the detection of small biological systems. Notably, our results evidence the possibility to discriminate among different structural conformation of the oligomers, one of which exhibiting a toxic behavior that leads to the formation of misfolded amyloid fibrils in Parkinson’s disease. Several possible developments are envisaged for both the LIQUISOR and the TERS methodologies. The LIQUISOR is of rapid use (few minutes), experimentally simple (standard micro-spectrometers and commercial nanorods are used), reliable and intrinsically scalable to lab-on-chip devices. Higher specificity can potentially be achieved by centrifuging functionalized NR mixed to the protein in order to separate free molecules in complex fluids from the target proteins interacting with aptamer functionalized NR or employing functionalized surfaces (instead of glass slides) to increase the affinity between BIO-NRCs and substrates and thus to speed up the aggregation process. On the other side higher sensitivity can be potentially achieved adopting silver nanoplatelets, as well as core-shell nanostructures. The use of laser beams in the optical transparency window of biological tissues could enable the application of our scheme in combination with optical injection of nanoparticles into living cells for in-vivo SERS biomolecular detection. TERS, with its high sensitivity and spatial resolution has already demonstrated potentialities in the field of DNA sequencing and can have unique applications in proteomics, allowing for the detection of single amino-acid alterations, e.g. phosphorylation, in complex proteins. The fabrication of new tips, more efficient, less expensive and more reproducible is another future challenge. Besides applications in the nanomedicine field, TERS can have important applications in cultural heritage field, for identification of inks on paper, dyes on statues which can be essential for dating, restoring and conserving the artwork.

Collagen fibrils are the main constituent of the extracellular matrix surrounding eukaryotic cells. Even though the assembly and structure of collagen fibrils is well characterized, very little is known about the physico-chemical properties of their surface which is one of the key determinants of their biological functions. One way to obtain surface sensitive structural and chemical data is to take advantage of the near field nature of surface and tip-enhanced Raman spectroscopy. Using Ag and Au nanoparticles bound to collagen type I fibrils, as well as tips coated with a Ag nanoparticles and a thin layer of Ag, we obtained Raman spectra characteristic of the first layer of collagen molecules at the surface of the fibrils. The most frequent Raman peaks were attributed to aromatic residues such as phenylalanine and tyrosine. We also observed in several instances Amide I bands with a full width at half maximum of 10-30 cm-1. The assignment of these Amide I bands positions suggests the presence of collagen-helices as well as alpha-helices and beta-sheets at the fibril’s surface. As a step towards in vivo characterization of collagen fibrils, fascicles removed from tendons were also examined with surface-enhanced Raman spectroscopy.

Przedmiotem niniejszej rozprawy doktorskiej było określenie struktury oscylacyjnej oraz procesów adsorpcji bradykininy (BK; NH2-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-COOH) i jej czterech antagonistów receptorów B2: [D-Arg0,Hyp3,Thi5,8,L-Pip7]BK, Aaa[D-Arg0,Hyp3,Thi5,8,L-Pip7]BK, [D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK i Aaa[D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK (gdzie: Aaa oznacza kwas 1-adamantylooctowy, L-Pip - kwas pipekolinowy, Thi - L-tienylalaninę, Hyp - L-hydroksyprolinę i D-Phe - D-fenyloalaninę; pozostałe aminokwasy to izomery typu L) na powierzchniach srebra, złota i miedzi, na styku powierzchni ciało stałe/roztwór. Bradykinina, jest hormonem peptydowym mającym wpływ na szereg stanów fizjologicznych i patologicznych m.in. zwiększenie przepuszczalności naczyń włosowatych, pobudzanie zakończeń nerwowych, reakcje zapalne. Dlatego też przedmiotem rozprawy były analogi posiadające zarówno własności antagonistyczne jak i wysokie powinowactwo do receptorów B2. W pracy wykorzystano metody spektroskopii rozproszenia Ramana: klasyczny efekt Ramana (RS), za pomocą którego zdefiniowano strukturę oscylacyjną badanych związków i powierzchniowo wzmocnionego efektu Ramana (powierzchniowo-wzmocnionego efektu Ramana (SERS) oraz rozproszenia Ramana wzmocnionego ostrzem (TERS)), dla związków zaadsorbowanych na odpowiednio przygotowanych w warunkach kontrolowanych (tj. rozmiar "schropowacenia", pH, stężenie) powierzchniach metalicznych. Dla BK określono proces adsorpcji na następujących powierzchniach: koloidalne Ag i Au; szklane płytki pokryte warstwą Au, schropowacona powierzchnia elektrody Ag, elektrochemicznie schropowacone powierzchnie Ag, Au i Cu w zależności od przyłożonego potencjału. Natomiast dla antagonistów receptorów B2 na powierzchniach koloidalnego Ag i Au, schropowaconej elektrodzie Ag oraz elektrochemicznie schropowaconych elektrodach Ag oraz Cu. Ponadto określono proces adsorpcji za pomocą TERS dla BK, [D-Arg0,Hyp3,Thi5,8,L-Pip7]BK oraz [D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK na powierzchniach Ag (koloidalny system i nanopręciki). W celu scharakteryzowania powierzchni metalicznej wykorzystano elektronowy mikroskop skaningowy (SEM) i mikroskop sił atomowych (AFM). W rozprawie, wykazano, że głównie aminokwasy/podstawniki znajdujące się w pozycji 5 i 8 sekwencji aminokwasowej peptydu (L-fenyloalanina (Phe) w przypadku BK; tienylalanina (Thi) lub reszta kwasu pipekolinowego (Pip) w przypadku analogów BK) i C-terminalna L-arginina (Arg9) biorą udział w adsorpcji peptydów na wykorzystanych SERS aktywnych substratach. Określono zmiany orientacji w położeniu pierścienia aromatycznego (fenylowy, tiofenu) oraz alifatycznego (piperydyny) w zależności od wykorzystanej powierzchni metalicznej i warunków eksperymentalnych. Wykazano, że na powierzchniach koloidalnego Au, schropowaconej elektrody Ag, oraz na powierzchniach wykorzystywanych w badaniach za pomocą techniki TERS, proces adsorpcji analogów jest selektywny, jedynie opisane powyżej fragmenty peptydów, zlokalizowane w jego C-końcowym fragmencie oddziałują z powierzchniami metalicznymi. Stwierdzono, że aminokwasy/podstawniki w tych samych pozycjach sekwencji aminokwasowej peptydu (w części C-terminalnej) biorą udział w oddziaływaniu z wykorzystywanymi SERS-aktywnymi substratami, podobnie jak biorą udział w oddziaływaniu z receptorem B2. W rozprawie zaproponowano ogólny model oddziaływania BK i jej analogów z powierzchnią metaliczną.

