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Adsorbuotų ant metalo paviršiaus monosluoksnių su piridinio funkcine grupe struktūros ir sąveikos su tirpalo komponentais tyrimas virpesinės spektroskopijos metodais / Vibrational spectroscopic study on the structure and interaction with solution components of monolayers with pyridinium functional group adsorbed on metal surfaceMatulaitienė, Ieva 06 January 2014 (has links)
Savitvarkiai monosluoksniai suteikia metalų paviršiams norimas savybes ir plačiai taikomi elektronų pernašos tyrimuose, konstruojant (bio)jutiklius, biotechnologinius bei fotoelektroninius procesus. Teigiamo krūvio monosluoksniai naudojami kuriant anijonų jutiklius ir (bio)technologinius procesus su adsorbuotomis neigiamo krūvio makromolekulėmis. Darbe buvo susintetinta bifunkcinė molekulė su galinėmis tiolio ir piridinio grupėmis, suformuotas monosluoksnis ir ištirtos jo struktūrinės ir funkcinės savybės. Pagrindiniai darbo tikslai buvo ištirti N-(6-merkapto)heksilpiridinio (MHP) adsorbuoto ant Au ir Ag elektrodų struktūrą ir nustatyti monosluoksnio sąveikos su neorganiniais anijonais, dodecilsulfato anijonu ir grafeno oksidu dėsningumus. Naudojant paviršiaus sustiprintos Ramano spektroskopijos, kvantų chemijos skaičiavimo ir izotopinio pakeitimo metodus nustatyti MHP struktūros ir orientacijos elektrodo atžvilgiu Ramano sklaidos žymenys. Tiriant juostų intensyvumo priklausomybes nuo potencialo ir žadinančios spinduliuotės bangos ilgio parodyta, kad Au/MHP sistemoje pasireiškia krūvio pernešimo Ramano spektrų stiprinimo mechanizmas. Nustatyta, kad adsorbuotų neorganinių anijonų pilnai simetrinio virpesio dažnis sumažėja lyginant su tirpalo spektru ir tas pokytis koreliuoja su Gibso dehidracijos energija. Parodyta, kad MHP pritraukia grafeno oksidą. Nustatyta, kad grafeno oksido C-C ryšio ilgis ir elektroninė struktūra gali būti keičiama, keičiant potencialą. / Self-assembled monolayers provide possibility of changing metal surface properties in controllable manner and are widely used in studies of electron transfer, construction of (bio)sensors, and biotechnological and photoelectronic processes. Positively charged monolayers are valuable in development of sensors for anions and (bio)technological processes with adsorbed negatively charged macromolecules. In this work the structural and functional properties of monolayer formed from synthesized molecule with terminal thiol and pyridinium groups have been studied. The main tasks were to assess the structure of N-(6-mercapto)hexylpyridinium (MHP) on Ag and Au electrodes, and to determine the peculiarities of interaction with inorganic anions, dodecylsulfate, and graphene oxide. Based on surface enhanced Raman spectroscopy (SERS), quantum chemical calculations, and isotopic substitution studies, the Raman marker bands for structure and orientation of MHP have been evaluated. Dependence of SERS intensity on potential and excitation wavelength has revealed the operation of charge transfer Raman enhancement mechanism. It was found that the frequency of symmetric stretching mode of adsorbed ions decreases comparing with solution value and the extent of the shift correlates with the Gibbs dehydration energy. Adsorption of graphene oxide at MHP monolayer has been demonstrated. The electronic structure and length of C-C bonds of graphene oxide was found to be altered by the potential.
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Vibrational spectroscopic study on the structure and interaction with solution components of monolayers with pyridinium functional group adsorbed on metal surface / Adsorbuotų ant metalo paviršiaus monosluoksnių su piridinio funkcine grupe struktūros ir sąveikos su tirpalo komponentais tyrimas virpesinės spektroskopijos metodaisMatulaitienė, Ieva 06 January 2014 (has links)
Self-assembled monolayers provide possibility of changing metal surface properties in controllable manner and are widely used in studies of electron transfer, construction of (bio)sensors, and biotechnological and photoelectronic processes. Positively charged monolayers are valuable in development of sensors for anions and (bio)technological processes with adsorbed negatively charged macromolecules. In this work the structural and functional properties of monolayer formed from synthesized molecule with terminal thiol and pyridinium groups have been studied. The main tasks were to assess the structure of N-(6-mercapto)hexylpyridinium (MHP) on Ag and Au electrodes, and to determine the peculiarities of interaction with inorganic anions, dodecylsulfate, and graphene oxide. Based on surface enhanced Raman spectroscopy (SERS), quantum chemical calculations, and isotopic substitution studies, the Raman marker bands for structure and orientation of MHP have been evaluated. Dependence of SERS intensity on potential and excitation wavelength has revealed the operation of charge transfer Raman enhancement mechanism. It was found that the frequency of symmetric stretching mode of adsorbed ions decreases comparing with solution value and the extent of the shift correlates with the Gibbs dehydration energy. Adsorption of graphene oxide at MHP monolayer has been demonstrated. The electronic structure and length of C-C bonds of graphene oxide was found to be altered by the potential. / Savitvarkiai monosluoksniai suteikia metalų paviršiams norimas savybes ir plačiai taikomi elektronų pernašos tyrimuose, konstruojant (bio)jutiklius, biotechnologinius bei fotoelektroninius procesus. Teigiamo krūvio monosluoksniai naudojami kuriant anijonų jutiklius ir (bio)technologinius procesus su adsorbuotomis neigiamo krūvio makromolekulėmis. Darbe buvo susintetinta bifunkcinė molekulė su galinėmis tiolio ir piridinio grupėmis, suformuotas monosluoksnis ir ištirtos jo struktūrinės ir funkcinės savybės. Pagrindiniai darbo tikslai buvo ištirti N-(6-merkapto)heksilpiridinio (MHP) adsorbuoto ant Au ir Ag elektrodų struktūrą ir nustatyti monosluoksnio sąveikos su neorganiniais anijonais, dodecilsulfato anijonu ir grafeno oksidu dėsningumus. Naudojant paviršiaus sustiprintos Ramano spektroskopijos, kvantų chemijos skaičiavimo ir izotopinio pakeitimo metodus nustatyti MHP struktūros ir orientacijos elektrodo atžvilgiu Ramano sklaidos žymenys. Tiriant juostų intensyvumo priklausomybes nuo potencialo ir žadinančios spinduliuotės bangos ilgio parodyta, kad Au/MHP sistemoje pasireiškia krūvio pernešimo Ramano spektrų stiprinimo mechanizmas. Nustatyta, kad adsorbuotų neorganinių anijonų pilnai simetrinio virpesio dažnis sumažėja lyginant su tirpalo spektru ir tas pokytis koreliuoja su Gibso dehidracijos energija. Parodyta, kad MHP pritraukia grafeno oksidą. Nustatyta, kad grafeno oksido CC ryšio ilgis ir elektroninė struktūra gali būti keičiama, keičiant potencialą.
