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.

Collagen is the most abundant protein in the animal kingdom and, together with elastin, forms extensive fibrous networks that constitute the primary structure of the mammalian extracellular matrix, respectively endowing it with the tensile and elastic properties that fulfil its principal role as the passive framework of the body. The fibrous proteins are distinctly hierarchically organised from the molecular scale upwards; for example, the nanoscale tropocollagen monomer assembles in arrays that form the micrometer scale microfibrils and fibrils, and thence into collections of millimetre scale collagen fibres, that in-turn, constitute functional tissues such as skin, tendon and bone. Much is known about the structure at each of these individual scales – collagen being the most extensively researched – and the macromechanics of the fibres are well established. However, far less is known about the micromechanics of these proteins, in particular how the monomers influence the functional mechanics of the macroscopic fibres. In this thesis, I explore the multiscale mechanics of collagen and elastin fibres over a range of hydrations – with fibres in direct contact with aqueous solution, and progressively dehydrated in humidity-controlled environments. I use quasi-static tensile testing to probe the macroscopic mechanical response (Young’s modulus and stress relaxation) of the fibres, and employ Brillouin and Raman microscopy to assess the longitudinal modulus in the GHz range and corresponding molecular properties of the proteins. Brillouin microscopy is an emerging technique in the biomedical field. It enables the all-optical, contact-free and non-destructive testing of tissue micromechanics through detection of frequency shifted light scattered off thermally excited acoustic waves or “phonons” in the GHz range. As one of the first studies of Brillouin light scattering in these fibres, it sets the basis for further investigation of tissue biomechanics. In particular, I provide the full description of the protein fibre micromechanics by performing angular measurements using a so-called platelet-like configuration with sample mounted onto a reflective substrate at 45° angle to the excitation beam. I derive the high-frequency longitudinal modulus, and discuss the results in comparison to the Young’s modulus, in terms of the different frequency and spatial scale of the measurements. I obtained a full description of elasticity using Brillouin spectroscopy applied to dried fibres; however, obtaining the same description in hydrated fibres is a challenge, as the Brillouin spectrum is dominated by water. An assessment of the mechanical differences between type-I and type-II collagens is also given here. Water is known to be a primary determinant of tissue biomechanics, and I identified for the first time, the critical hydration ranges between 100 and 85% relative humidity (RH) for collagen, and around 85% RH for elastin, at which point each macroscopic fibre switched from viscoelastic to plastic-like behaviour. Dehydration below these critical points was shown to severely diminish collagen fibrillar sliding, and completely rob elastin of its ability to reversibly deform under strain. The Young’s modulus increased markedly below these hydrations, and I observed a parallel increase in the longitudinal modulus at high frequencies in each protein, indicating a concomitant increase in stiffness at the two scales. The major difference observed between the two fibrous proteins is that, in the case of elastin, I observe a two-fold increase in the longitudinal modulus as the hydration is decreased from 100 to 21% RH, whilst the Young’s modulus increases by two orders of magnitude. This discrepancy was not observed in collagen, which confirmed that the protein maintained its long-range order in the form of the triple helix at all hydrations employed in this work, whilst the elastin ultrastructure experiences a liquid-to-solid state change at a critical hydration. I demonstrate through the analysis of the low-wavenumber region (< 500 cm-1) of the Raman spectrum, that the increase in molecular stiffness of both proteins, is reflected in an increase in torsional rigidity of the peptide backbone upon dehydration. Moreover in collagen, I observe a reduction in the number of inter-protein water bridges, which I propose causes a collapse of the lateral spacing between monomers and an increase in direct backbone-backbone hydrogen bonding, that further stiffens the fibre. Small strain induced reorientations of the amide III and C–C stretching modes in dehydrated collagen fibres suggest that macroscopic stresses may be transferred to the triple helix, otherwise left unperturbed in the hydrated state. I postulate that this is a result of the degraded intra- and interfibrillar sliding mechanism below the critical hydration. Hence in its dehydrated state, the collagen whole-fibre mechanics are similar to those at the molecular scale. The role of proteoglycans and glycosaminoglycans and their potential connection to hydration, is also discussed. In agreement with previous work, I found no Raman spectral changes as a result of stretching hydrated elastin fibres, indicating that even large strains e.g. 80%, have no significant effect on the structural scale probed by Raman microscopy, nor in the air-dried state where the brittle fibres break at low strains. I suggest this may imply a limited sensitivity of Raman bands to these changes, possibly an indication of elastin’s dynamic ultrastructure, or that stress is dissipated at a higher level of the fibre structure. On the macroscopic scale, it is the poroelastic nature of elastin which controls the stress relaxation under strain, and the elastic recovery is mediated by an interplay of hydrophobic interactions and hydration forces.

