Many environmental compartments are acknowledged transmission routes for infectious diseases, antibiotic resistance, and anthropogenic pollution. The need for environmental sensing has consistently been stressed as a means to minimize public health threats caused by such contaminants. Many analytical detection techniques have been developed and applied for environmental sensing. However, these techniques are often reliant upon centralized facilities and require intensive resources. For these reasons their use can be challenging under resource-constrained conditions characterized by poor water, sanitation, and hygiene (WASH) services. In this dissertation, we developed biotechnology- and/or nanotechnology-advanced analytical tools for environmental sensing that have potential for future application in regions with poor WASH services. First, loop-mediated isothermal amplification (LAMP) and nanopore sequencing were applied to develop assays for the detection of SARS-CoV-2, the causative agent of COVID-19, in wastewater samples. Second, surface-enhanced Raman spectroscopy (SERS) was applied for environmental detection of a range of analytes. Gold nanoparticle (AuNP)-based SERS substrates were fabricated by droplet evaporation-induced aggregation on a hydrophobic substrate. These SERS substrates were then applied for the detection of antibiotic resistance genes (ARGs) and other environmental contaminants (e.g., dye or hydrophobic organic contaminants). In a separate study, Au nanostructured SERS substrates were fabricated and applied for pH sensing in a range of environmental media. Finally, the environmental impact of an AuNP-based colorimetric detection assay was assessed via life cycle assessment. / Doctor of Philosophy / Environmental sensing is an important means to intervene against public health threats of infectious diseases and environmental contaminants. However, currently available analytical tools for environmental samples often require intensive resources that are not available in low- and middle-income countries. In this dissertation, we developed biotechnology and/or nanotechnology advanced analytical tools for environmental sensing that have potential future application applied under resource-constrained conditions. First, we applied loop-mediated isothermal amplification (LAMP) and nanopore sequencing to develop detection assays for SARS-CoV-2, the causative agent of COVID-19, in wastewater samples. Second, we applied surface-enhanced Raman spectroscopy (SERS) to develop assays for environmental analytes. We fabricated SERS substrates by evaporation-induced aggregation of gold nanoparticles (AuNPs) on a hydrophobic substrate and applied these for the detection of antibiotic resistance genes (ARGs) and other environmental contaminants. In addition, Au nanostructured SERS substrates were fabricated and applied for pH sensing in a range of environmental media. Finally, we used life cycle assessment to quantitatively evaluate the environmental impacts of an AuNP-based sensing applications.