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Desenvolvimento de um sistema contendo nanopartículas de ouro dispersas em material graxo e sua aplicação em espectroscopia raman intensificada por superfícies (SERS) para avaliação de moléculas orgânicas sulfuradas / Development of a system containing gold nanoparticles dispersed in a fatty materials and its application in surface enhanced raman spectroscopy (SERS) for evaluation of sulfur organic moleculesPacheco, Laís Henrique 07 April 2017 (has links)
In this study, we used anisotropic gold nanoparticles (AuNPs) dispersed in castor oil and immobilized in hydrogenated castor oil, for the construction of a material to be used in Surface Enhanced Raman Spectroscopy (SERS-active substrate). The AuNPs synthesized were characterized by absorption spectroscopy in the Ultraviolet-visible region (UV-vis), where it was possible to observe a displacement of the absorption band after preparation of the substrate, from 650 to 690 nm, and by Transmission Electron Microscopy (TEM), which showed obtaining AuNPs with star shape and average size of 100 nm. To investigate the potential SERS applications, the obtained substrates were tested using the sulfur molecules of 4-aminothiophenol (4-ATP), benzothiophene (BTF), thiophene (TF), cysteamine and cysteine as probe molecules. The SERS spectra were obtained by analyzing the substrate after the immersion, for 24 h, in solutions with different concentrations of probe molecules. Using 4-ATP as a probe molecule it was possible to obtain SERS spectra with a concentration range of 2,88x10-2 to 1x10-10 mol L-1, with BTF and TF it was possible to increase its Raman bands from 1x10-1 to 1x10-6 mol L-1. Cysteamine and cysteine were also used as probe molecules, but with only one concentration, 1x10-1 and 5x10-2 mol L-1, respectively. The results showed that the developed material has interesting sites with SERS activity, allowing us to study its interaction with different probe molecules, so it can be applied to different areas, both in qualitative and quantitative analysis, even for molecules in solutions with very small concentrations. The obtained substrate presented EF of the order of 102 to 103. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Neste trabalho, foram utilizadas nanopartículas anisotrópicas de ouro (AuNPs), dispersas em óleo de mamona e imobilizadas em óleo de mamona hidrogenado, para a construção de um material para ser utilizado em Espectroscopia Raman Intensificada por Superfície (substrato SERS-ativo). As AuNPs sintetizadas foram caracterizadas por espectroscopia de absorção na região do Ultravioleta-visível, onde foi possível a observação de um deslocamento da banda de absorção após a preparação do substrato, de 650 para 690 nm, e por Microscopia Eletrônica de Transmissão, as quais mostraram a obtenção de AuNPs na forma de estrelas com tamanho médio de 100 nm. Para a investigação de possíveis aplicações na área de SERS, o substrato obtido foi testado utilizando as moléculas sulfuradas: 4-aminotiofenol (4-ATP), benzotiofeno (BTF), tiofeno (TF), cisteamina e cisteína, como moléculas sonda de interesse. Os espectros SERS foram obtidos através da análise dos substratos após a imersão, por 24 h, em soluções com diferentes concentrações das moléculas sonda. Utilizando o 4-ATP como molécula sonda foi possível a obtenção de espectros SERS com uma faixa de concentração de 2,88x10-2 até 1x10-10 mol L-1, com o BTF e TF foi possível a ampliação de suas bandas Raman de 1x10-1 à 1x10-6 mol L-1. Foram utilizadas também cisteamina e cisteína como moléculas sonda, porém com apenas uma concentração, 1x10-1 e 5x10-2 mol L-1, respectivamente. Os resultados mostraram que o material desenvolvido possui sítios com atividade SERS, o que nos permitiu estudar sua interação com diferentes moléculas sonda, assim podendo ser aplicado em diversas áreas, tanto para análise qualitativa quanto quantitativa, mesmo para moléculas em soluções com concentrações muito pequenas. O substrato obtido apresentou EF da ordem de 102 a 103.
