Pathogenic microbial contamination of the environment poses a significant threat to human health. Accordingly, microbial surveillance is needed to ensure safe drinking water and air quality. Current analytical methods for microbes are generally either culture-based, gene amplification-based, or sequencing-based. However, these approaches require centralized facilities, well-trained personnel, and specialized instruments that result in high costs and long turnaround times. Surface-enhanced Raman spectroscopy (SERS)-based techniques have been proposed to overcome these limitations. In this dissertation, we discuss work conducted to develop novel SERS-based methods to enable both sensitive microbial quantification and analysis of the interactions of pathogens, their hosts, and the surrounding environment. We first developed a labeled SERS-based lateral flow test for virus quantification. Optimization of the lateral flow design and digital signal analysis enabled high sensitivity towards SARS-CoV-2. To elicit a comprehensive understanding of pathogen infection, label-free living-cell SERS sensors were engineered by incubating host cells with nanoparticles. SERS spectral changes in host cellular components and metabolites during infection were used for viral quantification and offered inherent insights into the temporal and spatial molecular-level mechanisms of infection. These biosensors were validated using bacteriophage Phi6 and then developed for infectious H1N1 influenza. To understand microbial survival in the environment, living-cell SERS methods were applied under various conditions. Results showed cell inactivation and antibiotic treatment induced significant cellular and metabolic responses in the living whole-cell sensors, implying their potential applicability to various environmental conditions. Our research achieves rapid and on-site pathogen quantification and infection mechanism identification. / Doctor of Philosophy / Pathogenic microbes, such as the SARS-CoV-2 virus, can spread through air and water and are potentially harmful to human health. Monitoring the concentrations of these microbes in the environment is crucial to track their presence and provide an early warning of their spread. Unfortunately, current microbial detection methods are often expensive and take a long time since they typically require professional facilities and expert elicitation. Our research relies on a technique called surface-enhanced Raman spectroscopy (SERS) to address these challenges. SERS enables identification and quantification of microbes by analyzing specific features (i.e., peak position, peak intensity) in the spectra. We first applied this technique by modifying a commercial SARS-CoV-2 antigen test kit with a label molecule that provides SERS signals. We achieve accurate and sensitive quantification, even in the presence of high levels of environmental interference. To better understand how these harmful microbes interact with our bodies, we developed sensors that can measure SERS signal changes in host cells before and after infection. These sensors were tested using the bacteriophage virus Phi6 that infects bacteria and infectious H1N1 influenza virus. Furthermore, we applied these sensors to study how bacteria respond to different environmental conditions, providing valuable insights into their survival and behavior under various conditions. In summary, our research introduces methods that are more accessible to identify and quantify harmful microbes that can be potentially used by the general public. The methods provide us with molecular level understanding of pathogen interactions with humans and the environment.

Les fibres optiques utilisées pour le transfert d’information présentent des pertes de signal pendant leur propagation. Ainsi, ces signaux ont besoin d’être régulièrement amplifiés. De nos jours, l’Amplification Raman, basée sur le principe de diffusion Raman stimulée, est une des techniques utilisées pour réaliser ces amplifications. Les nanoparticules de métaux nobles ont des propriétés optiques uniques provenant de l’oscillation collective des électrons lorsqu’elles interagissent avec une onde électromagnétique. Ces particules absorbent fortement le champ électromagnétique à une fréquence appelée fréquence de résonance de plasmon de surface. Ce travail de thèse concerne l’influence des nanoparticules métalliques sur l’amélioration de l’Amplification Raman. Il s’inscrit dans le cadre du projet ANR Fenoptic (2010-2012), réunissant l’entreprise Draka et plusieurs laboratoires français (ICB Dijon, CMCP Paris, LPCML Lyon), qui s’intéressent à l’intégration des nanoparticules de métaux nobles à l’intérieur des fibres optiques afin d’utiliser la résonance de plasmon de surface pour améliorer l’efficacité des amplificateurs optiques. Dans ce travail, différentes sources de nanoparticules métalliques ont été examinées (suspensions, couches, préformes de fibre optique). Les expériences ont porté sur la caractérisation (forme et position du plasmon) de nanoparticules de métaux nobles incluses en matrices vitreuses ainsi que sur des mesures de spectroscopie Raman au travers desquelles le phénomène de Diffusion Raman Exaltée de Surface (SERS) a particulièrement été étudié. Pour la première fois, nous avons mis en évidence l’exaltation du signal Raman d’une matrice vitreuse. / Signals in optical fibers used for the transfer of information are attenuated due to impurities, scattering, absorption… To compensate for these losses, several techniques were developed like Erbium Doped Fiber Amplifier (EDFA). An alternative to rare earth doped fiber amplifier is Raman Amplification, which results from stimulated Raman scattering. Noble metal nanoparticles have optical properties induced by the collective oscillation of their conduction electrons when they interact with an electromagnetic wave. These particles strongly absorb the electromagnetic field at a frequency called surface Plasmon resonance frequency. This work is mainly based on effects leading to the improvement of the Raman Amplification. The ANR project Fenoptic (2010-2012), gathering Draka and several French laboratories (ICB (Dijon), CMCP (Paris), LPCML (Lyon)) is interested in the integration of noble metal nanoparticles in optical fibers using properties of the surface Plasmon resonance to improve the efficiency of optical amplifiers. In this work, different kinds of samples (suspensions, layers, optical fiber performs) with metal nanoparticles were studied. The experiments were based on the characterization (form and position of the Plasmon band) of noble metal nanoparticles in amorphous matrix and Raman spectroscopy was used to study the Surface Enhanced Raman Spectroscopy (SERS) effect. For the first time, we found the Raman signal exaltation of an amorphous matrix.

Les études portent sur les propriétés optiques de nanofils d’or individuels et de réseaux de nanofils d’or. Malgré ses avantages, comme une sensibilité moindre à l’oxydation et sa biocompatibilité, les nanofils en or ont été peu étudiés par comparaison avec les nanofils en argent ou semiconducteurs. Les études sur ces substrats ont été réalisées par spectroscopie Raman exaltée de surface (SERS). Le régime de la molécule unique est atteint, ce que j’ai démontré en utilisant deux molécules différentes de colorant, co-déposées. J’ai étudié la propagation des plasmons de surface dans les nanofils ainsi que son évolution en fonction de la longueur d’onde. Une distance de propagation de 3,8 µm a été observée, plus grande que les valeurs précédemment rapportées. Les réseaux de nanofils ont également été étudiés en combinant la réponse de deux molécules pour démêler les différents processus contribuant au signal de la diffusion Raman. Les résultats obtenus par des études en polarisation et en fonction de la longueur d’onde sur l’évolution de l’intensité du signal SERS ont été confrontés aux résultats de simulations réalisées par la méthode de l’approximation des dipôles discrets (DDA). La microscopie de champ proche optique à balayage (SNOM) a été mise en œuvre pour étudier les effets d’exaltation locale sur les réseaux de nanofils. Ces études ont été réalisées avec deux polarisations croisées et à deux longueurs d’onde différentes. L’originalité des études SNOM repose sur l’utilisation de deux modes différents d’éclairement. L’un est utilisé pour cartographier l’exaltation des champs électromagnétiques, l’autre pour étudier les effets de propagation des plasmons dans les nanofils. / We have reported the optical properties of isolated gold nanowires and of nanowire arrays. Despite the advantages gold has to offer as it is less sensitive to oxidation and as an biocompatible metal, it has been scarcely studied than silver or semiconductors nanowires. We have made surface enhanced Raman spectroscopic (SERS) studies on the isolated nanowires and nanowire arrays. Single molecule regime is attained and has been proven with the aid of two dye molecules that are co-deposited. The propagation of surface plasmons in these nanowires and their evolution with the excitation wavelength have been studied. We report a propagation distance of 3.8 µm which is longer than the values previously reported in literature. Nanowire arrays have been investigated with two dyes again to disentangle the various factors contributing to SERS. Polarization studies and the evolution of enhancement in the nanowires with the wavelength have been reported and explained with the aid of simulations obtained by the discrete dipole approximation (DDA). The scanning near field optical microscopy (SNOM) has been done to investigate the local field enhancements on the nanowire arrays. Two different polarizations and two excitation wavelengths have been used. The original idea of the technique includes the use of two illumination modes which serves two purposes. One, to map the regions of enhanced field and the other to study the propagation effects seen on the nanowire.