Non-invasive and continuous spatiotemporal pathogen monitoring at biological interfaces (e.g., human tissue) holds promise for transformative applications in personalized healthcare (e.g., wound infection monitoring) and environmental surveillance (e.g., airborne virus surveillance). Despite notable progress, current receptor-based biosensors encounter inherent limitations, including inadequate long-term performance, restricted spatial resolutions and length scales, and challenges in obtaining multianalyte information. Surface-enhanced Raman spectroscopy (SERS) has emerged as a robust analytical method, merging the molecular specificity of Raman spectroscopy's vibrational fingerprinting with the enhanced detection sensitivity from strong light-matter interaction in plasmonic nanostructures. As a receptor-free and noninvasive detection tool capable of capturing multianalyte chemical information, SERS holds the potential to actualize bio-interfaced spatiotemporal pathogen monitoring. Nonetheless, several challenges must be addressed before practical adoption, including the development of plasmonic bio-interfaces, sensitive capture of multianalyte information from pathogens, regeneration of nanogap hotspots for long-term sensing, and extraction of meaningful information from spatiotemporal SERS datasets. This dissertation tackles these fundamental challenges. Plasmonic bio-interfaces were created using innovative nanoimprint lithography-based scalable nanofabrication methods for reliable bio-interfaced spatiotemporal measurements. These plasmonic bio-interfaces feature sensitive, dense, and uniformly distributed plasmonic transducers (e.g., plasmonic nano dome arrays, optically-coupled plasmonic nanodome and nanohole arrays, self-assembled nanoparticle micro patches) on ultra-flexible and porous platforms (e.g., biomimetic polymeric meshes, textiles). Using these plasmonic bio-interfaces, advancements were made in SERS signal transduction, machine-learning-enabled data analysis, and sensor regeneration. Large-area multianalyte spatiotemporal monitoring of bacterial biofilm components and pH was demonstrated in in-vitro biofilm models, crucial for wound biofilm diagnostics. Additionally, novel approaches for sensitive virus detection were introduced, including monitoring spectral changes during viral infection in living biofilms and direct detection of decomposed viral components. Spatiotemporal SERS datasets were analyzed using unsupervised machine-learning methods to extract biologically relevant spatiotemporal information and supervised machine-learning tools to classify and predict biological outcomes. Finally, a sensor regeneration method based on plasmon-induced nanocavitation was developed to enable long-term continuous detection in protein-rich backgrounds. Through continuous implementation of spatiotemporal SERS signal transduction, machine-learning-enabled data analysis, and sensor regeneration in a closed loop, our solution has the potential to enable spatiotemporal pathogen monitoring at the bio-interface. / Doctor of Philosophy / Continuous monitoring of pathogens within our bodies and surrounding environments is indispensable for various applications in personalized healthcare (e.g., monitoring wound infections) and environmental surveillance (e.g., airborne virus tracking). To accomplish this, we require sensors capable of seamlessly interfacing with biological systems, such as human tissue, and consistently providing pathogen-related information (e.g., spatial location and pathogen type) over prolonged periods. Our research relies on Surface-enhanced Raman spectroscopy (SERS) to address this challenge. SERS enables noninvasive sensing by providing unique fingerprints of molecules near the sensor's surface. SERS holds the potential to enable bio-interfaced spatiotemporal pathogen monitoring, but several challenges must be tackled before practical adoption. In this dissertation, we address various fundamental challenges in SERS, including constructing SERS devices that can seamlessly interface with biological systems while maintaining performance, sensitively capturing pathogen-related information, extracting meaningful insights from SERS datasets, and continuously regenerating the sensor surface to ensure long-term performance. We developed SERS devices capable of seamlessly interfacing with biological systems using innovative scalable nanofabrication methods. These devices contain sensitive, dense, and uniformly distributed SERS sensors on flexible and porous platforms, such as polymeric scaffolds and textiles. Leveraging these SERS devices, we made advancements in pathogen sensing, data analysis, and sensor regeneration. We demonstrated large-area spatiotemporal monitoring of biofilm components and pH in lab-grown biofilm models, critical for wound biofilm diagnostics. Additionally, we introduced novel approaches for sensitive virus detection, including monitoring changes in SERS signals during viral infection in living biofilms and directly detecting decomposed viral components. The SERS datasets were analyzed using machine learning models to extract biologically relevant spatial and temporal information, such as the spatial location of pathogen components and the temporal stage of pathogen growth, and to predict biological outcomes. Finally, we developed a sensor regeneration method to enable long-term continuous detection in complex backgrounds, such as blood. By continuously performing spatiotemporal pathogen sensing, data analysis, and sensor regeneration in a closed loop, our solution has the potential to realize bio-interfaced spatiotemporal pathogen monitoring.

Due to an ever-increasing global population and limited resource availability, there is a constant need for detection of both natural and anthropogenic hazards in water, air, food, and material goods. Traditionally a different instrument would be used to detect each class of contaminant, often after a concentration or separation protocol to extract the analyte from its matrix. Raman spectroscopy is unique in its ability to detect organic or inorganic, airborne or waterborne, and embedded or adsorbed analytes within environmental systems. This ability comes from the inherent abilities of the Raman spectrometer combined with concentration, separation, and signal enhancement provided by drop coating deposition Raman (DCDR) and surface-enhanced Raman spectroscopy (SERS). Herein the capacity of DCDR to differentiate between cyanotoxin variants in aqueous solutions was demonstrated using principal component analysis (PCA) to statistically demonstrate spectral differentiation. A set of rules was outlined based on Raman peak ratios to allow an inexperienced user to determine the toxin variant identity from its Raman spectrum. DCDR was also employed for microcystin-LR (MC-LR) detection in environmental waters at environmentally relevant concentrations, after pre-concentration with solid-phase extraction (SPE). In a cellulose matrix, SERS and normal Raman spectral imaging revealed nanoparticle transport and deposition patterns, illustrating that nanoparticle surface coating dictated the observed transport properties. Both SERS spectral imaging and insight into analyte transport in wax-printed paper microfluidic channels will ultimately be useful for microfluidic paper-based analytical device (𝜇PAD) development. Within algal cells, SERS produced 3D cellular images in the presence of intracellularly biosynthesized gold nanoparticles (AuNP), documenting in detail the molecular vibrations of biomolecules at the AuNP surfaces. Molecules involved in nanoparticle biosynthesis were identified at AuNP surfaces within algal cells, thus aiding in mechanism elucidation. The capabilities of Raman spectroscopy are endless, especially in light of SERS tag design, coordinating detection of analytes that do not inherently produce strong Raman vibrations. The increase in portable Raman spectrometer availability will only facilitate cheaper, more frequent application of Raman spectrometry both in the field and the lab. The tremendous detection power of the Raman spectrometer cannot be ignored. / Ph. D.