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Raman Spectroscopy Applications to High Energy MaterialsSil, Sanchita January 2014 (has links) (PDF)
Detection of explosives has always been a challenging issue all over the world. Different analytical techniques and instrumentation methods have been explored to obtain a 100% fail proof detector. Some technologies have matured and have been deployed in the field already. However, active research is still being pursued to make the ultimate explosive detection device. The present thesis broadly addresses the development of Raman spectroscopy based techniques for the detection of explosives. Although Raman spectroscopy has technologically developed and has become a regular tool for chemical identification, its use in the field of detection of explosives has been limited. Two aspects of detection were addressed in this thesis.
The first part consists of the detection of minute quantities or traces of explosives using a Raman based method. In order to approach this problem, surface enhanced Raman spectroscopy (SERS), an offshoot of Raman spectroscopy was explored. Chapters 2-4 deal with developing efficient SERS substrates. In this endeavour, the first and the most obvious choice as SERS substrates were silver (Ag) nanoparticles (NPs). However, we were exploring methods that could be simple one-pot synthesis methods, cost-effective and without employing strong reducing agents (green). Therefore, Ag NPs were synthesized using biosynthetic route. These nanoparticles were used to study their SERS efficiency. Sub-nano molar concentration of dye as well explosive like trinitrotoluene (TNT) and hexanitrohexaazaisowurtzitane (CL-20) could be obtained for both the clove reduced as well as pepper Ag nanoparticles. Hence Ag NPs are very efficient SERS substrates. In the second part of the work on SERS, bimetallic nanoparticles with core-shell (Agcore-Aushell) architecture were synthesized, characterized and tested for SERS activity. After successful synthesis and characterization of the bimetallic nanoparticles, these were tested for their SERS activities using a dye molecule and an explosive molecule. SERS spectra could be obtained for the bimetallic nanoparticles. It was observed that the sensitivity of these NPs were almost at par with the mono-metallic Ag NPs. In order to bring SERS from laboratory to field, a more practical approach was to prepare solid SERS substrates or SERS substrates on solid platform. In the next chapter, we ventured into the most abundant material which forms the backbone of the organic world, carbon. Various carbonaceous materials ranging from chemically synthesized graphene, graphene oxide, multi-walled carbon nanotube (MWCNT), graphite and activated charcoal were explored as potential substrates for surface enhanced Raman spectroscopic applications. The analytes chosen for this particular study were some fluorescent molecules such as rhodamine B (RB), rhodamine 6G (R6G), crystal violet (CV), Nile blue A (NBA) and a non-fluorescent molecule acetaminophen, commonly known as paracetamol. Enhanced Raman signals were observed for the fluorescent molecules, especially for the molecules whose absorbance maxima are near the excitation wavelength of the laser (514.5 nm). The most interesting outcome of this work was obtaining enhanced Raman signals of nanomolar concentration of R6G on activated charcoal. However, for the non-fluorescent molecule, paracetamol, Raman spectra could not be observed beyond
-5 10M concentration for all the carbon substrates including chemically synthesized graphene and MWCNT. This study was crucial in our quest for an ideal SERS substrate. Our observations let us to conclude that chemically synthesized graphene was not the only candidate for the preparation of SERS substrates. Since carbon materials efficiently adsorb and also provide a separate channel for energy decay (fluorescence quenching), even activated charcoal could be employed as a SERS platform. However, carbon alone could not provide an effective solution for the preparation of SERS substrates. Therefore, combining the plasmonic effect of the metal nanoparticles with the efficient adsorption and fluorescence quenching of carbon materials would be ideal. In the next part of the carbon studies, graphene-Ag composites which were either prepared by in situ reduction process or physically mixed were studied for SERS activity. An ideal SERS substrate should possess the following properties:
(i) Support plasmon, thereby provide SERS enhancement
(ii) Easy to fabricate or synthesize (large scale/bulk)
(iii) Ensure high reproducibility and sensitivity
(iv) Low false alarm from matrix chemicals
(v) Cost effective
(vi) Solid substrate (in the form of chip, pellet, slide etc.)
Hence, as a final study, carbon silver based composites were explored. R6G was chosen as an analyte again and SERS experiments were conducted. Raman signals at low concentration could be obtained for the carbon-Ag composites as well. In addition, feasibility experiments were also conducted for an explosive molecule, FOX-7. From these preliminary experiments we observed that carbon-metal NP composites can be efficient, cost-effective SERS substrates that will overcome the current issue.
The previous chapters dealt with the trace detection of explosives. The next part of the thesis deals with the development of the Raman spectroscopic methods for non-invasive detection of concealed objects. Chapters 4 and 5 primarily focus on explosives detection. Spatially offset Raman spectroscopy (SORS) instrumentation was developed in the laboratory for non-invasive detection solid and liquid explosives. Several experiments were carried out to detect concealed materials inside high density polyethylene (HDPE) containers, coloured glass bottles, envelopes etc. with this technique, Raman signals of materials could be retrieved even within 4 mm thick outer-layer. SORS imaging experiments were also performed on bilayered compounds, tablets etc. However, while performing the SORS experiments, it was observed that due to the restriction in geometry imposed by the method, the signals from the inner-layers could be obtained only up to a certain depth. This posed a serious limitation of SORS for practical scenarios, where the thickness of the outer layer may be tens of mm. In such situation, SORS may not be an effective method. We then performed Raman experiments using a transmission geometry using a series of samples. The transmission Raman (TR) experiments yielded better SNR for the inner (concealed) material as compared to the outer material. Although transmission Raman experiments yielded better signal but these experiments were again geometry dependent, hence, less flexible and TR experiments did not provide information about the position of the underlying materials.
