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).

The aim of the research documented in this thesis was to explore issues associated with the development of instrumentation for life detection and characterisation in a planetary exploration context. Within this aim, the following objectives had to be achieved: 1. To consider current and near-future single molecule detection (ultra-low lower limit of detection) analytical techniques that would be compatible with development into a Space qualifiable in situ analytical instrument for the detection of biomarkers in a planetary exploration context. 2. To practically consider the consequences of Planetary Protection and Contamination Control on the development of a sample return instrumentation in a planetary exploration context. 3. To consider the implications of flying an in situ instrument on-board a stratospheric balloon platform in order to apply them into a specific planetary exploration mission: In order to achieve the objectives described above, the following work was pursued:  A desk-based European Space Agency (ESA) study was carried out which entailed producing a literature review on single molecule detection technologies that had to be validated by the expert community. This was done by organising an International Workshop on Single Molecule Detection Technologies for Space Applications in March 2009 at Cranfield University, UK. The approved technologies then had to be analysed with standard analytical techniques (i.e., tradeoffs) in order to propose a specific technology for development and present its breadboard implementation and test plans at the end of the study.  A sample return experiment implementing PP&CC constraints and protocols was designed, built, tested and flown on-board the ESA, Swedish Space Corporation (SSC), Swedish National Space Board (SNSB) and German Space Agency (DLR) BEXUS stratospheric balloon platform. The biological and engineering results obtained from the sample return flight were then analysed and lessons learnt obtained for future flights.  Another desk-based study was performed to research future stratospheric balloon platforms for the exploration of Venus’ cloud layer. The in situ instrument previously proposed for the detection of biomarkers for planetary exploration missions was then put forward as a possible payload for a Venusian stratospheric balloon platform and approved by experts during the Venus Exploration Analysis Group (VEXAG) conference held in August 2011 in Washington D.C, USA. The first part of the research involved studying ultra-low lower limit of detection technologies as these have the potential to impact significantly on the technological and scientific requirements of future Space missions. Two systems were proposed: one based on Tandem Mass Spectrometry (with Cylindrical Ion Trap analysers) followed by Surface Enhanced Raman Scattering spectroscopy to create an MS/MS-SERS instrument for the detection of astrobiology biomarkers in Martian regolith, Europan ice and samples from Titan’s hydrocarbon lakes; and a second one as a Stand-Alone SERS system for the detection of biomarkers in Enceladean plumes, Venusian clouds and cometary coma. The second part of the research practically explored the design of instrumentation for stratospheric balloon platforms. CASS•E, the Cranfield Astrobiological Stratospheric Sampling Experiment, was a life detection experiment that aimed to be capable of detecting stratospheric microorganisms. The experiment consisted of a pump which drew air from the Stratosphere through a 0.2 μm collection filter which retained any microorganisms and >0.2 μm particulates present in the pumped air. Due to the expected rarity of microbes in the Stratosphere compared to the known levels of contamination at ground level, Planetary Protection and Contamination Control (PP&CC)constraints were introduced. Therefore PP&CC protocols were followed to implement Space qualified cleaning and sterilisation techniques; biobarrier technology was implemented to prevent re-contamination of the instrument after sterilisation; and cleanliness and contamination was monitored throughout assembly, integration and testing. The third part of the research demonstrated how an instrument from the first part of the study could be proposed as a payload on-board a stratospheric balloon platform with a focused mission context, i.e., a life detection mission for Venus. Therefore, the research concluded with the proposal of a payload for a Venus mission based on SERS technology on-board a stratospheric balloon platform to search for life above or in the mid Venusian cloud cover.

