It has been previously shown that SERS provides a rapid, confirmatory technique for the detection of blood, one of the most commonly found body fluids at a crime scene, from samples extracted with 1 µL of 50% acetic acid and placed on Au nanoparticle substrates developed by this laboratory. In recent results, the SERS spectra of blood extracted using a 50% acetic acid procedure is distinguished from 30 other reddish-brown stains (RBS) that do not contain blood, with 100% sensitivity and specificity using a partial least squares discriminant analysis (PLS-DA). A blind study was performed and 12 blinded samples were tested using the statistical method. All stains were identified as blood or non-blood with 100% accuracy. While peripheral blood and menstrual blood share many components, the complexity of menstrual blood is further enhanced by the addition of vaginal secretions. Further demonstrating SERS specificity, differential extraction procedures using water and acetic acid allow peripheral and menstrual blood to be distinguished. Given the sensitivity of SERS to identify bloodstains invisible to the naked eye, it is important to establish what limitations luminol might impose for SERS identification of bloodstains. SERS sensitivity allows blood diluted by a factor of at least 100 to be detected in the presence of undiluted luminol when using the acetic acid procedure. Current results demonstrate that the SERS detection limit is at least 103 diluted bloodstains when 1% luminol solutions, capable of producing the bright blue glow, are employed. In sexual assault cases, it may be necessary to identify the presence of semen prior to DNA analysis. Sexual assault cases may also require the identification of the presence of vaginal fluid in certain scenarios. A stain extraction method prior to SERS analysis was optimized for both semen and vaginal fluid by testing different extraction procedures with water and acetic acid. In semen, the acetic acid procedure was found to produce a signal intensity 4 times that of the water extraction procedure. Preliminary results show that for vaginal fluid, the water procedure was found to produce a SERS signal intensity more than 3 times that of the acetic acid procedure. Using differing solubilities of the chemical components of these body fluids, mixture deconvolution has been shown to be possible. The analysis of four swab types for SERS acquisition are compared in order to determine the optimal swab types for field collection of various trace body fluids. SERS analysis of trace body fluids for forensic work is a minimally destructive technique that can be applied for rapid and easy examination of evidence. The high sensitivity and specificity of SERS provides a robust spectroscopic technique for the rapid detection and identification of trace body fluids.

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SERS (surface-enhanced Raman spectroscopy) has the potential to be used in a variety of commercial and basic applications, which often rely on molecules that are bound to a nanostructured metal surface. Thiols are usually used as the intermediate to modify the substrate surface for SERS. In recent years, N-heterocyclic carbene (NHC) has been introduced as an alternative approach for metal surface modification. Nanostructured gold surfaces suitable for SERS had been modified by NHC species. Those studies showed the promising of the NHC modification route for the fabrication of a robust platform for SERS. The objective of this work is to explore the SERS characteristics of NHC species on silver surfaces. The interactions between two different NHC molecules and a nanostructured silver surface, instead of a gold surface, were studied for the first time. The experiments were realized in electrochemical conditions, using a three-electrodes system, to fully test the stability of the NHC-modified surfaces. The SERS spectra were compared to theoretical calculations and normal Raman in order to identify the vibrational characteristics of the NHC molecules. The effects of different NHC molecule substituents on the electrochemical stability of the surface were also discussed. The results showed that NHC molecules can be decomposed on the silver surface easily under electrochemical conditions. This contrast with the observations in gold, where the NHC monolayers showed a high level of stability. This work also discusses potential side products which may be derived from the decomposition of the NHC molecules. Raman spectra of potential side products were collected and compared to the NHC SERS collected under electrochemical control at different potentials. This study provides insights into the influence of the substituents at the NHC on their stability under the electrochemical condition, which should guide the development of future applications. / Graduate