O presente trabalho estuda a produção e modificações físico-químicas do grafeno frente a tratamentos térmicos. Em uma primeira etapa, foi investigada a síntese de grafeno pela técnica de Deposição Química a partir da fase Vapor (CVD) sobre fitas de cobre. Nós variamos quatro parâmetros que influenciam no crescimento de grafeno: fluxo de metano (CH4), fluxo de hidrogênio (H2), tempo de crescimento e grau de pureza do cobre. Usando as técnicas de caracterização de espectroscopia Raman e microscopia óptica, observamos que fluxo menor de H2 e fluxo intermediários de CH4 favorecem o crescimento de grafeno de alta qualidade. Além disso, vimos que 15 minutos de crescimento de grafeno é suficiente para cobertura do substrato de cobre com grafeno. Por fim, foi visto que o maior grau de pureza do cobre permite a produção de monocamadas de grafeno mais homogêneas. Numa segunda etapa, foi realizado um estudo com objetivo de entender a interação de hidrogênio com monocamadas de grafeno. Nós usamos amostras de grafeno depositadas em filmes de SiO2 (285 nm)/Si e tratadas termicamente em atmosfera controlada de deutério (99,8%) em temperaturas entre 200 e 800 °C. Nós também investigamos a dessorção de hidrogênio do grafeno usando amostras previamente tratadas em deutério a 600 °C e depois tratadas em atmosfera controlada de nitrogênio em temperaturas entre 200 e 800 °C. Após os tratamentos, análise por reação nuclear (NRA) foi realizada para quantificar o deutério, onde nós observamos uma grande incorporação de deutério no grafeno acima de 400 °C, tendo um aumento moderado até 800 °C. Nós também observamos que a dessorção do deutério do grafeno ocorre apenas em 800 °C, embora a dessorção de deutério do óxido de silício ocorra a partir de 600°C. Espectroscopia Raman também foi realizada após cada tratamento térmico. Os resultados mostram que os defeitos na estrutura do grafeno têm um grande aumento para as etapas de maior temperatura na incorporação de deutério. Análises realizadas com Espectroscopia de Fotoelétrons Induzidos por Raios X (XPS) mostraram que a incorporação de deutério para maiores temperaturas causa o "etching" do grafeno. Por fim, caracterizações usando Espectroscopia de Absorção de Raios X (NEXAFS) mostraram que o deutério liga-se ao grafeno sem orientação preferencial. / The present work studies the production and physical-chemical modifications of the graphene under thermal annealings. In a first study, the graphene synthesis by Chemical Vapor Deposition (CVD) on copper foils was investigated. We varied four parameters that influence the growth of graphene: methane flow (CH4), hydrogen flow (H2), growth time and copper purity. Using Raman spectroscopy and optical microscopy, we observed that lower flux of H2 and intermediate flux of CH4 leads to the growth of high quality graphene. In addition, we observed that 15 minutes growth of graphene is sufficient to cover the copper substrate. A higher copper purity allows the production of homogeneous graphene monolayers. In a second step, a study was carried out to understand the interaction of hydrogen with graphene monolayers. We used graphene samples deposited on SiO2 (285 nm)/Si films and annealed in a controlled atmosphere of deuterium (99.8%) at temperatures between 200 and 800 °C. We also investigated the hydrogen desorption of graphene using samples previously treated in deuterium at 600 °C and then annealed in a controlled atmosphere of nitrogen at temperatures between 200 and 800 °C. After the annealings, nuclear reaction analysis (NRA) was performed to quantify the deuterium, where we observed a large incorporation of deuterium in graphene above 400 °C, with a moderate increase up to 800 °C. We also observed that desorption of deuterium occurs only at 800 °C, although deuterium desorption from silicon oxide occurs at 600 °C. Raman spectroscopy was also performed after each annealing. The results show that defects in the structure of graphene have a large increase for deuterium incorporation. Analyzes carried out with X-ray Photoelectron Spectroscopy (XPS) showed that the deuterium incorporation at higher temperatures leads to graphene etching. Finally, characterizations using X-ray Absorption Spectroscopy (NEXAFS) showed that deuterium binds to graphene without preferential orientation.