Ce travail vise à utiliser des surfaces d'alumine nanostructurées pour guider la croissance et l'organisation de particules métalliques (Ag, Au et AgxAu1-x), et à les tester en tant que substrats SERS-actifs robustes et réutilisables. Nous avons utilisé la spectrophotométrie pour la caractérisation des propriétés optiques résultantes, l'ellipsométrie spectroscopique pour l'extraction des indices optiques et la microscopie électronique en transmission pour les caractérisations structurales. La diffusion Raman exaltée de surface (SERS) a été utilisée pour la détection de molécules de bipyridine adsorbées sur la surface des échantillons, en collaboration avec l’Institut des Matériaux Jean Rouxel de Nantes. Nous étudions d'abord des systèmes de nanoparticules monométalliques et bimétalliques afin de comprendre les modes de croissance de telles assemblées. Nous montrons que des arrangements différents de nanoparticules bimétalliques sont obtenus selon la séquence de dépôt utilisée et qu'un alliage est obtenu à l'issue de recuits ex situ sous vide. Les propriétés optiques en champ proche et lointain de nanoparticules d'alliage AgxAu1-x noyées dans une matrice d'Al2O3 sont comparées numériquement à celles des métaux purs, par la méthode de calcul des différences finies dans le domaine temporel. Les résultats indiquent que l’amplification du champ pour les nanoparticules de métal pur est plus élevée que pour les nanoparticules d’alliage. Enfin, les expériences SERS menées sur un système dichroïque de nanoparticules d’Ag plus ou moins couplées montrent que l'on peut obtenir un signal SERS intense avec des nanoparticules recouvertes. / The aim of this work is to use nanostructured alumina surfaces to guide the growth and to optimize the organization of metallic particles (Ag, Au and AgxAu1-x), and to test those systems as reusable SERS-active substrates. We used spectrophotometry to characterize the resulting optical properties, spectroscopic ellipsometry for the determination of the optical index and transmission electron microscopy for the structural characterizations. Surfaced-enhanced Raman spectroscopy (SERS) was used for the detection of adsorbed bipyridine molecules on the sample surface, in collaboration with the Institut des Matériaux Jean Rouxel at Nantes. We first study systems consisting of monometallic and bimetallic nanoparticles in order to understand the growth modes of such particle assemblies. A particular attention is paid to the influence of the sequential deposition of Au and Ag on the structural and optical properties. We show that different arrangements of bimetallic nanoparticles are obtained according to the deposition sequence used and that an alloy is obtained after ex situ annealing under vacuum. The near-field and far-field optical properties of AgxAu1-x nanoparticle alloys embedded in an alumina matrix are compared numerically by the finite difference time domain method, with those of pure metal nanoparticles. Our results indicate that pure metal nanoparticles exhibit a greater field enhancement than alloy nanoparticles. Finally, SERS experiments conducted with a dichroic system made of coupled Ag nanoparticles show that an intense SERS signal can be obtained with coated nanoparticles.

The light-matter interaction in the near-field of high-Q resonators in SOI and SiN platforms is studied. The interactions of high-Q traveling-wave resonators with both resonant and non-resonant nanoparticles are studied and different applications based on this enhanced interactions in near-field such as high-resolution imaging of mode profile of high-Q resonators, label-free sensing, optical trapping, and SERS sensing are investigated. A near-field imaging system for the investigation of the near-field phenomena in the near-field of high-Q resonators is realized. A new technique for high-resolution imaging of the optical modes in high-Q resonators based on the near-field perturbation is developed that enables to achieve a very high resolution (< 10 nm) near-field image. The prospect of the high Q resonators on SOI platform for highly multiplexed label-free sensing and the effect of different phenomena such as the analyte drift and diffusion and the binding kinetics are studied. Also, the possibility of enhancing nanoparticle binding to the sensor surface using optical trapping is investigated and the dynamic of a nanoparticle in the high-Q resonator optical trap is studied. Furthermore, the interaction between a resonant nanoparticle with a high-Q microdisk resonator and its application for SERS sensing is studied. A model for interaction of resonant nanoparticles with high-Q resonators is developed and the optimal parameters for the design of coupled microdisk resonator and a plasmonic nanoparticle are calculated. The possible of resonant plasmonic nanoparticle trapping and alignment in an SiN microdisk resonator optical trap is also shown.

