La concentration locale des messagers chimiques sécrétés par les cellules peut être mesurée afin de mieux comprendre les mécanismes moléculaires liés à diverses maladies, dont les métastases du cancer. De nouvelles techniques analytiques sont requises pour effectuer ces mesures locales de marqueurs biologiques à proximité des cellules. Ce mémoire présentera le développement d’une nouvelle technique basée sur la réponse plasmonique sur des leviers AFM, permettant d’étudier les réactions chimiques et biologiques à la surface des leviers grâce au phénomène de résonance des plasmons de surface (SPR), ainsi qu’à la diffusion Raman exaltée par effet de pointe (TERS). En effet, il est possible de localiser l’amplification du signal Raman à la pointe d’un levier AFM, tout comme le principe de la diffusion Raman exaltée par effet de surface (SERS) basée sur la diffusion de la lumière par des nanoparticules métalliques, et permettant une large amplification du signal Raman. La surface du levier est recouverte d’une nano-couche métallique d’or, suivi par des réactions biologiques pour l’immobilisation d’un récepteur moléculaire, créant ainsi un biocapteur sur la pointe du levier. Une détection secondaire utilisant des nanoparticules d’or conjuguées à un anticorps secondaire permet également une amplification du signal SPR et Raman lors de la détection d’antigène. Ce mémoire démontrera le développement et la validation de la détection de l’immunoglobuline G (IgG) sur la pointe du levier AFM.Dans des projets futurs, cette nouvelle technique d’instrumentation et d’imagerie sera optimisée grâce à la création d’un micro-détecteur protéique généralement adapté pour l’étude de la communication cellulaire. En intégrant le signal SPR à la microscopie AFM, il sera alors possible de développer des biocapteurs SPR couplés à une sonde à balayage, ce qui permettra d’effectuer une analyse topographique et de l’environnement chimique d’échantillons cellulaires en temps réel, pour la mesure des messagers moléculaires sécrétés dans la matrice extracellulaire, lors de la communication cellulaire. / Measurement of the local concentration of chemical messengers secreted by cells may give a better understanding of molecular mechanisms related to different diseases, such as cancer metastasis. Current techniques are not suited to perform such measurements and thus, new analytical techniques must be developed. This Master’s thesis reports the development of a new technique based on the plasmonic response of atomic force microscopy (AFM) tips, which will ultimately allow monitoring of chemical and biological molecules on the surface of a cantilever by use of surface plasmon resonance (SPR) and tip-enhanced Raman scattering (TERS). Indeed, it is possible to localize the enhancement of the Raman signal on the AFM tip using principles associated to surface-enhanced Raman spectroscopy (SERS), based on the absorption of light by nanometer-sized metal particles, resulting in a large enhancement of the Raman signal. The AFM tip was constructed by the deposition of a nanometer-size gold layer, followed by the assembly of a biosensor with a biomolecular receptor. Gold nanoparticles (AuNPs) conjugated with a secondary antibody served as the secondary detection step. In addition, the use of the gold nanoparticles for antigen detection allows an amplification of the SPR and Raman signals. This Master’s thesis will demonstrate the development and validation of a biosensor for immunoglobuline G (IgG) at the tip of an AFM cantilever.This thesis sets the basis for future projects, where this new imaging technique will be developed for monitoring cellular communication by exploiting the plasmonic signal at the AFM tip. Different biosensors will then be developed and coupled to an AFM probe for scanning the chemical environment and detect in real-time chemical messengers secreted in the extracellular matrix in cellular communication.

