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Traditionally, hydrogen sulfide (H2S) has been regarded as toxic. It can affect the various human systems and even cause death. However, research in the 1990s has shown that H2S can be endogenously generated by many cells and tissues in mammalian bodies, and that H2S also may play physiological roles such as those of neuromodulator and vasorelaxant in the biological system. As such, the precise measurement of the amount of H2S in a mammalian body has generated researchers strong interest. The ultimate goal of such a measurement should be conducted in-vivo and in real time.<p>The existing methods for H2S measurement require both a large quantity of tissue samples and a complex procedure, so they are not highly practicable for the purpose of achieving the aforementioned goal. In this dissertation, a new method that uses carbon nanotube as an absorbent or transducer and laser-based microscopy techniques (Raman and confocal laser scanning microscopy) as signal excitation and acquisition is proposed and developed. Experimental studies are described of using this new method for analysis of both distilled water samples and serum samples in which a group of proteins are present. <p>The study concludes that the new method (1) can measure H2S in water solutions down to a low level of concentration of 10 µM, (2) can measure H2S in sera down to a low concentration of approximately 20 µM), and (3) has a high feasibility for being used in the clinical context. Regarding (3), this is confirmed by presenting a control system that allows the laser microscopy to track carbon nanotube in a solution that has Brownian motion.<p>While not having reached the ultimate goal as mentioned above, this work advances the state-of-the-art of the measurement of low concentration and nano-quantity of H2S in water and serum samples, in particular providing a promise toward a real-time and in-vivo H2S measurement.

Traditionally, hydrogen sulfide (H2S) has been regarded as toxic. It can affect the various human systems and even cause death. However, research in the 1990s has shown that H2S can be endogenously generated by many cells and tissues in mammalian bodies, and that H2S also may play physiological roles such as those of neuromodulator and vasorelaxant in the biological system. As such, the precise measurement of the amount of H2S in a mammalian body has generated researchers strong interest. The ultimate goal of such a measurement should be conducted in-vivo and in real time.<p>The existing methods for H2S measurement require both a large quantity of tissue samples and a complex procedure, so they are not highly practicable for the purpose of achieving the aforementioned goal. In this dissertation, a new method that uses carbon nanotube as an absorbent or transducer and laser-based microscopy techniques (Raman and confocal laser scanning microscopy) as signal excitation and acquisition is proposed and developed. Experimental studies are described of using this new method for analysis of both distilled water samples and serum samples in which a group of proteins are present. <p>The study concludes that the new method (1) can measure H2S in water solutions down to a low level of concentration of 10 µM, (2) can measure H2S in sera down to a low concentration of approximately 20 µM), and (3) has a high feasibility for being used in the clinical context. Regarding (3), this is confirmed by presenting a control system that allows the laser microscopy to track carbon nanotube in a solution that has Brownian motion.<p>While not having reached the ultimate goal as mentioned above, this work advances the state-of-the-art of the measurement of low concentration and nano-quantity of H2S in water and serum samples, in particular providing a promise toward a real-time and in-vivo H2S measurement.