Técnicas espectroscópicas vibracionais Raman e infravermelho foram utilizadas no estudo conformacional de algumas proteínas de interesse biológico, no que se refere a aspectos estruturais de seus esqueletos polipeptídicos e microambientes de cadeias laterais de certos resíduos de aminoácidos. O trabalho foi dividido em dois grupos, de acordo com os sistemas em estudo: a) Toxina &#947; do veneno de escorpião brasileiro Tityus serrulatus. A análise vibracional revelou que o esqueleto polipeptídico dessa proteína consiste de diferentes estruturas secundárias, com predominância de folhas &#946;, seguida por estruturas do tipo não regular e &#945;-hélice, com alguma evidência de dobras &#946;. Conformação gauche-gauche-gauche foi detectada para os segmentos CCSSCC das quatro pontes dissulfeto. A intensidade do dubleto Raman da Tyr a 853 e 828 cm-1 indicou que 4 dentre 5 resíduos de Tyr encontram-se expostos na superficie molecular. Também há indicações de que os 3 resíduos de Trp apresentem localização externa. Sob o ponto de vista qualitativo, as características conformacionais da toxina no estado sólido amorfo e em solução são virtualmente as mesmas. b) Fosfolipases A2 de pâncreas de porco e de Crotalus durissus terrificus. Mudanças confarmacionais foram detectadas para as moléculas de fosfolipase como consequência de diferentes condições experimentais tais como mudança de estado físico, presença de certas espécies iônicas e interação com um análogo de substrato e com o próprio substrato. Características conformacionais discrepantes foram observadas para a forma sólida amorfa e forma cristalina da fosfolipase pancreática. Transições conformacionais foram detectadas para a transformação zimogênio &#8594; fosfolipase A2 e diferentes conteúdos estruturais foram calculados para a forma tóxica e atóxica dessa enzima. Todas essas mudanças conformacionais envolveram basicamente a arquitetura do esqueleto polipeptídico, não afetando a conformação das cadeias laterais dos resíduos de amino ácidos. As pontes dissulfeto apresentaram consistentemente uma conformação ggg a qual não foi perturbada por nenhuma das condições experimentais empregadas. A ocorrência externa de resíduos de triptofano constituiu uma característica comum para os sistemas ensaiados, assim como a localização predominante de residuos de tirosina em microambientes hidrofílicos, provavelmente na superficie molecular. / Raman and infrared spectroscopies were used to investigate conformational features of some proteins of biological interest, in what concerns structural aspects of their polypeptide backbones and microenvironments of certains amino acid residue side-chains. The work has been divided in two groups as related to the systems studied: a) Toxin &#947; from the venom of Brazilian scorpion Tityus serrulatus. The vibrational analysis has revealed that the protein polypeptide backbone consists of different secondary structures, with predominance of &#946;-sheet, followed by unordered structure and &#945;-helix, with some evidence of &#946;-turns. A gauche-gauche-gauche (ggg) conformation for the CCSSCC fragments of the four dissulfide bridges has been detected. The intensity of the Tyr Raman doublet at 853 and 828 cm-1 indicated that 4 out of the 5 Tyr residues are exposed at the molecular surface. External localization of the 3 Trp residues has also been indicated. Under a qualitative point of view, conformational features of the toxin the amorphous solid state and in solution were virtually the same. B) Crotalus durissus terrificus and porcine pancreatic phospholipases A2. Conformational changes were detected for the phospholipases molecules as a consequence of different conditions such as change of physical state, presence of certain ionic species and interaction with a model substrate analog and with the substrate itself. Amorphous and crystalline solid pancreatic phospholipases presented discrepant conformational features. Conformational transitions were detected for the pancreatic zymogen &#8594; phospholipase A2 transformation and different secondary structures contents were observed for the toxic and the non toxic phospholipase melecules. All those structural changes heve been shown to involve primarily the architecture of the polypeptide backbone rather than the conformation of amino acid residue side-chains. Disulfide bridges have shown consistently a ggg conformation which has not been disturbed by any of the experimental conditions employed. The external occurrence of tryptophan residues has been a common feature for the systems assayed, as well as the predominant localization of tyrosine residues in hydrophylic environments, probably at the molecular surface.