Detection and identification of pesticide residues on objects of cultural heritage is a serious and urgent challenge that currently faces many museums, Native American communities, and private collections worldwide. Organic artifact materials, such as wood, animal hide, basketry, textiles, paper, horn and bone, have traditionally been treated with pesticides to eradicate and prevent infestation by insects, rodents, and mold. These poisonous substances can persist for years in the controlled environment of a museum storeroom and present a potential poisoning risk to people who come in contact with the objects. Surface-enhanced Raman spectroscopy (SERS) has the potential to detect volatile organic pesticides in this context. The technique can overcome the insensitivity of normal Raman spectroscopy and fluorescence interference, and make possible detection of many organic compounds in parts per million concentration. This investigation is aimed at evaluating SERS for the detection and identification of volatiles in museums, with emphasis on naphthalene vapor. The potential of several SERS-active materials; Tollens mirrors, gold film over nanosphere arrays, citrate-stabilized colloidal silver, and nanoporous gold; to detect Rhodamine B and naphthalene is investigated. The research also highlights the mechanisms that underlie SERS, and the relationship between substrate nanostructure and SERS performance.

This thesis describes the construction of an Optical Tweezer apparatus to be used in conjunction with a confocal Raman spectrometer. The tweezer utilizes an infrared (λ=1064 nm) laser directed into an inverted microscope with NA=1.4 oil immersion 100x objective lens that strongly focuses the laser light into a sample to function as a single-beam gradient force trap. The long term goal of this research program is to develop a single molecule Raman tweezers apparatus that allows one to control the position of a Raman nanoplasmonic amplifier. This thesis describes the construction of the Raman tweezer apparatus along with several Raman spectra obtained from optically trapped samples of polystyrene fluorescent orange, amine-modified latex beads. In addition, I explored the Raman spectra of bulk cytochrome c mixed with or injected onto Ag aggregates for SERs enhancement.

The advent of nanoscience and nanotechnology has sparked many research forefronts in the creation of materials with control over size, shape, composition, and surface properties.1,2 However, for most of the applications, nanoscale materials need to be assembled into functional nanostructures that exhibit useful and controllable physical properties. Therefore, numerous efforts on the assembly of nanoparticles (NPs) using organic ligands, polymers and polyelectrolytes have been reported.3,4 However, the interactions between NPs are mediated by intervening ligands, which are detrimental to charge transport and limit the thermal stability. Hence, developing a new method to produce solid state nanostructures with direct NP linkage has become a significant challenge. To avoid the bridging ligands and improve the conductivity of NP based solid state structures, a novel strategy has been developed in which colloidal NPs undergo condensation to wet “jello-like” hydrogel with direct interfacial linkage. Then hydrogels can be dried supercritically to produce aerogels.5 Resultant nanostructures exhibit low densities, large open interconnected pores, and high internal surface areas and are containing entirely of colloidal metal NPs.6 Since noble metal NPs have been widely used in applications such as catalysts, sensors, and novel electrochemical device components, we herein expanded the sol-gel method to noble metal NPs to produce a new class of metal aerogels. In the dissertation, the synthesis of hollow Ag hollow NPs, Au/Ag alloy NPs, and Au/Pt/Ag alloy hollow NPs with tunable sizes and physical properties, and their oxidative-assembly into high-surface-area, mesoporous, self-supported gel framework has been achieved. The gelation kinetics have been controlled by tuning the oxidant/thiolate molar ratio that governs the rate of NP condensation, which in turn determines the morphology, optical transparency, surface area, and porosity of the gel frameworks. These low-density mesoporous nano-architectures displaying optical transparency or opacity, enormously surface area, and interconnected meso-to-macro pore structure are promising candidates for catalytic, electrocatalytic, and SERS-based sensing applications. The SERS activity of Au/Ag alloy aerogels has been studied and significant signal enhancement was achieved. The performance of the Au/Pt/Ag aerogel towards methanol oxidation reaction has been studied via cyclic voltammetry and significant electro-catalytic activity was observed.