This work describes a master’s thesis in engineering physics at Umeå University carried out during the spring semester of 2014. In the thesis the student has constructed and tested a temperature controlled cell for cooling/heating of surface-enhanced-Raman-spectroscopy (SERS) substrates for rapid detection of volatile substances. The thesis was carried out at the Swedish Defence Research Agency (FOI) in Umeå, Sweden. A Linkam Scientific Instruments TS1500 cell was equipped with a Peltier element for cooling/heating and a thermistor temperature sensor. A control system was constructed, based on an Arduino Uno microcontroller board and a pulse-width-modulated (PWM) H-bridge motor driver to control the Peltier element using a proportional-integral (PI) control algorithm. The temperature controlled cell was able to regulate the temperature of a SERS substrate within -15 to +110 °C and maintain the temperature over prolonged periods at ±0.22 °C of the set point temperature. Gas phase of 2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane (isoflurane) was flowed through the cell and SERS spectra were collected at different temperatures and concentrations. This test showed that the signal is increased when the substrate is cooled and reversibly decreased when the substrate was heated. Keywords: temperature control, Raman scattering, surface enhanced Raman spectroscopy SERS, SERS substrate, volatile substances, Peltier module, thermistor, PWM, H-bridge, PI(D) control. / Detta dokument beskriver ett examensarbete för civilingenjörsexamen i teknisk fysik vid Umeå Universitet som utförts under vårterminen 2014. I examensarbetet har en kyl-/värmecell för temperaturkontroll av substratytor för ytförstärkt ramanspektroskopi (SERS) för snabb detektion av farliga flyktiga ämnen konstruerats och testats. Arbetet utfördes vid Totalförsvarets forskningsinstitut (FOI) i Umeå, Sverige. Utgångspunkten var ett Linkam Scientific Instruments TS1500 mikroskopsteg, vilket utrustades med ett Peltierelement för kylning/värmning och en termistor för temperaturövervakning. Ett styrsystem baserat på ett Arduino Uno mikrostyrenhetskort konstruerades med ett motordrivkort (H-brygga) vilket använder pulsbreddsmodulering (PWM) för att reglera spänningen till Peltierelementet utifrån en PI-regulator. Den färdiga cellen klarade att reglera temperaturen på ett SERS-substrat i ett temperaturspann på ungefär -15 till +110 °C med en temperaturstabilitet på ±0.22 °C av måltemperaturen. Cellen testades sedan på flyktiga ämnen för att visa dess funktion. Difluorometyl-2,2,2-trifluoro-1-kloroetyleter (isofluran) i gasfas, med instrumentluft som bärargas, flödades genom cellen och SERS-spektra erhölls vid olika koncentrationer och temperaturer. Vid samtliga koncentrationer visades att lägre temperatur ger ökad signalstyrka. När ytan sedan värmdes upp sjönk signalen reversibelt tillbaka till ursprungsvärdet. Nyckelord: temperaturkontroll, ytförstärkt ramanspektroskopi, SERS, flyktiga ämnen, Peltierelement, thermistor, PWM, H-brygga, PI(D)-regulator.

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.

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.