Reducing equivalents were extracted from in vitro photosynthesis and used to drive cell-free and enzyme-free biochemical reduction reactions in this research. To investigate photosynthetic electron flow, an algal extract dense in chloroplasts was made from the microalga Scenedesmus sp. A6. The algal extract was subjugated to a variety of environmental parameters and exogenous quinones in order to optimize electron extraction. To monitor electron extraction and donation to metabolites, a novel assay was created that monitored the chemiluminescence (CL) produced by superoxide radicals formed during the process. In particular, these formed when a reduced exogenous quinone oxidized spontaneously. Our studies found that calcium chloride improved the reduction of low redox potential mediators along with prolonged exposure to red light. Other salts and environmental conditions examined had diverse effects on the quinones based on structure, redox potential, and site of electron extraction. We next applied our assay for monitoring the reduction of different metabolites. The CL recorded for different metabolites was compared to the Gibbs free energy of reduction and a highly correlated relationship was found. The assay was then applied to the reduction of metabolites via the oxidation of glucose in an alkaline environment. To exhibit the diverse application of the CL assay, urine of healthy individuals, patients with chronic kidney disease (CKD), and patients with bladder cancer (BCa) were characterized through their interactions with different quinones. The CL output was compared to that of SurineTM and urea followed by ANOVA analysis. Statistically significant differences were found for all quinones with 1,2-napthoquinone-4-sulfonate (NQS) producing significant differences between all groups examined. Monitoring algal phenotypes for biofuels or photosynthetic output requires arduous protocols and advanced instrumentation. Both of these energy producing options were explored along with rapid, high-throughput protocols for measuring reduction reactions. To monitor the phenotypes and health of our microalgae, Raman microscopy was applied to algal cultures of Scenedesmus sp. A6 grown under stress. Statistically unique phenotypes were found based on environmental factors during cell growth. ANOVA analysis determined the effect of stressors that caused significant change to algal phenotypes related to photosynthesis and lipids. / Doctor of Philosophy / Photosynthesis is the process by which plants and algae harness sunlight to convert CO2 to plant mass. Photosynthesis is performed in the chloroplast which can excite electrons and use them to generate energy. Detecting how much energy a chloroplast can produce and what chemicals effect the chloroplast requires complex procedures with complicated instruments. In this thesis the chloroplast from the microalgae Scenedesmus sp. A6 were isolated to evaluate how they are affected by different chemicals in the environment using a new, rapid and robust assay. Then, a group of chemicals called quinones were used to steal electrons (plant energy), and this process was optimized in this research. The purpose of stealing this plant energy from photosynthesis was so it could be re-directed into synthesizing valuable chemicals that are normally produced from fossil fuels. A new sensor was also developed in this research that would "light-up" the environment whenever this plant energy (electron) stealing process was successful allowing us to measure the efficiency of this energy transfer. Once a quinone stole an electron, it would spontaneously give up the electron to oxygen, creating an unstable molecule that could then react with the chemical luminol, forming a strong luminescence (light) signal. We found that calcium chloride greatly enhanced a quinone's ability to harvest electrons from the chloroplast. We also reported unique effects caused by salt, magnesium, phosphate, a mild detergent, and changing the amount of light the chloroplast would receive. This information was then used to transfer electrons from the chloroplast to make new valuable chemicals. We found that electrons could be donated to multiple chemicals using a quinone, chloroplasts, and light. We were also able to take electrons from glucose with our quinones when glucose was in an environment with a high pH. Electrons from glucose could also be donated to chemicals of interest using quinones. In addition, Quinones were used once more to find differences in the urine composition of healthy individuals and those with chronic kidney disease or bladder cancer. The urine from healthy individuals produced a unique luminescence signal when interacting with the quinones. Thus, quinones could be used for rapidly detecting changes in a patient's kidney and bladder function. We also developed a new method for detecting changes in the health of an algal culture. Algal cultures are used for producing biofuel, food, and pharmaceuticals, therefore it is imperative to track the growth of a culture to avoid contamination and algal death. Scenedesmus sp. A6 was exposed to chemicals harmful to algal health to see how these chemicals caused the algae to grow differently. Raman spectroscopy was used to collect data on algae grown under different conditions. The Raman spectra obtained then underwent statistical analysis to determine the chemicals that had the greatest impact on algal function. Methyl viologen, nickel sulfate, salt, and light exposure had the greatest impact on the algae.