In the search for the conformation of extant or extinct life in an extraterrestrial setting the detection of organic molecular species which may be considered diagnostic of life is a key objective. These molecular targets comprise a range of distinct chemical species, with recognisable spectroscopic features. This project aims to use these features to develop an in-situ molecular specific Raman spectroscopic methodology which can provide structural information about the organic–inorganic interface. The development of this methodology identified a surface enhanced Raman spectroscopic technique, that required minimal sample preparation, allowed for the detection of selected organic species immobilised on an inorganic matrix and was effective for quantities below those which conventional dispersive Raman spectroscopy would detect. For the first time spectral information was gained which allowed analysis of the organic–inorganic interface to be carried out, this gave an insight into the orientation with which molecules arrange on the surfaces of the matrices. Additionally a method for the detection of organic residues intercalated into the interlamellar space of smectite type clays was developed. An evaluation of the effectiveness of uni and multivariate methods for the analysis of large datasets containing a small number of organic features was also carried out, with a view to develop an unsupervised methodology capable of performing with minimal user interaction. It has been shown that a novel use of the Hotellings T2 test when applied to the principal component analysis of the datasets combined with SERS allows identification of a small number of organic features in an otherwise inorganic dominated dataset. Both the SERS and PCA methods hold relevance for the detection of organic residues within interplanetary exploration but may also be applied to terrestrial environmental chemistry.

Nesta tese foi estudado o espalhamento Raman intensificado pela superfície (SERS) em regime de detecção de uma molécula em eletrodo de prata ativado por ciclos de oxidação e redução. Neste regime, de baixas concentrações, são observadas intensas flutuações de intensidade SERS as quais foram controladas neste substrato pela aplicação de potencial ao eletrodo, o que foi associado a alterações na concentração de moléculas adsorvidas na superfície do eletrodo. Além da dependência com o potencial aplicado, foram estudadas através de simulações Monte Carlo, a contribuição nestas flutuações da constante de adsorção das moléculas, do número de \"hot spots\" (regiões de altas intensificações SERS) e do tipo de \"hot spot\" (em termos de eficiência para detecção de espectros de uma molécula). Através destas simulações foram verificadas flutuações de intensidade muito semelhantes às observadas experimentalmente. Além das flutuações de intensidade foram também observadas flutuações de intensidades relativas, como por exemplo, das relações de intensidades anti-Stokes/Stokes, as quais foram interpretadas segundo um modelo de ressonância, através do qual foi possível estimar as energias de ressonância nos \"hot spots\". Alguns dos resultados indicaram a contribuição de ressonâncias finas, as quais foram interpretadas como resultado de interferências entre ressonâncias de plasmon de superfície. Interferências como estas foram demonstradas através de simulações pelo método DDA (\"Discrete Dipole appoximation\") em modelos simples de \"hot spots\" formados por nanobastões de Au. / In this thesis it was studied surface-enhanced Raman scattering (SERS) at single-molecule detection on Ag electrode activated by oxidation and reduction cycles. At this low concentration limit it was observed strong SERS intensity fluctuations that were controlled by the applied potential to the electrode and this control was associated to changes in surface concentration of adsorbed molecules. Furthermore, it was studied through Monte Carlo simulations the influence of adsorption constant, number of \"hot spots\" (regions of high SERS enhancements) and type of \"hot pot\" (in terms of efficiency for single-molecule detection). With such simulations, it was verified fluctuations of SERS intensities very similar to experimental observations. Besides absolute intensity fluctuations, we also observed fluctuations of relative intensities as, for instance, the. anti-Stokes to Stokes intensity ratios. These fluctuations were interpreted according to a resonance model, which made possible the estimative of resonance energies at the SERS \"hot spots\". Some of these results indicated the existence of sharp resonances that were interpreted as a result of interferences among surface plasmon resonances, which were demonstrated through DDA (Discrete Dipole Approximation) simulations in simple models of \"hot spots\" formed by Au nanorods