In this study, we used anisotropic gold nanoparticles (AuNPs) dispersed in castor oil and immobilized in hydrogenated castor oil, for the construction of a material to be used in Surface Enhanced Raman Spectroscopy (SERS-active substrate). The AuNPs synthesized were characterized by absorption spectroscopy in the Ultraviolet-visible region (UV-vis), where it was possible to observe a displacement of the absorption band after preparation of the substrate, from 650 to 690 nm, and by Transmission Electron Microscopy (TEM), which showed obtaining AuNPs with star shape and average size of 100 nm. To investigate the potential SERS applications, the obtained substrates were tested using the sulfur molecules of 4-aminothiophenol (4-ATP), benzothiophene (BTF), thiophene (TF), cysteamine and cysteine as probe molecules. The SERS spectra were obtained by analyzing the substrate after the immersion, for 24 h, in solutions with different concentrations of probe molecules. Using 4-ATP as a probe molecule it was possible to obtain SERS spectra with a concentration range of 2,88x10-2 to 1x10-10 mol L-1, with BTF and TF it was possible to increase its Raman bands from 1x10-1 to 1x10-6 mol L-1. Cysteamine and cysteine were also used as probe molecules, but with only one concentration, 1x10-1 and 5x10-2 mol L-1, respectively. The results showed that the developed material has interesting sites with SERS activity, allowing us to study its interaction with different probe molecules, so it can be applied to different areas, both in qualitative and quantitative analysis, even for molecules in solutions with very small concentrations. The obtained substrate presented EF of the order of 102 to 103. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Neste trabalho, foram utilizadas nanopartículas anisotrópicas de ouro (AuNPs), dispersas em óleo de mamona e imobilizadas em óleo de mamona hidrogenado, para a construção de um material para ser utilizado em Espectroscopia Raman Intensificada por Superfície (substrato SERS-ativo). As AuNPs sintetizadas foram caracterizadas por espectroscopia de absorção na região do Ultravioleta-visível, onde foi possível a observação de um deslocamento da banda de absorção após a preparação do substrato, de 650 para 690 nm, e por Microscopia Eletrônica de Transmissão, as quais mostraram a obtenção de AuNPs na forma de estrelas com tamanho médio de 100 nm. Para a investigação de possíveis aplicações na área de SERS, o substrato obtido foi testado utilizando as moléculas sulfuradas: 4-aminotiofenol (4-ATP), benzotiofeno (BTF), tiofeno (TF), cisteamina e cisteína, como moléculas sonda de interesse. Os espectros SERS foram obtidos através da análise dos substratos após a imersão, por 24 h, em soluções com diferentes concentrações das moléculas sonda. Utilizando o 4-ATP como molécula sonda foi possível a obtenção de espectros SERS com uma faixa de concentração de 2,88x10-2 até 1x10-10 mol L-1, com o BTF e TF foi possível a ampliação de suas bandas Raman de 1x10-1 à 1x10-6 mol L-1. Foram utilizadas também cisteamina e cisteína como moléculas sonda, porém com apenas uma concentração, 1x10-1 e 5x10-2 mol L-1, respectivamente. Os resultados mostraram que o material desenvolvido possui sítios com atividade SERS, o que nos permitiu estudar sua interação com diferentes moléculas sonda, assim podendo ser aplicado em diversas áreas, tanto para análise qualitativa quanto quantitativa, mesmo para moléculas em soluções com concentrações muito pequenas. O substrato obtido apresentou EF da ordem de 102 a 103.