Stabilization of highly sensitive noble metal nanoparticles is essential for their practical application. Bionanocomposites in which various types of noble metal nanoparticles, especially anisotropic noble metal nanoparticles, are immobilized into a macroscopic biomaterial membrane show promising applications in biomedical, catalytic, and environmental fields. This research focuses on developing two fabrication methods to generate novel bionanocomposite materials by immobilizing gold (Au) or silver (Ag) nanoparticles onto a "green" biomaterial, namely an eggshell membrane (ESM). Furthermore, the applications of the resulting bionanocomposite materials were demonstrated by studying their use as catalysts for environmental pollutant conversion and for the detection of two pollutant chemicals. The first fabrication method immobilizes ex situ synthesized nanoparticles onto a chemically modified ESM. Disulfide originating from the ESM was reduced by dithiothreitol into free thiol groups for binding to Au nanoparticles with different morphologies. The immobilization of Au nanoparticles greatly enhances their stability, making it possible to apply the resulting bionanocomposites for catalyzing the reduction reaction to convert pollutant p-nitrophenol (PNP) to p-aminophenol (PAP), with a great increase in their lifetime use from 2 to 10 reaction cycles. The second fabrication method utilizes the zwitterionic property of the protein based ESM for binding with Ag nanoparticles to form bionanocomposites. A seed mediated nanoparticle synthesis method originally performed in suspension was modified and adapted for the in situ synthesis of Ag nanodisks in this research. Ag nanoseeds were first immobilized onto an eggshell membrane using the static interaction between the nanoseeds and the membrane. Subsequently, Ag nanodisks were further grown directly on the Ag nanoseeds on the ESM. The final distribution density of Ag nanodisks can be adjusted by tuning the distribution density of Ag nanoseeds immobilized on the ESM. The performance of the resulting bionanocomposites were evaluated for both catalysis, and their application as substrates for surface enhanced Raman spectroscopy (SERS). The material performance was found to depend on the final distribution density of the Ag nanodisks on the ESM, offering the possibility to optimize bionanocomposite material performance by adjusting this density. A SERS based technique was further developed for detecting pollutant chemical species using the Ag nanodisks/ESM bionanocomposite material as a SERS substrate. Direct detection of thiram, a commonly used pesticide, was achieved at a concentration that is lower than that regulated by the US EPA. By using crystal violet as a SERS probe molecule, mercury, a heavy metal without an intrinsic Raman fingerprint, was indirectly detected not only at a limit of detection lower than most reported in the scientific literature, but also with a selectivity against a group of metal ions including Ba, Cu, Ca, Co, Mg, Mn, Ni, and Zn. It was also found that the detection sensitivity can be optimized by adjusting the Ag nanodisk distribution density on the ESM. The development of the fabrication approach and the use of ESM as a matrix material for immobilizing noble metal nanoparticles to form bionanocomposite materials demonstrates a novel strategy for meeting the needs of a variety of applications. The development of bionanocomposites for detecting pollutant species with different SERS activities by simply tuning the nanoparticle distribution density on the surface of the substrate, is a novel discovery, as it does not appear to have been previously reported in the literature. / Doctor of Philosophy / Noble metal nanoparticles exhibit special physical and chemical properties, which are totally different from the bulk material, making them promising candidates for use as novel materials in broad applications, such as catalysis, pollutant detection, antibacterial materials, etc. However, due to their high activity and poor colloidal stability (having high tendency to aggregate and lose activity), the nanoparticles require stabilization when being exploited for practical applications. A promising method to achieve this goal is to immobilize highly active noble metal nanoparticles onto a macroscopic membrane to form a nanocomposite. In this research, a "green" biomaterial, eggshell membrane (ESM), was utilized to immobilize noble metal nanoparticles. The resulting bionanocomposite materials were applied for catalyzing a reduction reaction to convert an environmental pollutant p-nitrophenol (PNP) to p-aminophenol (PAP) for environmental cleaning purposes, as well as detecting pollutant chemicals such as pesticide thiram and heavy metal mercury. General physical and chemical properties of the proteins in the ESM include rich chemical functional groups on the amino acid residue, and a zwitterionic property that allows the surface charge of the ESM to be changed under different pH levels. These properties, which have not been unleashed to immobilize noble metal nanoparticles in this field as of yet, were exploited in this research to create strong interactions between the noble metal nanoparticles and the ESM. This resulted in the formation of a bionanocomposite where the ESM served as a matrix for stably immobilizing the nanoparticles. Different bionanocomposites were fabricated using gold (Au) or silver (Ag) nanoparticles. The resulting bionanocomposite materials with gold nanoparticles were applied for catalyzing a reduction reaction for the conversion of p-nitrophenol, a commonly used chemical in the pharmaceutical photographic industries. The immobilized nanoparticles exhibited catalytic activity for ten