The Warburg effect describes the survival advantage of cancer cells in that they can proliferate under low oxygen/hypoxic conditions via a less efficient pathway known as glycolysis. It has not yet been documented at which point, in an oxygen gradient, phenotypic changes occur. Measuring the intracellular redox potential (IRP) and its impact on cellular dynamics would provide greater insight into how disruption of redox homeostasis caused by changes in oxygen concentration leads to aberrant cell signalling and diseases such as cancer. Current techniques in measuring IRP include redox-sensitive fluorescent proteins such as roGFP which is glutathione-specific. Measuring the concentration of one redox couple is, however, not an accurate representation of IRP as it does not necessarily inform about the state of other redox couples. Furthermore, fluorescent biosensors can suffer from photobleaching and may interact with other oxidants. The IRP was measured, in this work, using our newly developed novel-class of surface enhanced Raman scattering nanoparticles which can quantitatively measure the redox potential of cells in vitro. A 'homemade' device was created to keep the cells under fixed pO2 whilst obtaining measurements. The IRP was correlated with the transcriptomic and downstream metabolic profiles of MCF7 breast cancer cells, under perturbed pO2, using 1H NMR spectroscopy (NMR), mass spectrometry (MS) and RNA-sequencing. Discriminatory metabolites were all associated with energy and glucose metabolism. Discriminatory microRNAs were all affiliated with the hallmarks of cancer; the regulation of some is controlled by transcription factors containing redox-sensitive motifs in their DNA binding domains. Multivariate analysis techniques were used to analyse the different data streams in a holistic way that allows the correlation of redox potential, metabolism and transcription.

Vaginal fluid is most often found at crime scenes where a sexual assault has taken place or on clothing or other items collected from sexual assault victims or perpetrators. Because the victim is generally known in these cases, detection of vaginal fluid is not a matter of individual identification, as it might be for semen identification. Instead, linkages can be made between victim and suspect if the sexual assault was carried out digitally or with a foreign object (e.g., bottle, pool cue, cigarette, handle of a hammer or other tool, etc.). If such an object is only analyzed for DNA and the victim is identified, the suspect may claim that the victim’s DNA is present because she handled and/or is the owner of the object and not because it was used to sexually assault her; identification of vaginal fluid residue would alleviate such uncertainty. Most of the research conducted thus far regarding methods for the identification of vaginal fluid involves mRNA biomarkers and identification of various bacterial strains.1-3 However, these approaches require extensive sample preparation and laboratory analysis and have not fully explored the genomic differences among all body fluid RNAs. No existing methods of vaginal fluid identification incorporate both high specificity and rapid analysis.4 Therefore, a new rapid detection method is required. Surface-enhanced Raman spectroscopy (SERS) is an emerging technique with high sensitivity for the forensic analysis of various body fluids. This technique has the potential to improve current vaginal fluid identification techniques due to its ease-of-use, rapid analysis time, portability, and non-destructive nature. For this experiment, all vaginal fluid samples were collected from anonymous donors by saturation of a cotton swab via vaginal insertion. Samples were analyzed on gold nanoparticle chips.4 This nanostructured metal substrate is essential for the large signal-enhancement effect of SERS and also quenches any background fluorescence that sometimes interferes with normal Raman spectroscopy measurements.5 Vaginal fluid SERS signal variation of a single sample over a six-month period was evaluated under both ambient and frozen storage conditions. Vaginal fluid samples were also taken from 10 individuals over the course of a single menstrual cycle. Four samples collected at one-week intervals were obtained from each individual and analyzed using SERS. The SERS vaginal fluid signals showed very little variation as a function of time and storage conditions, indicating that the spectral pattern of vaginal fluid is not likely to change over time. The samples analyzed over the span of one menstrual cycle showed slight intra-donor differences, however, the overall spectral patterns remained consistent and reproducible. When cycle spectra were compared between individuals, very little donor-to-donor variation was observed indicating the potential for a universal vaginal fluid signature spectrum. A cross-validated, partial least squares – discriminant analysis (PLS-DA) model was built to classify all body fluids, where vaginal fluid was identified with 95.0% sensitivity and 96.6% specificity, which indicates that the spectral pattern of vaginal fluid was successfully distinguished from semen and blood. Thus, SERS has a high potential for application in the field of forensic science for vaginal fluid analysis.