"L'étude des cellules vivantes et la dentine humaine par microscopie confocale Raman" La microscopie confocale Raman est utilisée pour suivre des médicaments et des nanoparticules dans les cellules et dans les tissus durs. La microscopie Raman est non-invasive, ne nécessite aucun marqueur et permet une imagerie à haute résolution. Dans la première partie de l'étude cette méthode est utilisée pour suivre un médicament anticancéreux, le paclitaxel, au sein d'une lignée de cellules cancéreuses vivantes Michigan Cancer Foundation-7 (MCF-7). Les images Raman ont été traitées par un algorithme de partitionnement des données par k-moyennes pour détecter le paclitaxel dans les cellules. La distribution du paclitaxel dans les cellules est vérifiée par le calcul du coefficient de corrélation de Pearson entre le spectre de référence du traitement et les spectres de l'image entière. Le temps progressif de diffusion du paclitaxel dans toute la cellule est observé. Cette observation demande une étude complémentaire sur l'action pharmaceutique du produit, basé sur la liaison rapide de la tubuline libre au paclitaxel cristallisé. L'apoptose dans les cellules a été suivies par partitionnement de données et par corrélation. Le partitionnement de données a été utilisé pour déterminer la position de mitochondries dans les cellules ; le cytochrome C de distribution à l'intérieur des cellules est basé sur l'analyse de corrélation. L'apoptose des cellules est défini par le cytochrome C dans le cytoplasme de diffusion. Le cytochrome C agit comme un déclencheur pour l'activation en cascade des caspases, et sa libération par les mitochondries est un signe d'apoptose. La Co-localisation de cytochrome C est effectuée après incubation de cellules avec une concentration différente de paclitaxel. L'autre produit étudié est le dioxyde de titane. Le titane est largement utilisé pour les matériaux orthopédiques et dentaires implantés dans le corps humain. Il est inévitable que le sang prenne contact avec la surface de l'implant et des nanoparticules. Les nanoparticules de dioxyde de titane ont été suivies en intracellulaire dans les cellules MCF-7 et TERT épithéliales humaines (lignée orale cellulaire de kératinocytes OKF6/TERT-2). La détection des nanoparticules et leur toxicité ont été étudiées en utilisant deux méthodes d'analyse. La microscopie confocale Raman a également été utilisée pour réaliser l'analyse structurale et chimique de la jonction émail-dentine-résine et de la carie dentaire, grâce à une analyse précise des constituants minéraux et organiques. La microscopie Raman associée à des méthodes d'analyse de données ouvre de nouvelles portes pour la recherche en biologie-santé et en particulier en odontologie. / "The Study of living cells and human dentin by confocal Raman microscopy"Confocal Raman microscopy is employed to trace drugs and nanoparticles intracellular and in hard tissues. Raman spectroscopy a non-invasive, label-free and high spatial resolution imaging technique in first part of the study is being used to trace the anticancer drug paclitaxel in living Michigan Cancer Foundation-7 (MCF-7) cells. An analytical method was developed and applied to Raman data acquired. The Raman images were treated by K-mean cluster analysis to detect the drug in cells. Distribution of paclitaxel in cells is verified by calculating the Pearson correlation coefficient between the reference spectrum of the drug and the whole Raman image spectra. A time dependent gradual diffusion of paclitaxel all over the cell is observed suggesting a complementary picture of the pharmaceutical action of this drug based on rapid binding of free tubulin to crystallized paclitaxel. The apoptosis in the cells were followed by post-measurement analysis including K-mean clustering and Pearson correlation coefficient. K-mean clustering was used to determine mitochondria position in cells and cytochrome c distribution inside the cells was based on correlation analysis. Cell apoptosis is defined as cytochrome c diffusion in cytoplasm. Cytochrome c acts as a trigger for the activation of the caspase cascade, and its release from mitochondria is a sign of apoptosis. Co-localization of cytochrome c is done after cell incubation with different concentration of paclitaxel. The other product used was titanium dioxide. Titanium has been widely used for orthopedic and dental implant materials. When biomaterial is implanted into the human body, it is unavoidable that blood will contact the implant surface and nanoparticles. The question is: do these nanoparticles cause toxicity? Titanium dioxide nanoparticles were followed intracellular in MCF-7 cells and TERT epithelial human oral keratinocyte cell line (OKF6/TERT-2). Detection of nanoparticles and their toxicity were studied using two analytical methods. Confocal Raman microscopy were also used to obtain Structural analysis and chemical profile of Enamel – Dentine- Resin and Raman map of decay and sound dentin samples, through accurate analysis of the mineral and organic components. The Raman spectroscopy combined with this novel method developed in this study, will provide accurate finger prints of chemical composition and by post-measurement analysis of the data acquired more information would be obtained, which might open new gates in pharmaceutical and dentistry researches.