In order to obtain complete information, it was necessary to understand photon migration in a multiple scattering medium. It is known that a photon in a multiple scattering medium may be approximated to undergo a random-walk. Statistically, the photon that undergoes multiple scattering in a medium loses its sense of origin (direction), hence, there is a finite probability to observe the exiting photon in any direction. Rayleigh and NIR based imaging modalities have been conducted using this model. Diffuse optical tomographic (DOT) measurements also deal with measuring the photons that have exited the sample after undergoing multiple scattering in a turbid medium. If it was possible to collect the Rayleigh photons or the diffuse photons in DOT experiments, in principle, Raman photons could also be collected from several directions. It was then proposed that if Rayleigh scattered photons can exit at 4π solid angle from a sample, then it can be assumed that some Rayleigh photons may convert to Raman photons, which in turn, shall have a finite probability to exit the sample from all the sides (4π solid angles). This idea of collecting Raman photons has never been discussed before! Thus, as expected based on the above principles, we were able to record Raman scattered photons at all angles and on all sides. This new technique has been
termed as ‘Universal Multiple Angle Raman Spectroscopy (UMARS)’. Monte Carlo
simulation studies were also performed to understand the distribution of photons in a multiple scattering medium. Simulation studies also revealed that Raman photons exited from all sides of the medium at varying percentages. Hence, several fiber optic probes were designed for illumination and collection to perform the UMARS experiments for samples concealed at depths beyond 20 mm. UMARS was not only applied successfully for the detection of concealed explosives, but also for biologically relevant samples as well. In fact a pharmaceutical tablet as thick as 7 mm was also tested with UMARS and signals could be successfully obtained. Since the UMARS signals were obtained from all possible angles, imaging experiments were also conducted to obtain sample specific information. Frequency-specific images of bilayer materials could be obtained. In the case where one material was concealed within another, the reconstruction of the frequency-specific intensities in a contour plot revealed the position of the concealed layer. One of the most challenging and exciting studies that was conducted was to use UMARS to obtain shapes of hidden materials. Several shapes such as dumbbell, ellipsoid etc were fabricated (made of glass) and were filled with a test chemical, trans-stilbene (TS). This shape was placed inside an outer material like ammonium nitrate (AN) that was taken in a glass beaker. The diameter of the beaker was varied from 25 mm to 60 mm. A series of UMARS measurement was carried out with 10
collection fiber optic probes. The spatial resolution (vertical) was varied from 200 μm to 1 mm. Series of UMARS images were obtained which were then processed and the intensity of the individual fibers were averaged (CCD row pixels) based on the image of the individual fiber on the CCD. The frequency specific intensity of the materials was utilized to reconstruct 2D or a 3D shape. The shapes of the objects could be clearly discerned using UMARS imaging. This marks a major step for the development of UMARS as a 3D imaging modality. UMARS experiments conducted so far have affirmed our belief that this technology can be used as an effective technique for screening solid and liquid samples at airports, railway stations and other entry points. 3D imaging for biomedical diagnostics will provide molecular information in addition to the location and shape of an object inside a tissue such as calcified masses and bones.
In the final part of the thesis, 2D Raman correlation spectroscopic method was applied to understand the dynamics of a system that was subjected to external perturbation. In the field of explosive processing and formulations, large batches are generally prepared. However, it is very difficult to ascertain the molecular or structural changes that occur during the processing of these formulations in situ. Analytical methods to monitor the changes online are limited. Raman spectroscopy can be an effective technique for such measurements. This process however, generates a large number of spectra. In such cases, it becomes cumbersome to handle such large number of data and obtain meaningful information. 2D correlation spectroscopy can be applied under such situations. 2D correlation analysis generates essentially two maps, synchronous and asynchronous. In this study, 2D Raman correlation spectroscopy was applied to ammonium nitrate that was subjected to temperature variations. 2D maps were constructed to obtain information about the structural changes associated with temperature. The synchronous map reveals the overall similarity of the intensity changes. Whereas, the 2D asynchronous maps provide the sequence of changes that occur. Based on the set of well defined rules proposed by Isao Noda, the synchronous and the asynchronous correlation maps were analysed. Hence, generalized 2D correlation spectroscopy can be extended to any kind of perturbation and will prove useful in understanding the structural dynamics.
The objective of the thesis was to explore various facets of Raman spectroscopy that would be useful in the field of high energy materials specifically in the detection of explosives. Attempts were made for the development of trace detection of explosives using Raman based technique, SERS. In addition, bulk detection of concealed explosives was performed non-invasively using SORS and UMARS. In the field of high energy materials, these techniques will find immense applications. Raman spectroscopy, as we saw is a very important technique that can be used as a stand-alone method and can also be interfaced with other analytical or imaging modalities. This treatise is an example where the strength of this powerful spectroscopic method has been explored to some extent.