This dissertation comprises experimental studies on the synthesis and applications of metallic nanoshells. These are a class of nanoparticles composed of a dielectric core and a thin metallic shell. Metallic nanoshells play an important role in nanotechnology, particularly in nanomedicine, due to their peculiar optical properties. The overall objectives of the dissertation were to improve the fabrication of these nanoparticles, and to demonstrate new applications of these materials in cancer research and spectroscopy. The fabrication of nanoshells is a multi-step process. Previously to our work, the procedures for the synthesis of nanoshells reported in the literature lacked systematic characterization of the various steps. The procedure was extremely time-consuming and the results demonstrated a high degree of size variation. In Chapter 3, we have developed characterization tools that provide checkpoints for each step of the synthesis. We demonstrated that it is possible to control the degree of coverage on the shell for a fixed amount of reagents, and also showed important differences on the shell growth phase for gold and silver. The synthetic optimization presented in Chapter 3 led to an overall faster protocol than those previously reported. Although the improvements presented in Chapter 3 led to a higher degree of control on the synthesis of nanoshells, the variations in the resulting particle population were still too large for applications in single particle spectroscopy and imaging. In Chapter 4, the synthesis was completely reformulated, aiming to narrow the size distribution of the nanoshell colloids. Through the use of a reverse microemulsion, we were able to fabricate ultramonodisperse silica (SiO2) cores, which translate into nanoshell colloids with narrow extinction bands that are comparable to those of a single nanoshell. We then fabricate a library of colloids with different core sizes, shell thicknesses and composition (gold or silver). The localized surface plasmon resonance (LSPR) of these colloids span across the visible range. From this library, two nanoshells (18nm silver on a 50nm SiO2 core, and 18nm gold on a 72nm SiO2 core) were selected for a proof of principle cell imaging experiment. The silver nanoshells were coated with a nuclear localization signal, allowing it to target the nuclear membrane. The gold nanoshells were coated with an antibody that binds to a receptor on the plasma membrane of MCF-7 human breast cancer cells. The nanoshells were easily distinguishable by eye in a dark field microscope and successful targeting was demonstrated by hyperspectral dark field microscopy. A comparison was made between fluorescent phalloidin and nanoshells, showing the superior photostability of the nanoparticles for long-term cell imaging. The results from Chapter 4 suggest that the nanoshells obtained by our new synthetic route present acceptable particle-to-particle variations in their optical properties that enables single particle extinction spectroscopy for cell imaging. In Chapter 5 we explored the use of these nanoshells for single-particle Surface-enhanced Raman spectroscopy (SERS). Notice that particle-to-particle variations in SERS are expected to be more significant than in extinction spectroscopy. This is because particle-to-particle SERS variabilities are driven by subtle changes in geometric parameters (particle size, shape, roughness). Two types of gold nanoshells were prepared and different excitation wavelengths (λex) were evaluated, respective to the LSPR of the nanoshells. Individual scattering spectra were acquired for each particle, for a total of 163 nanoshells, at two laser excitation wavelengths (632.8 nm and 785 nm). The particle-to-particle variations in SERS intensity were evaluated and correlated to the efficiency of the scattering at the LSPR peak. Chapter 6 finally shows the application of gold nanoshells as a platform for the direct visualization of circulating tumor cells (CTCs). 4T1 breast cancer cells were transduced with a non-native target protein (Thy1.1) and an anti-Thy1.1 antibody was conjugated to gold nanoshells. The use of a transduced target creates the ideal scenario for the assessment of nonspecific binding. On the in vitro phase of the study, non-transduced cells were used as a negative control. In this phase, parameters such as incubation times and nanoshell concentration were established. A murine model was then developed with the transduced 4T1 cells for the ex vivo portion of the work. Non-transduced cells were implanted in a control group. Blood was drawn from mice in both groups over the course of 29 days. Antibody-conjugated nanoshells were incubated with the blood samples and detection of single CTCs was achieved in a dark field microscope. Low levels of nonspecific binding were observed in the control group for non-transduced cells and across different cell types normally found in peripheral blood (e.g. lymphocytes). All positive and negative subjects were successfully identified. Chapter 7 provides an outlook of the work presented here and elaborates on possible directions to further develop the use of nanoshells in bioapplications and spectroscopy. / Graduate / 2019-05-03