Cette thèse concerne le développement d’approches spectroscopiques infrarouge et Raman exaltées de surface: la spectroscopie infrarouge exaltée de surface (SEIRAS) combinée avec une cellule de perfusion et la spectroscopie Raman exaltée de surface (SERS) couplée avec l’électrochimie. Dans le cadre du premier projet, différentes protéines ont été étudiées : lactose perméase (LacY), complexe I et IM30. Nous avons déterminé le pKa de Glu325 dans LacY sauvage et dans différents mutants portant des mutations dans le centre actif de translocation des protons. Sauvage complexe I a été oxydé avec différents agents oxydants et réduit avec NADH. Spectres différentiels correspondants ont été analysés. Des changements conformationnels dans la protéine IM30, induits par la présence des ions Mg2+, ont été observés.Dans le cadre du deuxième projet, une cellule spectroélectrochimique contenant une grille d’or a été adaptée pour étudier des protéines redox actives. Cette grille d’or sert à la fois de substrat SERS et d’électrode de travail. Cyt c, Hb et Mb ont d'abord été utilisés pour valider la configuration, puis l'approche a été étendue pour étudier une protéine membranaire. / This thesis concerns the development of surface-enhanced infrared and Raman spectroscopic approaches: surface-enhanced infrared absorption spectroscopy (SEIRAS) combined with perfusion cell and surface-enhanced Raman spectroscopy (SERS) combined with electrochemistry. Within the first project different proteins were studied: Lactose Permease (LacY), complex I and IM30.The pKa of Glu325 in LacY WT and in different mutants carrying mutations in the proton translocation active center was determined. WT complex I was oxidized with different oxidizing agents and reduced with NADH. Corresponding redox-induced conformational changes were studied. The evidence was given that Mg2+ ions induce conformational changes in the protein IM30.Within the second project the spectroelectrochemical cell containing gold grid electrode was adopted for the studies of redox active proteins. This gold grid serves both as working electrode and as SERS active substrate. First Cyt c, Hb and Mb were used to validate the setup and then the approach was extended to study a membrane protein.

L'objectif de cette thèse est d'étudier des variations de composition de verres ou l'intégration de nanostructures métalliques pouvant présenter un intérêt pour les capteurs à fibres optiques répartis (CFO). Ces capteurs utilisent le coeur des fibres optiques constitué majoritairement de silice (SiO2) pour sonder la température ou les déformations sur des parcours plurikilométriques de fibres. A cette fin, les rétrodiffusions Raman et Brillouin dans les fibres optiques sont utilisées, la diffusion Raman étant sensible aux variations de température et la diffusion Brillouin aux variations de température et de déformation. L'intensité de diffusion Raman peut-être exaltée au contact de nanostructures de métaux nobles, par l'intermédiaire de l'effet SERS (Diffusion Raman Exaltée de Surface) faisant intervenir notamment la résonance plasmon de surface (SPR), oscillation collective des électrons libres à la surface métallique générant une large amplification du champ électrique local. L'intégration de nanostructures métalliques dans les fibres optiques pourrait permettre d'augmenter l'intensité de rétrodiffusion Raman et améliorer les performances des CFO (sensibilité, temps d'intégration…). Une étude sur des échantillons modèles, constitués de substrats SERS nanostructurés d'or et recouvert de silice par dépôt sol-gel, est présentée dans ce manuscrit. Les résultats SERS ainsi obtenus permettent de caractériser le phénomène physique du SERS de SiO2 et de prédire sa potentielle utilité dans les CFO Raman / The purpose of this thesis is to study composition variations of silicate glasses or the integration of metallic nanostructures that may be relevant for distributed fiber optic sensors (FOS). These sensors use optical fiber cores mainly composed of silica (SiO2) to probe temperature or strain on multi-kilometer route fibers. To measure these effects, Raman and Brillouin backscattering in optical fibers are used, Raman scattering being sensitive to temperature variations and Brillouin scattering to temperature and strain variations. Raman