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Vibrational Microspectroscopic Studies of Biomedical Conditions Using Model SystemsGautam, Rekha January 2014 (has links) (PDF)
Over the last century, despite enormous advancements in biomedical research and the development of sophisticated analytical instruments many diseases continue to be a burden on humankind particularly on the aged. This is because of a lack of complete understanding of the pathogenesis and specific therapies. Due to the complexity involved, we need to explore all facets of diagnosis and therapies. Therefore, there is a requirement for different strategies to combat these diseases. A quick diagnosis is the primary step towards improving treatment and increasing the chance of survival. To realize this goal we entail to monitor multiple biomarkers which will also help us to understand the progression of disease. Mid-Infrared (MIR) and Raman spectroscopic techniques are well established analytical methods to understand the molecular structure and chemical composition of heterogeneous systems. These techniques are rapid, non-destructive and offer multiple component analysis (global/multiplex) in a single measurement without any labels. Importantly, biological materials like proteins, carbohydrates, lipids, nucleic acids etc. have unique structures and therefore we can obtain unique spectral fingerprints of these molecules in different physiological and pathological conditions. This will provide a potential route to obtain diagnostic markers for diseases. Also, to improve the ability to diagnose and treat human diseases much more efficiently, understanding the mechanisms involved in the progression of disease is necessary. It would be time consuming and often unethical to perform these studies directly on humans. Therefore, there is a need for model organisms to explore the complexity of various diseases. A model organism is an animal, plant or microbe that is being studied to understand a range of biological phenomena. They should meet certain criteria such as short life cycles, easy to breed and maintain in large numbers under laboratory conditions, and the data generated through use of the model should be applicable to other higher organisms like humans. The microbial system, mouse, rat, Drosophila (fruitfly), C Elegans (nematode worm) and zebrafish are being used extensively for this purpose. The most adaptable organisms to study diseases in humans are the mice as they share almost 99% of their genes with humans. Mice are similar to humans in most physiological and pathological features such as nervous, cardiovascular, immune, liver etc. In addition to mice, Drosophila melanogaster (fruitfly) has been used for years as an attractive model organism to understand the mechanisms of underlying human diseases. This is because 75% of human disease genes have counterparts in Drosophila and it meets the above mentioned criteria to be a model organism. It also plays an important role
for studying genetics and development biology. The average life span of Drosophila is 60-80 days; therefore it is a suitable model to study age related diseases.
In the present thesis, the ability to probe low-micrometer domains using Raman and Fourier Transform Infrared (FTIR) microspectroscopy was utilized to monitor the chemical changes during various biomedical conditions using model systems. Chapter 1 of the thesis discusses about the origin of Raman and FTIR microspectroscopy along with instrumentation and applications. Various data analysis methods (both univariate & multivariate) and the validation criterion are described in chapter 2. Depending on the objective of the study and based on the technique (Raman or FTIR) used, one (or more) of these methods can be applied for effective interpretation of the data. Further, the thesis includes four different investigations; a) the FTIR spectroscopic study of hepatotoxicity due to acetaminophen using mice as model, b) the Raman spectroscopic studies of muscle-related disorders using Drosophila as a model, c) Vibrational spectroscopic study of septic shock using mice as model, d) Surface Enhanced Raman Spectroscopy (SERS) study of serum components using Lab-on-a-chip (LOC).
The first part comprises mainly the FTIR microspectroscopy study of hepatotoxicity in mice post oral dosing of acetaminophen (paracetamol), which is extensively used worldwide as an analgesic and antipyretic drug (chapter 3). The infrared spectra of acetaminophen treated livers in BALB/c mice show a decrease in glycogen and an increase in amounts of cholesteryl esters and DNA. Importantly, analysis of sera identified the lowering of glycogen and increase in DNA and chlolesteryl esters earlier than the increase in alanine aminotransferase, which is routinely used to diagnose liver damage. Similar changes are also observed in C57BL/6 and Nos2−/− mice. Revert experiments using an antidote (L-methionine) demonstrate that depletion in glycogen and increase in DNA are abrogated with pre-treatment, but not post-treatment, with L-methionine.
In the second study Raman spectroscopy is applied to discriminate between various muscle defects in Drosophila, since it can provide a unique molecular fingerprint of tissues on the basis of their biochemical composition (chapter 4). Raman spectra were collected from Indirect Flight Muscles (IFM) of mutants upheld1 (up1), heldup2 (hdp2), Myosin heavy chain7 (Mhc7), Actin88FKM88 (Act88FKM88), upheld101 (up101) and Canton-S (CS) for both 2 and 12-days old flies.
The difference spectra (mutant minus CS) of all mutants have shown an increase in nucleic acids (DNA/RNA) content along with an increase in β-sheet and/or random coil content at the expense of α-helix. Interestingly, 12th day sample of up1 & Act88FKM88 exhibit significantly higher levels of glycogen and carotenoids than CS. A Principal Components based Linear Discriminant Analysis (PC-LDA) classification model was developed, which classifies the mutants according to their pathophysiology and yielded overall accuracy (OA) of 97% and 93% for 2 and 12-days old flies respectively. up1 & Act88FKM88 (nemaline myopathy phenotypes) form a group which is clearly separated in a Linear Discriminant (LD) Plane from up101 & hdp2 (cardiomyopathy phenotypes).
In the third part we investigated septic shock, a life threatening condition associated with multiple organ dysfunctions, in mice (chapter 5). Salmonella typhimurium were given to BALB/c and 129/SvJ mice via the intraperitoneal route to induce infection. Liver, spleen and sera samples were studied using FTIR microspectroscopy. The infrared spectra of liver, spleen and serum samples in BALB/c (Nramp1-deficient) mice show significant spectral changes as early as 1 hour post infection but spleen shows changes only after 6 hour. Interestingly, 129/SvJ (Nramp1-sufficient) mice were resistant to sepsis and show significant spectral changes only at 12 hour post infection. This study demonstrates that suppression of Nramp-1, a renowned gene known to control susceptibility to infections by intracellular bacteria can be an effective cure for sepsis.