Uma questão ambiental preocupante é a contaminação do meio ambiente por substâncias que interagem com os sistemas endócrinos de seres humanos e animais. Tais substâncias são denominadas desreguladores endócrinos e, como outros poluentes ambientais, apresentam uma variedade de fontes e grande potencial agressor à saúde humana. Neste trabalho, buscamos a aplicação de técnicas de espectroscopia vibracional e técnicas eletroquímicas ao estudo de desreguladores endócrinos, em termos de sua detecção, caracterização, estudo de sua reatividade, desenvolvimento de métodos para sua degradação fotoquímica, estudo dos mecanismos envolvidos e características dos produtos de degradação. O enfoque é voltado ao uso de técnicas espectroscópicas, com especial destaque para o desenvolvimento de metodologias de espectroscopia vibracional intensificadas, associadas ou não a sistemas eletroquímicos. Dentre os compostos considerados desreguladores endócrinos, o nosso trabalho é dedicado ao estudo fotoquímico e espectroeletroquímico das seguintes substâncias: o difenilalcano bisfenol A (BPA), os herbicidas triazínicos ametrina e atrazina, e os compostos organoclorados ácido 2,4-diclorofenoxiacético (2,4-D) e 2,4-diclorofenol (2,4-DCF). O estudo de processos de fotólise e degradação fotocatalítica mediada por TiO2 foram realizados para o BPA e para a ametrina. Tais processos mostram-se importantes para a remoção destes compostos do meio ambiente. Os estudos fotocatalíticos foram estendidos ainda para o uso de compósitos TiO2-Pr como catalisadores. Foi observado que a dopagem do TiO2 pelo íon terra rara praseodímio resulta em alterações nas propriedades físico-químicas do material semicondutor, sendo um método valioso para o aumento da atividade fotocatalítica do TiO2. Conclui-se ainda pela necessidade do controle da porcentagem de modificação química para a otimização do processo fotocatalítico, sendo a proporção de 1% praseodímio considerada ótima. O estudo de processos de fotólise e degradação fotocatalítica do BPA mediado por TiO2 mostra diferenças significativas entre o processo de degradação e mineralização, relacionadas à formação de intermediários orgânicos recalcitrantes. Para a ametrina observa-se que a fotólise e a fotocatálise envolvem mecanismos de degradação distintos. Estudos espectroeletroquímicos dos desreguladores endócrinos (DEC) foram conduzidos através de espectroscopia Raman intensificada (efeito SERS) visando a compreensão da natureza da interação química entre os adsorbatos e superfícies metálicas nanoestruturadas, bem como a caracterização vibracional dos produtos de processos faradáicos desses compostos sobre eletrodos. Produtos de reações químicas de oxidação e redução dos DEC foram identificados através de modificações espectrais em função de potenciais eletroquímicos anódicos e catódicos. / An environmental issue of concern is contamination of the environment by substances which interact with the endocrine systems of humans and animals. Such substances are called endocrine disruptors (ED) and, like other environmental pollutants, may produce adverse effects in human health. In this work, we explore the application of vibrational spectroscopy and electrochemical techniques in the study of ED, in terms of their detection, characterization, study of their reactivity, development of methods for their photochemical degradation, study of the involved mechanisms and characteristic of degradation products. The approach employs the use of advanced spectroscopic techniques, with special attention for the development of methodology of surface-enhanced vibrational spectroscopy, associated or not with electrochemical systems. Among the compounds thought to cause endocrine disruption, the present study is dedicated to the photochemical and electrochemical study of the following substances: the plasticizer bisphenol A (BPA), the triazinic pesticides ametrine and atrazine, and the organochlorine compounds 2,4-dichlorophenoxyacetic acid and 2,4-dichlorophenol. The study of photolytic and TiO2-mediated photocatalytic degradation was carried out for BPA and ametrine. Such processes are important for the removal of such substances from the environment. The photocatalytic studies were extended to include the use of TiO2-Pr composite as catalysts. The doping of TiO2 by the rare earth ion praseodymium results in changes in the physicochemical properties of semiconducting material, being a valuable method for enhancing the photocatalytic activity of TiO2. Our results showed the need