scattering intensity may be enhanced in conjunction with noble metal nanostructures via the SERS effect (Surface Enhanced Raman Scattering) which involves surface plasmon resonance (SPR), a collective oscillation of free electrons at the metal surface generating a large amplification of the local electric field. The integration of metallic nanostructures in optical fibers could increase Raman backscattering intensity and improve FOS performance (sensitivity, integration time…). A study on model samples has been performed with SERS substrates consisting of gold nanostructures and coated with a sol-gel oxide deposition (TiO2, SiO2). The obtained SERS results are used to predict the contribution of metallic nanostructures in Raman distributed temperature FOS

Die Charakterisierung der Protein-Nanopartikel-Wechselwirkungen in komplexen biomolekularen Systemen wie einer lebenden Zelle ist für die Pharma-, Medizin- und Umweltforschung von entscheidender Bedeutung. In solchen biomolekularen Systemen adsorbieren Proteine leicht auf der Oberfläche von Nanopartikeln, die die Proteinkorona bilden. Diese Arbeit konzentriert sich auf die Charakterisierung der Proteinkorona in lebenden Zellen, wobei verschiedene analytische Ansätze kombiniert werden. Experimente mit oberflächenverstärkter Raman-Streuung (SERS) an reinen Proteinlösungen zeigten die Konzentrationsabhängigkeit der Protein-Gold-Nanopartikel-Wechselwirkungen, die zu unterschiedlichen SERS-Spektren führten und ermöglichten die Bestimmung von Proteinsegmenten, die an Citrat-stabilisierte Gold-Nanopartikel binden. In SERS-Experimenten mit lebenden Zellen wurde die Anwesenheit von Proteinfragmenten in der innersten Schicht der Proteinkorona, die als harte Proteinkorona bezeichnet wird, festgestellt. Eine analytische Methode, die Natriumdodecylsulfat-Polyacrylamid-Gelelektrophorese und Hochleistungs-Flüssigchromatographie-gekoppelte Elektrospray-Ionisations-Massenspektrometrie kombiniert, wurde entwickelt, um die Bestandteile der Hartproteinkorona zu identifizieren. Die Proteomics-, SERS- und Cryo-Soft-X-Ray-Nanotomographiedaten, wobei letztere Informationen über die dreidimensionale Ultrastruktur der Zelle liefern, zeigen den Aufnahmemechanismus, die Verarbeitung, die Akkumulationsstelle, die molekulare Umgebung und die induzierten zellulären Reaktionen internalisierter Goldnanopartikel. Diese Arbeit validiert die Verwendung von SERS bei der Analyse der Proteinkorona in der Lösung von Modellproteinen und in lebenden Zellen und präsentiert eine geeignete Methode zur Analyse der unveränderten harten Proteinkorona, die in lebenden Zellen gebildet wird. / The characterization of the protein-nanoparticle interactions in complex biomolecular systems such as a living cell is vital for pharmaceutical, medical, and environmental research fields. In such biomolecular systems, proteins readily adsorb on the surface of nanoparticles forming the protein corona. This thesis focuses on the characterization of the protein corona in living cells combining different analytical approaches. Surface-enhanced Raman scattering (SERS) experiments on pure protein solutions revealed the concentration dependence of the protein-gold nanoparticle interactions resulting in different SERS spectra, and allowed for the determination of protein segments binding to citrate-stabilized gold nanoparticles. In live cell SERS experiments, the presence of protein fragments in the innermost layer of the protein corona, called the hard protein corona, was revealed. An analytical method combining sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography-coupled electrospray ionization mass spectrometry was developed to identify the constituents of the hard protein corona. The proteomics, SERS, and cryo soft X-ray nanotomography data, the latter providing information of the three dimensional ultrastructure of the cell, reveal the uptake mechanism, processing, accumulation site, molecular environment, and the induced cellular responses of internalized gold nanoparticles. This work validates the use of SERS in the analysis of the protein corona in the solution of model proteins and in living cells, and presents a suitable method for the analysis of the unaltered hard protein corona formed in living cells.

No description available.