Cette thèse considère de nouvelles architectures prometteuses des métamatériaux plasmoniques pour biosensing, comprenant: (I) des réseaux périodiques 2D de nanoparticules d'Au, qui peuvent supporter des résonances des réseaux de surface couplées de manière diffractive; (II) Reseaux 3D à base de cristaux plasmoniques du type d'assemblage de bois. Une étude systématique des conditions d'excitation plasmonique, des propriétés et de la sensibilité à l'environnement local dans ces géométries métamatérielles est présentée. On montre que de tels réseaux peuvent combiner une très haute sensibilité spectrale (400 nm / RIU et 2600 nm / RIU, ensemble respectivement) et une sensibilité de phase exceptionnellement élevée (> 105 deg./RIU) et peuvent être utilisés pour améliorer l'état actuel de la technologie de biosensing the-art. Enfin, on propose une méthode de sondage du champ électrique excité par des nanostructures plasmoniques (nanoparticules uniques, dimères). On suppose que cette méthode aidera à concevoir des structures pour SERS (La spectroscopie du type Raman à surface renforcée), qui peut être utilisée comme une chaîne d'information supplémentaire à un biocapteur de transduction optique. / This thesis consideres novel promissing architechtures of plasmonic metamaterial for biosensing, including: (I) 2D periodic arrays of Au nanoparticles, which can support diffractively coupled surface lattice resonances; (II) 3D periodic arrays based on woodpile-assembly plasmonic crystals, which can support novel delocalized plasmonic modes over 3D structure. A systematic study of conditions of plasmon excitation, properties and sensitivity to local environment is presented. It is shown that such arrays can combine very high spectral sensitivity (400nm/RIU and 2600 nm/RIU, respectively) and exceptionally high phase sensitivity (> 105 deg./RIU) and can be used for the improvement of current state-of-the-art biosensing technology. Finally, a method for probing electric field excited by plasmonic nanostructures (single nanoparticles, dimers) is proposed. It is implied that this method will help to design structures for SERS, which will later be used as an additional informational channel for biosensing.

<p>Chromatography is a powerful technique, essential in chemical analyses and preparative separation in industry and research. Many different kinds of chromatographic material are needed, due to the large variety of applications. Detailed methods of characterisation are needed to design new chromatographic materials and understand their properties. In this thesis, confocal Raman spectroscopy and surface enhanced Raman spectroscopy (SERS) have been applied to micrometer-size chromatographic beads, for which these techniques have not been used earlier. New methodology, optimized for use with the chromatographic beads, has been developed and evaluated. </p><p>Confocal spectroscopy has been used to determine distributions of functional groups within single chromatographic beads. This distribution is of great importance in determining the chromatographic properties, since the material is porous and the solute molecules can diffuse inside the beads. Most of the confocal experiments have been performed with Raman spectroscopy; fluorescence spectroscopy, using Nd³⁺ ions or dye-labelled proteins as fluorescence probes, has been used for comparison. </p><p>The concentration of adsorbed analytes is very low within the beads. SERS was therefore used to enhance the Raman signal. SERS-active surfaces were prepared by incorporating gold nano-particles into the interior of the bead. TEM measurements showed that the gold nano-particles could be observed throughout, and it was possible to record analyte spectra from different positions within the bead. Enhanced spectra could be obtained both for small test molecules and for larger bio-molecules, although the spectra for the smaller analytes were much more intense.</p>

Chromatography is a powerful technique, essential in chemical analyses and preparative separation in industry and research. Many different kinds of chromatographic material are needed, due to the large variety of applications. Detailed methods of characterisation are needed to design new chromatographic materials and understand their properties. In this thesis, confocal Raman spectroscopy and surface enhanced Raman spectroscopy (SERS) have been applied to micrometer-size chromatographic beads, for which these techniques have not been used earlier. New methodology, optimized for use with the chromatographic beads, has been developed and evaluated. Confocal spectroscopy has been used to determine distributions of functional groups within single chromatographic beads. This distribution is of great importance in determining the chromatographic properties, since the material is porous and the solute molecules can diffuse inside the beads. Most of the confocal experiments have been performed with Raman spectroscopy; fluorescence spectroscopy, using Nd3+ ions or dye-labelled proteins as fluorescence probes, has been used for comparison. The concentration of adsorbed analytes is very low within the beads. SERS was therefore used to enhance the Raman signal. SERS-active surfaces were prepared by incorporating gold nano-particles into the interior of the bead. TEM measurements showed that the gold nano-particles could be observed throughout, and it was possible to record analyte spectra from different positions within the bead. Enhanced spectra could be obtained both for small test molecules and for larger bio-molecules, although the spectra for the smaller analytes were much more intense.