The final study presented in this thesis demonstrates the use and benefits of lab-on-a-chip (LOC) devices in surface enhanced Raman spectroscopy (SERS) which is used to enhance the weak Raman signals (chapter 6). Most of the diseases have related proteins or analytes present in serum although in early stages their concentration in blood are low. The idea is to detect at low concentration using SERS the serum components which are related to progression of disease. Here, we have compared the effect of different aggregating agents on silver colloids and the resulting enhancement in Raman signals for tryptophan and Bovine Serum Albumin (BSA). Reproducibility issues, the key concern of static phase SERS, can be overcome by performing SERS spectroscopic measurements in automated flow cells. Further, pyridine and tryptophan were used to demonstrate SERS in a segmented flow system. The spectra from different drops were compared and demonstrate the high reproducibility in comparision to static SERS.
Lastly, chapter 7 summarizes the entire work of the present thesis with future prospects of Raman and FTIR microspectroscopy to study the progression mechanism of various diseases like neurodegenerative diseases which is easy to follow in drosophila due to their short life span. Also, technological developments in the field of nanotechnology and micro-fluidics will enable the detection of early biochemical changes in bodily fluids such as urine, cerebral spinal fluid, tears etc. Building on the results demonstrated in this thesis, hopefully label-free vibrational (Raman and FTIR) microspectroscopic studies using model organisms would help in understanding the underlying mechanisms of progression of various other diseases which in turn would facilitate the development of effective therapies.
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Advanced vibrational spectroscopic studies of biological moleculesOstovar Pour, Saeideh January 2012 (has links)
Raman optical activity (ROA) is a powerful probe of the structure and behaviour of biomolecules in aqueous solution for a number of important problems in molecular biology. Although ROA is a very sensitive technique for studying biological samples, it is a very weak effect and the conditions of high concentration and long data collection time required limit its application for a wide range of biological samples. These limitations could possibly be overcome using the principle of surface enhanced Raman scattering (SERS). The combination of ROA with SERS in the form of surface enhanced ROA (SEROA) could be a solution for widening the application of ROA. In the last few years, the generation of reliable SEROA spectra of biomolecules has been problematic due to non-homogenous colloidal systems forming and low signal-to-noise ratios which complicated detection of the true SEROA signal from the analyte. L- and D-enantiomers give full or partially mirror image chiroptical spectra, this property of enantiomers can be employed to prove the chiroptical activity of the SEROA technique. In this thesis we employed a hydrophilic polycarbopol polymer as stabilising media which has led to the first report of mirror image SEROA bands for enantiomeric structures. This new technique of incorporating the hydrogel polymer as a means to stabilise the colloidal system has proven to be reliable in obtaining high quality SEROA spectra of D- and L-enantiomers of ribose and tryptophan. In an extension of the hydrogel-stabilised SEROA work, we also demonstrate that single nanoparticle plasmonic substrate such as silver silica nanotags can enhance the weak ROA effect. These dye tagged silica coated silver nanoparticles have enabled a chiral response to be transmitted from a chiral analyte to the plasmon resonance of an achiral metallic nanostructure. The measurement of mirror image SERROA bands for the two enantiomers of each of ribose and tryptophan was confirmed for this system. The generation of SEROA for both systems was achieved and confirmed SEROA as a new sensitive tool for analysis of biomolecular structure. In a related project, Raman and ROA spectra were measured for adenosine and seven of its derivative ribonucleotides. Both of these spectroscopic techniques are shown to be sensitive to the site and degree of phosphorylation, with a considerable number of marker bands being identified for these ribonucleotides. Moreover, the SERS studies of these ribonucleotides were also performed. The obtained SERS spectra were shown similar features that confirm these analytes interact with the surface in a similar manner, hence limiting the structural sensitivity of this method towards phosphate position. Short dipeptides such as diketopiperazine (DKP) have been investigated during the last decades as both natural and synthetic DKPs have a wide variety of biological activities. Raman and ROA spectra of linear and cyclic dialanine and diserine were measured to charecterize their solution structures. Density functional theory (DFT) calculations were carried out by a collaborator to assist in making vibrational band assignments. Considerable differences were observed between the ROA bands for the cyclic and linear forms of both dialanine and diserine that reflect large differences in the vibrational modes of the polypeptide backbone upon cyclicization. In this study, the ROA spectra of cyclic dialanine and diserine have been reported for the first time which demonstrated that ROA spectroscopy when utilised in combination with computational modelling clearly provides a potential tool for characterization of cyclic peptides.
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Synthesis, Physiochemical And Electrochemical Studies On Iridium, Osmium And Graphene Oxide-Based NanostructuresKalapu, Chakrapani 10 1900 (has links) (PDF)
Nanoscience dominates almost all areas of science and technology in the 21st century. Nanoparticles are of fundamental interest since they possess unique size dependent properties (optical, electrical, mechanical, chemical, magnetic etc.), which are quite different from the bulk and the atomic state. The research work presented in the thesis is on the preparation, characterization and studies on Ir, Os and graphene oxide-based systems. Interconnected Ir and Os nanochains are prepared under environmentally friendly conditions in aqueous media and subsequently used as substrates for surface enhanced Raman scaterring studies and also as electrocatalysts for oxygen reduction and formaldehyde oxidation. Ir and IrOx nanostructures are prepared using borohydride at different temperatures. The nature of interaction of heme proteins with IrOx is studied using spectroscopic techniques. Electrochemical studies on reduced graphene oxide include sensing of biomolecules with high sensitivity and oxygen reduction reaction (ORR) in aqueous alkaline medium. rGO is also used as support for anchoring Ir nanoparticles and the catalyst is used for the oxidation of benzyl amines to corresponding imines. The thesis is divided in to seven chapters and details are given below.