碩士
國立中興大學
化學系所
102
Because biological samples are usually only available with limited sample volumes, a technique is essential needed to determine the interested species in the small amount of samples non-destructively. Suface enhanced Raman spectroscopy (SERS) has the potential to be this technique as it is non-destructive method and also possible to analyze a sample with few µLs in volume. Therefore, we proposed a SERS detection method based on optical fibers. In this proposal, a thin layer of silver nanoparticles (AgNPs) immobilized on the tip of the optical fiber was used as SERS substrate. Due to the small tip size, only a small volume of sample is required for detection. To prepare these substrates, photo-reduction method was used to grow AgNPs on commercial available plastic optical fibers. The morphologies of AgNPs to match the requirement for SERS measurements were controlled by both the irradiation condition and the chemical reaction system. By probing with scanning electron microscope and Raman microscope,the relationships between the preparation condition, morphologies and the SERS intensities were built. By the optimized condition, the prepared substrates can detect samples with a volume less than 30 nL and the enhancement factor reached 6 order in magnitude.

La concentration locale des messagers chimiques sécrétés par les cellules peut être mesurée afin de mieux comprendre les mécanismes moléculaires liés à diverses maladies, dont les métastases du cancer. De nouvelles techniques analytiques sont requises pour effectuer ces mesures locales de marqueurs biologiques à proximité des cellules. Ce mémoire présentera le développement d’une nouvelle technique basée sur la réponse plasmonique sur des leviers AFM, permettant d’étudier les réactions chimiques et biologiques à la surface des leviers grâce au phénomène de résonance des plasmons de surface (SPR), ainsi qu’à la diffusion Raman exaltée par effet de pointe (TERS). En effet, il est possible de localiser l’amplification du signal Raman à la pointe d’un levier AFM, tout comme le principe de la diffusion Raman exaltée par effet de surface (SERS) basée sur la diffusion de la lumière par des nanoparticules métalliques, et permettant une large amplification du signal Raman. La surface du levier est recouverte d’une nano-couche métallique d’or, suivi par des réactions biologiques pour l’immobilisation d’un récepteur moléculaire, créant ainsi un biocapteur sur la pointe du levier. Une détection secondaire utilisant des nanoparticules d’or conjuguées à un anticorps secondaire permet également une amplification du signal SPR et Raman lors de la détection d’antigène. Ce mémoire démontrera le développement et la validation de la détection de l’immunoglobuline G (IgG) sur la pointe du levier AFM.Dans des projets futurs, cette nouvelle technique d’instrumentation et d’imagerie sera optimisée grâce à la création d’un micro-détecteur protéique généralement adapté pour l’étude de la communication cellulaire. En intégrant le signal SPR à la microscopie AFM, il sera alors possible de développer des biocapteurs SPR couplés à une sonde à balayage, ce qui permettra d’effectuer une analyse topographique et de l’environnement chimique d’échantillons cellulaires en temps réel, pour la mesure des messagers moléculaires sécrétés dans la matrice extracellulaire, lors de la communication cellulaire.
Measurement of the local concentration of chemical messengers secreted by cells may give a better understanding of molecular mechanisms related to different diseases, such as cancer metastasis. Current techniques are not suited to perform such measurements and thus, new analytical techniques must be developed. This Master’s thesis reports the development of a new technique based on the plasmonic response of atomic force microscopy (AFM) tips, which will ultimately allow monitoring of chemical and biological molecules on the surface of a cantilever by use of surface plasmon resonance (SPR) and tip-enhanced Raman scattering (TERS). Indeed, it is possible to localize the enhancement of the Raman signal on the AFM tip using principles associated to surface-enhanced Raman spectroscopy (SERS), based on the absorption of light by nanometer-sized metal particles, resulting in a large enhancement of the Raman signal. The AFM tip was constructed by the deposition of a nanometer-size gold layer, followed by the assembly of a biosensor with a biomolecular receptor. Gold nanoparticles (AuNPs) conjugated with a secondary antibody served as the secondary detection step. In addition, the use of the gold nanoparticles for antigen detection allows an amplification of the SPR and Raman signals. This Master’s thesis will demonstrate the development and validation of a biosensor for immunoglobuline G (IgG) at the tip of an AFM cantilever.This thesis sets the basis for future projects, where this new imaging technique will be developed for monitoring cellular communication by exploiting the plasmonic signal at the AFM tip. Different biosensors will then be developed and coupled to an AFM probe for scanning the chemical environment and detect in real-time chemical messengers secreted in the extracellular matrix in cellular communication.