Die Elektrochemie und die Spektroelektrochemie von selbst-organisiererten Monoschichten (selfassembled monolayers, SAMs) gebildet aus 2-Mercaptopyridin (2MP) und 4-Mercaptopyridin (4MP) wurden an polykristallinen Goldelektroden in wässrigen Elektrolytlösungen untersucht. Folgende Untersuchungsmethoden wurden angewandt: zyklische Voltammetrie, elektrochemische Impedanzmessungen (EIM) und oberflächenverstärkte Raman Streuung (surface-enhanced Raman scattering, SERS). Die elektrochemischen Untersuchungen von 2MP und 4MP in wässriger saurer Lösung (0.5 M H2SO4) zeigten, dass 2MP stärker adsorbiert wird als 4MP aufgrund der Bildung eines S-Au-N Chelates, wobei die S-Au-Interaktionen bei 4MP stärker sind. Die Bildung eines Chelates im Falle von 2MP verringert die Wahrscheinlichkeit der Bildung eines Dimers. In sauren Lösungen wird das N-Atom von 2MP protoniert, was zu einer schwächeren Bindung von 2MP-Molekülen zur Substratoberfläche führt. Die Ergebnisse der SERS-Untersuchungen stimmen mit den Resultaten aus der zyklischen Voltammetrie überein. Man erhält eine Au-S-Streckschwingungsbande für 2MP zwischen 225 bis 250 cm-1 bei Abscheidung aus wässriger oder saurer Lösung und für 4MP bei ca. 263 cm-1 in beiden Lösungen. Die SERS-Experimente ergaben eine senkrechte Orientierung zur Goldoberfläche sowohl für 2MP als auch für 4MP. Die Thion-Thiol-Tautomerie von 2- Mercaptopyridinen wurde ebenfalls in Betracht gezogen. Die Unter- und Überpotentialabscheidung von Kupfer auf einer polykristallinen Goldelektrode in wässriger 0.1 M Schwefelsäure in An- und Abwesenheit von SAMs von 2- und 4- Mercaptopyridin wurde mit zyklischer Voltammetrie untersucht. Es zeigte sich, daß bei Vorhandensein der SAMs die Elektrodeposition von Kupfer verhindert wird, was auf starke Wechselwirkungskräfte zwischen dem Adsorbat (MP) und der Goldoberfläche zurückzuführen ist. 2MP zeigt eine grössere Inhibierung, was höchstwahrscheinlich auf die Bildung der Chelatstruktur zurückzuführen ist. Es wurden ebenso Untersuchungen zum Einfluss von 2MP und 4MP auf die abgeschiedene Kupfermonolage auf der Goldelektrode durchgeführt. Es zeigte sich, daß die Kupfermonolage teilweise durch 2MP oder 4MP ersetzt wird. Die Elektronenaustauschgeschwindigkeit für das Fe2+/Fe3+-Redoxsystem in An- und Abwesenheit von 2MP- oder 4MP-Monolagen wurde mit zyklischer Voltammetrie und elektrochemischen Impedanzmessungen (EIM) untersucht. Es stellte sich heraus, dass der Elektronenaustausch höchstwahrscheinlich über Defektstellen in der Monolage (Pinholes) erfolgt. In einer wässrigen Lösung verringert 4MP den Elektronenaustausch stärker als 2MP. Da die Packungsdichte bei 4MP größer ist als bei 2MP ist wahrscheinlich auch die Zahl der Pinholes geringer in der 4MP-Monolage. In saurer Lösung liegen die N-Atome protoniert vor. Man kann davon ausgehen, dass in saurer Lösung zwei Prozesse gleichzeitig ablaufen, die für den Elektronenaustausch entscheidend sind. Erstens kommt es zu einer Abstoßung zwischen der positiv geladenen Monolage und den positiv geladenen Redoxionen. Und zweitens erfolgt eine Abstoßung zwischen den positiv geladenen Molekülen der SAMs, was zu einer geringeren Packungsdichte führt. Der Ladungsaustausch wird dominiert durch den zweiten Effekt. Mit Hilfe von EIM wurden die Elektronenaustauschgeschwindigkeit und der Bedeckungsgrad bestimmt. Die korrosionshemmende Wirkung von 2MP und 4MP auf Stahl in 3.5 % wässriger NaCl-Lösung wurde mit Hilfe der EIM untersucht. 2MP zeigte eine grössere Hemmung als 4MP. / The electrochemistry and spectroelectrochemistry of the self-assembled monolayers (SAMs) prepared of 2-mercaptopyridine (2MP) and 4-mercaptopyridine (4MP) dissolved either in water or 0.1 M H2SO4 have been investigated at polycrystalline gold electrodes in aqueous electrolyte solutions using cyclic voltammetry, electrochemical impedance measurements (EIM) and surface enhanced Raman spectroscopy (SERS). Electrochemical studies of 2MP and 4MP monolayers in aqueous acidic solution (0.5 M H2SO4) suggest that 2MP is adsorbed more strongly than 4MP due to the formation of S-Au-N chelate. However, the S-Au bond was found to be stronger in 4MP as compared with 2MP. The formation of the chelate in case of 2MP diminishes the probability of dimer formation. In the acidic solvent, the N-atom of 2MP molecule will be protonated leading to a weaker interaction of 2MP molecules with the substrate surface. The SERS results are in good agreement with the cyclic voltammetry results. The Au-S stretching band was obtained in the region from 215 to 245 cm-1 for 2MP deposited from water and acidic solvent and around 263 cm-1 for 4MP in both solvents. The SERS measurements showed also a perpendicular orientation of both 2MP and 4MP on the gold surface. In explaining the SERS results, the thione-thiol tautomerisations of the mercaptopyridines were also taken into consideration. The under- and overpotential deposition of copper on a polycrystalline gold electrode in aqueous 0.1 M sulfuric acid in the presence and in the absence of SAMs of 2- and 4-mercaptopyridine has been studied using cyclic voltammetry. In general, the presence of these SAMs has been found to inhibit the electrodeposition process of copper, suggesting very strong interactions between these adsorbates and the Au surface. 2MP shows a higher degree of inhibition, which is due to a stronger interaction probably due to the formation of the chelate structure. Studies have also been made of the influence of mercaptopyridines SAMs on the copper monolayer electrodeposited on the gold surface. The copper adlayer was found to be partially displaced by 2MP and 4MP monolayers. The rate of electron transfer for the Fe3+/2+ redox system on the gold electrode has been probed in the absence and presence of 2MP and 4MP monolayers by cyclic voltammetry and electrochemical impedance measurements (EIM). The charge transfer process was suggested to occur through the defects (pinholes) in the monolayer. In case of aqueous solvent 4MP decreases the electron transfer reaction stronger than 2MP. Since the packing density for 4MP is higher than that of 2MP the number of pinholes might be lower in 4MP monolayer. In acidic solvent the N-atoms of the mercaptopyridines will be protonated. It is proposed that two effects, which exist at the same time, are responsible for the electron transfer process in acidic solution. First, there will be a repulsive interaction between the positively charged monolayer and the positively charged redox probe. Second, there is a repulsion among the positively charged monolayer molecules that results in a less compact monolayer. The charge transfer is dominated due to the latter effect. With the EIM the rate of electron transfer and the surface coverage were determined. 2MP and 4MP were examined as steel corrosion inhibitors in 3.5% aqueous NaCl solution using EIM. 2MP shows higher inhibition efficiency than 4MP.