of percentage control of the chemical modification for the optimization of the photocatalytic process, with the 1% praseodymium/titanium ration the optimal composition. The study of photolysis and TiO2-mediated photocatalytic degradation processes of BPA shows significant differences between the degradation and mineralization, suggesting the production of recalcitrant organic compounds. For ametrine, photolysis and photocatalytic degradation processes were found to occur via different mechanisms. Spectroelectrochemical studies of endocrine disruptors (ED) were carried out through surface-enhanced Raman spectroscopy (SERS effect) aiming at the understanding of the nature of the chemical interaction between these molecules and nanostructured metallic surfaces, and at the vibrational characterization of the products formed by faradaic charge transfer processes. Products of oxidation and reduction chemical reactions were identified through spectral changes observed in the SERS spectra as a function of cathodic and anodic potentials.

Micro-mechanical Raman spectroscopy is an excellent tool for direct stress measurements in the structure. The presence of mechanical stress changes the Raman frequency of each Raman modes compared to the Raman frequencies in absence of stress. This difference in Raman frequency is linearly related to stress induced and can be calibrated to stress by uniaxial or biaxial tension/compression experiments. This relationship is not generally linear for non-linear behavior of the materials which limits its use to experimentally study flow stress and plastic deformation behavior of the material. In this work strontium titanate ceramic particles dispersed inside epoxy resin matrix were used to measure stress in epoxy resin matrix with non-linear material behavior around it. The stress concentration factor between stress induced inside ceramic particles and epoxy resin matrix was obtained by non-linear constitutive finite element model. The results of finite element model were used for training a machine learning model to predict stress in epoxy resin matrix based on stress inside ceramic particles. By measuring stress inside ceramic particles using micro-mechanical Raman spectroscopy, the stress inside epoxy matrix was obtained by pre-determined stress concentration factor.

Neste trabalho foi realizado o estudo espectroscópico (UV-VIS-NIR, FTIR, Raman e RMN) dos produtos de polimerização da anilina com persulfato de amônio em condições de síntese diferentes da utilizada na síntese padrão da polianilina (pH inicial = 1). A reação foi realizada em meio de HCl (pH inicial = 3) e em meio tamponado (pH = 3). No primeiro caso foi observada a formação majoritária de oligômeros de anilina tipo cabeça-cauda, enquanto que em meio tamponado são formadas macromoléculas com segmentos do tipo 1,4 de Michael de anilina e benzoquinona monoimina com diferentes graus de hidrólise/oxidação, além de unidades tipo fenazina. Esta caracterização está de acordo com as propostas distintas de dois grupos (Surwade et al. e Stejskal et al.), que a princípio pareciam contraditórias. Ainda com relação aos produtos obtidos em meio tamponado (pH = 3), é observada a formação de microesferas com diâmetro entre 3-7 &#181;m. A microscopia Raman juntamente com a espectroscopia Raman ressonante revelou uma correlação entre estrutura tipo fenazina e a formação das microesferas. / In this work it was performed the spectroscopic study (UV-VIS-NIR, FTIR, Raman e NMR) of the polymerization products of aniline and ammonium persulfate using different reaction conditions in relation to the standard polyaniline synthesis (initial pH = 1). The reaction was performed in HCl medium (initial pH =3) and in buffered medium (pH = 3) In the first case it was observed the major formation of aniline oligomers with head-to-tail coupling, while in buffered medium macromolecules with 1,4 Michael-type segments between aniline and benzoquinone monoimine with different hydrolysis/oxidation ratio of iminic groups are formed, in addition to phenazine-like units. This characterization is in accordance to the distinct proposals of two groups (Surwade et al. e Stejskal et al.), which at first seemed contradictory. Again, relative to the products obtained in buffered solution (pH = 3), it is observed the formation of microspheres whose diameter range is 3-7 &#181;m. The use of Raman microscopy and resonance Raman spectroscopy revealed a correlation between phenazine-like structure and the microspheres formation.