Usefulness of a particular clinical assay is directly correlated with its ability to extract highest possible signal from available data. This is particularly relevant for personalized radiation therapy since early plan modifications confer greater benefits to treatment outcome. Recent studies have demonstrated capability of single-cell Raman microscopy to detect cellular radiation response at clinical (below 10Gy) doses, but only in certain strongly responding cell lines and after at least two day incubation. One possible cause is rather unoptimized signal processing used. This work investigates application of several advanced multivariate methods - weighted principal component analysis (WPCA), robust PCA, probabilistic PCA, and nonlinear PCA to increase radiation response signal. Representative datasets from strongly (H460 - human lung) and weakly (LNCaP - human prostate) responding cell lines were analysed in 0-50Gy and 0-10Gy dose ranges and results quantified to determine relative and absolute algorithm performance. It was found that with careful tuning, significant improvements in sensitivity and better signal separation could be achieved as compared to conventional PCA. / Graduate

<p>Second harmonic generation (SHG) microscopy and Raman microscopy were used for qualitative and quantitative analysis of pharmaceutical materials. Prototype instruments and algorithms for sampling strategies and data analyses were developed to achieve pharmaceutical materials analysis with low limits of detection and short measurement times<br></p><p>Manufacturing an amorphous solid dispersion (ASD), in which an amorphous active pharmaceutical ingredient (API) within polymer matrix, is an effective approach to improve the solubility and bioavailability of a drug. However, since ASDs are generally metastable materials, they can often transform to produce crystalline API with higher thermodynamic stability. Analytical methods with low limits of detection for crystalline APIs were used to assess the stability of ASDs. With high selectivity to noncentrosymmetric crystals, SHG microscopy was demonstrated as an analytical tool, which exhibited a limit of detection of 10 ppm for ritonavir Form II crystals. SHG microscopy was employed for accelerated stability testing of ASDs, which provided a four-decade dynamic range of crystallinity for kinetic modeling. An established model was validated by investigating nucleation and crystal growth based on SHG images. To achieve <i>in situ</i> accelerated stability testing, controlled environment for in situstability testing (CEiST) was designed and built to provide elevated temperature and humidity, which is compatible with a commercial SHG microscope based on our research prototype. The combination of CEiST and SHG microscopy enabled assessment of individual crystal growth rates by single-particle tracking and nucleation rates for individual fields of view with low Poisson noise. In addition, SHG microscopy coupled with CEiST enabled the study of heterogeneity of crystallization kinetics within pharmaceutical materials.<br></p><p>Polymorphism of APIs plays an important role in drug formulation development. Different polymorphs of identical APIs may exhibit different physiochemical properties, e.g., solubility, stability, and bioavailability, due to their crystal structures. Moreover, polymorph transitions may take place during the manufacturing process and storage. Therefore, analytical methods with high speed for polymorph characterization, which can provide real-time feedback for the polymorphic transition, have broad applications in pharmaceutical materials characterization. Raman spectroscopy is able to determine the API polymorphism, but is hampered by the long measurement times. In this study, two analytical methods with high speed were developed to characterize API polymorphs. One is SHGmicroscopy-guided Raman spectroscopy, which achieved the speed of 10 ms/particle for clopidogrel bisulfate. Initial classification of two different polymorphs was based on SHG images, followed acquisition of Raman spectroscopyat the selected positions to determine the API crystal form. Another approach is implementing of dynamic sampling into confocal Raman microscopy to accelerate Raman image acquisition for 6-folds. Instead of raster scanning, dynamic sampling algorithm enabled acquiring Raman spectra at the most informative locations. The reconstructed Raman image of pharmaceutical materials has <0.5% loss of image quality with 15.8% sampling rate.<br></p>