Chapter 1 gives an introduction about the synthetic strategies and properties of metal nanostructures. This is followed by literature survey on Ir, Os and graphene oxide-based systems relevant to the present study. Aim and scope of the present investigation is given at the end. Chapter 2 discusses the experimental procedures and characterization techniques used in the present study.
Chapter 3 involves the preparation, characterization and studies on interconnected Ir nanochains. Assemblies of small sized nanoparticles forming network-like structures have attracted enormous interest and different metal nanoassemblies have been reported using different procedures. Ir3+ reduction is kinetically not a very favourable process and hence there are not many attempts to synthesize Ir-based nanostructures. Assemblies of interconnected Ir nanoparticles have been synthesized in the present studies using borohydride as reducing agent and ascorbic acid as capping agent, at high temperatures. Polyfunctional capping molecules such as ascorbic acid and vitamin P play important role for the formation of network- like Ir nanostructures. Optical properties of the networks are probed using UV-Vis spectroscopy and evolution of coupled plasmon of Ir nanochains at 418 nm (figure 1) is observed. The nanochains are used as substrates for SERS studies while the catalytic activity is followed for the reduction of nitroaromatics. Electrocatalytic activity of Ir nanochains is exemplified using oxygen reduction and formaldehyde oxidation. Ir nanochains show better electrocatalytic activities than nanoparticles as shown in figure 2.
Figure 1. Time dependent UV-Vis absorption spectra of Ir nanoparticles recorded at various time intervals of (a) 5; (b) 15; (c) 30 and (d) 60 minutes of reduction of Ir3+ using borohydride and the corresponding TEM images.
Figure 2. Polarization curves for oxygen reduction on (i) Ir nanochains and (ii) Ir nanoparticles in (A) 0.5 M H2SO4 and (B) 0.1 M KOH at a scan rate of 0.005 V/s. Rotation speed used is 1000 rpm.
Chapter 4 discusses the preparation of Ir and IrOx using borohydride. The reaction temperature determines the product. Various physicochemical, microscopic and spectroscopic techniques have been used to understand the evolution of nanostructures. Borohydride reduces Ir3+ at high temperatures to form high surface area foams, while at 25oC, it results in an alkaline environment that helps in the hydrolysis of the Ir precursor to form IrOx nanoparticles.
Porous IrOx is formed when Ir foams are annealed at high temperatures. Water oxidation has been demonstrated using IrOx nanoparticles and foams. Biocompatibility of IrOx is used to study the nature of interaction of heme proteins and the formation of bioconjugates using spectroscopic techniques. IrOx forms bioconjugates with substantial changes observed in secondary and tertiary structures of proteins.
Chapter 5 explores the synthesis of interconnected ultrafine Os nanoclusters and the nanostructured materials are used as SERS substrates. Os nanochains are prepared under environmentally friendly conditions using polyfunctional molecules like ascorbic acid and vitamin P as both reducing agent and capping agent in aqueous media. Small sized (1-1.5 nm) Os nanoparticles spontaneously self-assemble to form clusters of few tens of nm that in turn self-organize to form branched nanochains of several microns in size. The as-formed nanochains show surface plasmon absorption in the visible region 540 nm which make them active substrates for surface enhanced Raman scattering (SERS) studies. High SERS activity is observed for fluorescent analyte, rhodamine 6G and non-fluorescent analyte, mercaptopyridine, with different laser excitation sources. Efficient energy transfer from fluorescent R6G dye to Os nanochains is observed based on steady state and time resolved fluorescence measurements.Figure 3. (I) Time dependent UV-Vis absorption spectra of Os nanochains recorded at different time intervals of (a) 5; (b) 7; (c) 15; (d) 30 and (e) 60 minutes. Inset shows the TEM images of Os nanochains after 60 minutes of reduction. (II) SERS spectra of 4-MPy adsorbed on Os nanochains from (a) 1 mM; (b) 10 µM and (c) 1 µM solutions using 514 nm laser excitation.
Chapter 6 discusses the studies based on reduced graphene oxide. Reduced graphene oxide (rGO) is explored as electrodes for simultaneous determination of dopamine (DA), ascorbic acid (AA) and uric acid (UA) at low concentrations useful in medical diagnostics (figure 4A). It is also used as metal-free electrocatalyst for ORR (figure 4B). The use of rGO as a support for anchoring Ir nanoparticles is probed and subsequently the Ir/rGO is used as catalyst for direct aerobic oxidation of benzyl amine derivatives to corresponding imines.
Chapter 7 describes the summary of the work and scope for further studies.