Novel biocompatible nanoprobes for optical imaging of Epidermal Growth Factor receptor (EGFR) were created. 5 and 18 nm gold nanoparticles (AuNPs) and 5 and 45 nm diameter silver nanoparticles (AgNPs) were conjugated to EGF protein via ?-lipoic acid. AgNPs were not previously attached to EGF. TOF-MS confirms EGF-linker formation. ELISA verifies the linked-EGF activity alone and with EGF-NPs. Core-shell silver-gold nanoparticles (AgAuNPs) gave similar results. TEM staining with uranyl acetate exhibits a bright ring, smaller than EGF, around nanoparticles. Dark field microscopy shows localized, intense cytoplasmic scattering, possibly lipid droplets, in cancer cells incubated with or without nanoprobes. Following injection, mice organs were harvested for EGF-NP immune response determination. Sterilization likely inactivated EGF before ICP-MS. Intense surface enhanced Raman scattering (SERS, 632.8 nm) follows MgSO4 induced EGF-AgNPs aggregation. Pelleted EGF-AgNP tagged cancer cells lack SERS indicative intensity contrast. AgAuNPs could provide increased stability, brighter SERS, and reduced silver biocompatibility concerns.

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.

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 CC ryšio ilgis ir elektroninė struktūra gali būti keičiama, keičiant potencialą.

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.

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.