Neste trabalho foi estudado o efeito sinérgico do microambiente e de óxidos na degradação de corantes e aglutinantes. Para avaliar o efeito de MnO2 e &#945;- Fe2O3 na degradação de aglutinantes em câmaras climáticas à temperatura de 35°C e UR de 50% foi utilizado linoleato de metila como molécula modelo e observou-se que MnO2 atua catalisando reações de auto-oxidação, que ocorrem preferencialmente na degradação desses aglutinantes. Já &#945;- Fe2O3 apresentou comportamento ambíguo, sugerindo mecanismo de ação mais complexo desse óxido. Experimentos para verificar o efeito de luz UV foram conduzidos e reafirmaram os resultados sobre falta de reprodutibilidade dos resultados na presença de &#945;- Fe2O3 e sobre o efeito da presença de MnO2 ser mais significativa para a degradação de linoleato de metila do que a luz. Para gordura vegetal e animal foram conduzidos experimentos com maior tempo de exposição (22 dias), uma vez que não foram observadas mudanças espectrais significativas com 8 dias de exposição. No caso das gorduras animal e vegetal a presença de óxidos de Mn e Fe teve um efeito menos expressivo, mas mesmo assim foi possível observar que o comportamento foi o mesmo do linoleato de metila. No caso dos corantes foi feita uma caracterização espectroscópica de 6 corantes sintéticos semelhantes aos encontrados em têxteis pré-colombianos e avaliou-se a interação de índigo carmim com metais visando mimetizar o comportamento de mordentes proveniente de sais usados como mordentes. Para os corantes índigo carmim, purpurina e alizarina foram estudados os espectros SERS e SERRS, e observou-se que os dois últimos formam complexos com o substrato metálico usado para o obtenção do efeito tendo uma variação significativa em seus espectros Raman. A análise de fibras arqueológicas Huari permitiu identificar que se trata de lã e que o corante da fibra vermelha era carmim. / This work aimed the investigation of the microenvironment and metal ions synergic effect on the degradation of binders and dyes. The role of MnO2 and &#945;- Fe2O3 was assessed using environment chambers at 35°C and 50 % RH and methyl linoleate was chosen as a model binder since fats present very complex chemical composition. It was find that MnO2 catalyses auto-oxidation reactions which are the main routes for binders degradation. On the other hand, the results using &#945;- Fe2O3 were not reproducible, suggesting that this oxide present a more complex mechanism of action. The same behavior was observed when methyl linoleato -metal oxides mixtures were irradiated with UV light and confirms that a radical mechanism mediated the MnO2 catalytic effect. Vegetal and animal fats, although not as sensitive as methyl linoleate to metal oxide, presented the same behavior. The preliminary results on dyes degradation involved the spectroscopic characterization of 6 synthetic dyes, selected from those found in pre-Colombian textiles and indigo carmine metal complexes aiming to mimic mordants. Fe, Cu and Al complexes were prepared and analyzed and complexation only caused minor changes in their spectra compared to indigo carmine. SERS and SERRS were used in the investigation of indigo carmine, purpurin and alizarin and except for the former, the spectra indicated that chemical adsorption to the SERS active surface (Ag and Cu) with a significant change in their spectrum. The analysis of the archaeological Huari fibers allowed to identify that it was wool and the dyes were carmine ( red fiber) and indigo carmine (blue fiber).

Neste trabalho foi estudado o efeito sinérgico do microambiente e de óxidos na degradação de corantes e aglutinantes. Para avaliar o efeito de MnO2 e &#945;- Fe2O3 na degradação de aglutinantes em câmaras climáticas à temperatura de 35°C e UR de 50% foi utilizado linoleato de metila como molécula modelo e observou-se que MnO2 atua catalisando reações de auto-oxidação, que ocorrem preferencialmente na degradação desses aglutinantes. Já &#945;- Fe2O3 apresentou comportamento ambíguo, sugerindo mecanismo de ação mais complexo desse óxido. Experimentos para verificar o efeito de luz UV foram conduzidos e reafirmaram os resultados sobre falta de reprodutibilidade dos resultados na presença de &#945;- Fe2O3 e sobre o efeito da presença de MnO2 ser mais significativa para a degradação de linoleato de metila do que a luz. Para gordura vegetal e animal foram conduzidos experimentos com maior tempo de exposição (22 dias), uma vez que não foram observadas mudanças espectrais significativas com 8 dias de exposição. No caso das gorduras animal e vegetal a presença de óxidos de Mn e Fe teve um efeito menos expressivo, mas mesmo assim foi possível observar que o comportamento foi o mesmo do linoleato de metila. No caso dos corantes foi feita uma caracterização espectroscópica de 6 corantes sintéticos semelhantes aos encontrados em têxteis pré-colombianos e avaliou-se a interação de índigo carmim com metais visando mimetizar o comportamento de mordentes proveniente de sais usados