Several traditional methods have been used to characterise bacteria, such as biochemical, morphological and molecular tests; however, these methods are time-consuming and not always reliable. Recently, modern analytical techniques have emerged as powerful tools offering high-throughput, reliable and rapid analysis in applications, such as clinical and microbiology studies. A variety of modern analytical techniques have been employed for bacterial characterisation, including matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), liquid chromatography-mass spectrometry (LC-MS), Fourier transform infrared (FT-IR) spectroscopy and Raman spectroscopy. This thesis focused on developing a robust MALDI-TOF-MS methodology to generate mass spectra profiles for the discrimination of clinically-significant bacteria. The data generated from MALDI-TOF-MS analysis are significantly influenced by a number of experimental factors, namely instrument settings, sample preparation, the choice of matrix, matrix additives and matrix preparation as well as sample-matrix deposition methods. The need to optimise experimental variables for bacterial analysis using MALDI-TOF-MS was evident despite the increased application of this analytical tool for clinical microbiology. Experimental optimisation revealed that the choice of matrix is the most important element in MALDI-TOF-MS analysis. Based on this study, a number of different matrices were used to obtain more reproducible mass spectra to classify bacterial samples using a rapid and effective approach. Studies in this thesis indicated that sinapinic acid (SA) is the best matrix for the analysis of proteins from intact bacteria, while 6-aza-2-thiothymine (ATT) and 2,5-dihydroxybenzoic acid (DHB) produced promising results for the analysis of lipid extracts from bacteria. Analytical techniques in combination with multivariate analysis, such as principal components analysis (PCA) and principal component-discriminant function analysis (PC-DFA), were used for bacterial discrimination. Classification was initially undertaken using MALDI-TOF-MS analysis, and subsequently FT-IR spectroscopy, Raman spectroscopy and LC-MS were performed to confirm the classification results. Two main types of bacteria were used for this analysis: 34 strains from seven Bacillus and Brevibacillus species and 35 isolates from 12 Enterococcus faecium strains. The findings showed that the four analytical techniques provide clear discrimination between bacteria at these different levels. Classification of different Bacillus and Brevibacillus bacteria using MALDI-TOF-MS analysis of extracted lipids was confirmed by LC-MS data. In addition, MALDI-TOF-MS data based on extracted lipids and intact bacterial cell proteins were very similar. MALD-TOF-MS analysis of intact enterococci cells produced successful classification with 78% correct classification rate (CCR) at the strain level. FT-IR and Raman spectroscopic data produced very similar bacterial classification with CCR of 89% and 69% at the strain level, respectively. However, classification based on MALDI-TOF-MS data and that based on spectroscopic data were slightly different (Procrustes distance of 0.81, p < 0.001, at the species level). Overall, the findings in this thesis indicate the potential of MALDI-TOF-MS as a rapid, robust and reliable method for the classification of bacteria based on different bacterial preparations.