Appendix 1 discusses the preparation of different Ir nanostructures using simple
galvanic displacement reaction on copper foil while appendix 2 describes the preparation of
different sized Ir nanoparticles and their electrocatalytic activity towards oxygen reduction
reaction
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Détection de l’ADN par spectrométrie de diffusion Raman exaltée de surface couplée à la microfluidique / DNA detection by surface enhanced Raman spectroscopy coupled with microfluidicPrado, Enora 10 November 2011 (has links)
Ce travail présente une méthode originale de détection et de quantification, sans étape de marquage, de la proportion de bases libres contenues dans des acides nucléiques. La spectrométrie de diffusion Raman exaltée de surface (DRES ou SERS en anglais) nous a permis d’obtenir la signature spectrale spécifique des nucléotides caractéristiques des ARN (adénosine, cytosine, guanosine et uridine), en utilisant des colloïdes d’argent comme substrat-DRES et des ajouts de MgCl2 comme agent d’agrégation. Les conditions de détection ont été optimisées pour établir un protocole de quantification de la proportion des nucléobases non-appariées par spectrométrie DRES. Les limites de détection obtenues sont de l’ordre de quelques dizaines de picomoles. L’amélioration de la reproductibilité des mesures par spectrométrie DRES passe par le contrôle précis des temps de réaction (adsorption et agrégation), qui peut être contrôlé grâce à l’utilisation de plateformes microfluidiques adaptées. Nous avons mis en œuvre deux types de plateformes microfluidiques, l’une basée sur des écoulements monophasiques et l’autre sur la génération de gouttes. Les espèces à analyser sont contenus dans les gouttes, permettant la détection in situ par spectrométrie DRES des divers nucléotides. / This work deals with the development of an original label-free method for free bases proportions detection and quantification of nucleic acids. The surface enhanced Raman spectroscopy (SERS) allowed obtaining the specific spectral signature of characteristic nucleotides of RNA (adenosine, cytosine, guanosine and uridine), using silver colloids as SERS substrate and MgCl2 addition as aggregating agent. Then, the condition detection have optimizing to establish a label-free quantification protocol of free nucleobases proportion by SERS spectroscopy. The detection limits obtained are order of few picomoles. The reproducibility improvement of SERS detection requires the precise control of time reaction (adsorption and aggregation), which could be control thanks to microfluidic chips use. We have implemented two different microfluidic chips, one based on single-phase flows and one other based on droplets generation. The analyzed species are containing in droplets, allowing in situ detection by spectroscopy SERS of various nucleotides.
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Structure and Composition of the Protein Corona in Animal CellsSzekeres, Gergő Péter 17 August 2020 (has links)
Die Charakterisierung der Protein-Nanopartikel-Wechselwirkungen in komplexen biomolekularen Systemen wie einer lebenden Zelle ist für die Pharma-, Medizin- und Umweltforschung von entscheidender Bedeutung. In solchen biomolekularen Systemen adsorbieren Proteine leicht auf der Oberfläche von Nanopartikeln, die die Proteinkorona bilden. Diese Arbeit konzentriert sich auf die Charakterisierung der Proteinkorona in lebenden Zellen, wobei verschiedene analytische Ansätze kombiniert werden. Experimente mit oberflächenverstärkter Raman-Streuung (SERS) an reinen Proteinlösungen zeigten die Konzentrationsabhängigkeit der Protein-Gold-Nanopartikel-Wechselwirkungen, die zu unterschiedlichen SERS-Spektren führten und ermöglichten die Bestimmung von Proteinsegmenten, die an Citrat-stabilisierte Gold-Nanopartikel binden. In SERS-Experimenten mit lebenden Zellen wurde die Anwesenheit von Proteinfragmenten in der innersten Schicht der Proteinkorona, die als harte Proteinkorona bezeichnet wird, festgestellt. Eine analytische Methode, die Natriumdodecylsulfat-Polyacrylamid-Gelelektrophorese und Hochleistungs-Flüssigchromatographie-gekoppelte Elektrospray-Ionisations-Massenspektrometrie kombiniert, wurde entwickelt, um die Bestandteile der Hartproteinkorona zu identifizieren. Die Proteomics-, SERS- und Cryo-Soft-X-Ray-Nanotomographiedaten, wobei letztere Informationen über die dreidimensionale Ultrastruktur der Zelle liefern, zeigen den Aufnahmemechanismus, die Verarbeitung, die Akkumulationsstelle, die molekulare Umgebung und die induzierten zellulären Reaktionen internalisierter Goldnanopartikel. Diese Arbeit validiert die Verwendung von SERS bei der Analyse der Proteinkorona in der Lösung von Modellproteinen und in lebenden Zellen und präsentiert eine geeignete Methode zur Analyse der unveränderten harten Proteinkorona, die in lebenden Zellen gebildet wird. / The characterization of the protein-nanoparticle interactions in complex biomolecular systems such as a living cell is vital for pharmaceutical, medical, and environmental research fields. In such biomolecular systems, proteins readily adsorb on the surface of nanoparticles forming the protein corona. This thesis focuses on the characterization of the protein corona in living cells combining different analytical approaches. Surface-enhanced Raman scattering (SERS) experiments on pure protein solutions revealed the concentration dependence of the protein-gold nanoparticle interactions resulting in different SERS spectra, and allowed for the determination of protein segments binding to citrate-stabilized gold nanoparticles. In live cell SERS experiments, the presence of protein fragments in the innermost layer of the protein corona, called the hard protein corona, was revealed. An analytical method combining sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography-coupled electrospray ionization mass spectrometry was developed to identify the constituents of the hard protein corona. The proteomics, SERS, and cryo soft X-ray nanotomography data, the latter providing information of the three dimensional ultrastructure of the cell, reveal the uptake mechanism, processing, accumulation site, molecular environment, and the induced cellular responses of internalized gold nanoparticles. This work validates the use of SERS in the analysis of the protein corona in the solution of model proteins and in living cells, and presents a suitable method for the analysis of the unaltered hard protein corona formed in living cells.
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Optophysiologie SERS : analyse in vitro d’environnement cellulaire en Raman exalté par les surfacesLussier, Félix 03 1900 (has links)
No description available.
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