como mordentes. Para os corantes índigo carmim, purpurina e alizarina foram estudados os espectros SERS e SERRS, e observou-se que os dois últimos formam complexos com o substrato metálico usado para o obtenção do efeito tendo uma variação significativa em seus espectros Raman. A análise de fibras arqueológicas Huari permitiu identificar que se trata de lã e que o corante da fibra vermelha era carmim. / This work aimed the investigation of the microenvironment and metal ions synergic effect on the degradation of binders and dyes. The role of MnO2 and &#945;- Fe2O3 was assessed using environment chambers at 35°C and 50 % RH and methyl linoleate was chosen as a model binder since fats present very complex chemical composition. It was find that MnO2 catalyses auto-oxidation reactions which are the main routes for binders degradation. On the other hand, the results using &#945;- Fe2O3 were not reproducible, suggesting that this oxide present a more complex mechanism of action. The same behavior was observed when methyl linoleato -metal oxides mixtures were irradiated with UV light and confirms that a radical mechanism mediated the MnO2 catalytic effect. Vegetal and animal fats, although not as sensitive as methyl linoleate to metal oxide, presented the same behavior. The preliminary results on dyes degradation involved the spectroscopic characterization of 6 synthetic dyes, selected from those found in pre-Colombian textiles and indigo carmine metal complexes aiming to mimic mordants. Fe, Cu and Al complexes were prepared and analyzed and complexation only caused minor changes in their spectra compared to indigo carmine. SERS and SERRS were used in the investigation of indigo carmine, purpurin and alizarin and except for the former, the spectra indicated that chemical adsorption to the SERS active surface (Ag and Cu) with a significant change in their spectrum. The analysis of the archaeological Huari fibers allowed to identify that it was wool and the dyes were carmine ( red fiber) and indigo carmine (blue fiber).

This dissertation evaluates how gold nanoparticle structure and local environment influence resulting sensor function when using these nanomaterials for complex sample analysis. Molecular imprinted polymers (MIPs), a class of plastic antibodies, are engineered and incorporated into these nanosensors thereby facilitating the quantitative detection of a variety of small molecules when Raman spectroscopy and surface enhanced Raman scattering (SERS) are used for detection. First, homogeneous seeded growth gold nanosphere synthesis is evaluated as a function of ionic double layer composition and thickness. Systematically increasing the citrate concentration during synthesis improves nanomaterial shape homogeneity; however, further elevations of citrate concentration increase the number of internal and/or external atomic defects in the nanomaterials which leads to decreasing solution-phase stability. Next, spherical gold nanoparticles are modified with self-assembled monolayer (SAM), modeled using interfacial energy calculations, and experimental characterized using transmission electron microscopy, NMR, extinction spectroscopy, zeta potential, X-ray photoelectron spectroscopy, and flocculation studies to assess the morphology, surface chemistry, optical properties, surface charge, SAM packing density, and nanoparticle stability, respectively. The number of molecules on the nanostructures increases with increasing ionic strength (by decreasing the electrostatic interfacial energy between assembled molecules) which subsequently promotes nanoparticle stability. Third, plastic antibodies that recognize three drugs commonly used to treat migraines are engineered. These methacrylate-based MIPs are synthesized, extracted, characterized, and used to quantitatively and directly detect over-the-counter drugs in complex samples using Raman microscopy. These results along with numerical approximation methods to estimate drug binding site densities and dissociation constants with the MIPs serve as a foundation for understanding how modest recognition selectivity of MIPs coupled with shifts in the vibrational energy modes from the drugs upon hydrogen binding to the polymer backbone promote sensitive and selective drug detection in complex samples. Finally, nanomaterial incorporation into MIPs for applications in SERS-based biosensors is evaluated. Importantly, gold nanorod concentration increases the detectability of the same drugs using MIPs as pre-concentration and recognition elements. This combination of materials, theory, and applications forms a solid foundation which should aid in the design and development of MIP nanobiosensors for specific and sensitive detection of small molecules in complex matrices.