Protein engineering strategies, such as directed evolution, generate large libraries of enzyme variants, typically in the range of 106-108 variants. However, the availability of rapid, robust high-throughput screening methods has often limited the impact of directed evolution in discovering enzymes with enhanced catalyst performance. Raman spectroscopy is an established analytical technique, providing molecular specific information, permitting analysis in aqueous solutions and as such is an attractive, alternative screening method for biological systems. Although an inherently weak physical phenomenon, enhanced Raman scattering techniques, such as surface enhanced Raman scattering (SERS) and ultraviolet resonance Raman (UVRR) spectroscopy, can be used to overcome the associated sensitivity issues. Herein, we successfully monitored xanthine oxidase (XO) catalysed conversions of xanthine to uric acid, before extending to hypoxanthine, using two contrasting Raman scattering enhanced approaches. Firstly, a SERS-based assay was developed utilising silver nanoparticles to measure analytes directly and quantitatively on micromolar scale, in the absence of chromogenic substrates or lengthy chromatography. Secondly, a UVRR approach was developed enabling monitoring of the XO-mediated reaction in real-time and without the need to quench the system. Significantly, both methods demonstrated over &gt;30 fold reduction in acquisition times (when compared to conventional HPLC analysis), and offered excellent medium-term reproducibility and accuracy of results over significant time periods. Furthermore, investigations were made into developing this SERS-based assay into an enantiomeric screen using another vibrational spectroscopy approach, Raman optical activity (ROA), along with circular dichroism (CD). Successful chiral reduced nanoparticles were synthesised, with multiple characterisation techniques employed, affording enantiopure Au-cysteine and Ag-tyrosine colloids. However, it was not possible to generate consistent and reproducible SEROA responses, with these techniques ultimately being unsuccessful in analysing these chiral sensitive nanoprobes, and thus differentiating between the D- and L- forms. Finally, a novel SERS-based approach, in combination with the standard addition method (SAM), was developed for the routine analysis of uric acid (end product in XO catalysed reaction(s) and biomarker for various diseases), at clinically relevant levels in urine samples from patients. Results were highly comparable and in very good agreement with HPLC analyses, with an average < 9% difference in predictions between the two analytical approaches across all samples analysed, and a 60-fold reduction in acquisition time (when compared with HPLC). Together, the research presented in this thesis demonstrates the suitability of Raman enhanced techniques for quantitative analysis, measuring the analytes directly using a portable Raman instrument and, most importantly, offering significant reductions in acquisition times when compared to established analytical techniques.

Graphene, a single layer of graphite, owing to its excellent mechanical, electrical, and thermal properties, has evolved as an exceptional nanomaterial in the past decade. It holds great promise in developing various novel applications from biomedical to structural composites. However, several challenges remain in realising the great potential of this material; one being the bulk scale production of graphene. This thesis has been concerned with production of pristine few-layer graphene (FLG) using liquid phase exfoliation (LPE) of graphite in various solvent media and exploring the applications of graphene-based composite coatings as optical Raman-strain sensors. LPE of natural graphite using bath sonication was used to produce highly stable pristine FLG in 1-methyl-2-pyrrolidinone (NMP) and N,N-dimethylformamide (DMF). Atomic force microscope (AFM) was used to analyse the exfoliation efficiency and lateral dimensions, while Raman spectroscopy provided an insight about the quality of the graphene flakes. Moreover, the potential for dynamic light scattering (DLS) as an efficient in situ characterisation technique for estimating the lateral dimensions of graphene flakes in dispersions was demonstrated. LPE was also employed to explore various routes to produce pristine graphene in aqueous media which can be used for toxicity studies. Aqueous dispersions were prepared by a solvent exchange method of graphene originally in organic solvents (NMP and DMF) using dialysis, achieving 0.1 v/v% organic solvent levels. Pristine aqueous graphene dispersions were also prepared by directly exfoliating graphite in biocompatible surfactant (TDOC- Sodium taurodeoxycholate) and biomolecules (Phosphatidylcholine and human serum albumin) solutions. Cell culture studies by collaborators revealed that solvent-exchanged and TDOC-exfoliated pristine FLG displayed minimal toxicity and albumin-exfoliated FLG hardly any cytotoxicity, whereas phosphatidylcholine-exfoliated FLG was cytotoxic. Raman spectroscopy is a well-established technique used to study the local deformation of carbon-based composites by following the shift rates of the Raman 2D band with strain. Raman active strain coatings were produced from epoxy composites made with the FLG produced by LPE in organic solvents and by electrochemical exfoliation method. The deformation experiments on these coatings revealed little or no strain sensitivity, due to several factors such as length of flakes, processing history, graphene loading, defects in graphene and alignment of flakes within the composites. As an alternative, composite coatings made from chemical vapour deposition (CVD) graphene were investigated. Excellent strain sensitivity was observed upon various cyclic deformational sequences and Raman mapping over 100 × 100 µm area. In comparison to the commercially available wide area strain sensors, CVD graphene composite coatings with a calculated absolute accuracy of ~ ± 0.01 % strain and absolute resolution of ~ 27 microstrains show promise for wide area Raman-based strains sensors.