reaction cycles and one hundred days after they were fabricated, while the colloidal nanoparticles (not immobilized nanoparticles) have catalytic activity for only two reaction cycles. For the chemical detection application, bionanocomposites with immobilized silver nanodisks were used as substrates for surface enhanced Raman spectroscopy. Different detection strategies were developed for detecting thiram with intrinsic Raman fingerprints and mercury without intrinsic Raman fingerprints. Outstanding detection sensitivities were achieved compared to those reported in the literature. For detection of mercury, a good selectivity was also obtained against a group of metal ions including Ba, Cu, Ca, Co, Mg, Mn, Ni, and Zn. The development of the fabrication approach and the use of ESM as a matrix material for immobilizing noble metal nanoparticles to form bionanocomposite materials demonstrates a good strategy for meeting the needs of a variety of applications

Currently, it is very challenging to accurately monitor the response of patients to radiation therapy over the course of treatment. The initial response to ionizing radiation occurs in the cells at a molecular level, and effects of the response are not typically noticeable on short time scales. Surface-enhanced Raman Spectroscopy, or SERS, has proven to be a useful technique in the analysis of tissues and cells at a molecular level. Specifically, the use of targeted SERS probes allows for the detection of specific proteins on the cell membrane. The work presented here looks to assess the feasibility of using targeted SERS probes and two-dimensional SERS microscopy to measure the response of tumour cells to ionizing radiation, by identifying changes in the distribution of membrane proteins following exposure to clinically relevant doses of ionizing radiation (≤ 60Gy). Two different types of targeted SERS probes were investigated, based on the work of Grubisha et al. ([1]; Type I) and Qian et al. ([2]; Type II), both containing a gold nanoparticle core. In a simplified cellular experiment, biotin on the surface of biotinylated OVCAR5 cells was targeted with streptavidin-SERS probes, and the Type-II SERS probes showed the most promising results. However, SERS maps still provided less characteristic spectral signal than expected, and challenges remain in the development of a reproducible cellular imaging technique. Despite difficulties in cellular imaging, the functionality of the Type-II SERS probes was verified separately, using gold slides with a biotin monolayer in place of cells. Following verification, the SERS intensities provided by differently sized clusters of the SERS probes were characterized. To begin, both SERS maps and scanning electron microscope (SEM) images of gold slides were acquired after incubation with Type-II SERS probes for multiple times (1hr, 2hr, 3hr, 12hr). Data analysis of the SEM images provided a measure of the physical distribution of the SERS probes on the surface of the slide, while analysis of the SERS maps provided information about the spectral distribution of the probes. By relating the information provided by the SEM images and SERS maps, a simple polynomial relationship between SERS intensity and the number of clustered SERS probes providing the enhancement was determined, providing a framework for quantifiable SERS imaging. Finally, an independent experiment was devised to ensure that exposure to clinically relevant doses of ionizing radiation would affect the ability of the targeted protein to bind to SERS probes, thus leading to measurable differences in SERS maps of irradiated and unirradiated cells. A series of experiments utilizing the enzyme-linked immunosorbant assay (ELISA) was performed to test the effect of ionizing radiation-induced damage on the ability of streptavidin to bind to biotin, and the results confirmed that a noticeable reduction in binding could be detected at doses as low as 10 Gy. The results of this work demonstrate that following the development of a suitable cell/SERS probe incubation technique, Type-II SERS probes would be appropriate for use in quantifiable SERS imaging. Also, it is suggested that a measurable change in protein function will be present when comparing SERS maps of control cells to those of cells irradiated to clinically relevant doses. / Graduate

Dans cette thèse, des réseaux de nano- et micro-capteurs électrochimiques et opto-électrochimiques sont fabriqués en utilisant la technique de microfabrication « template synthesis ». Dans une première partie, des ensembles de nanoélectrodes (NEEs) sont utilisés comme plate-forme pour obtenir un biocapteur. Les NEEs sont préparés par déposition d’or dans une membrane poreuse en polycarbonate. L’originalité de notre approche a été de modifier la membrane de polycarbonate entourant les NEEs et non les NEEs elles-mêmes. La peroxydase de raifort (HRP) qui est fixée sur un anticorps secondaire a servi comme marqueur. Cette enzyme catalyse la réduction de H2O2 qui est ajouté en solution. En utilisant un système de détection dérivé de l'approche ELISA (Enzyme- Linked Immuno-Sorbants Assay), le récepteur de la protéine HER2 a été pris comme analyte cible. Il s’agit d’une protéine très importante puisqu’elle permet de dépister le cancer du sein. Dans une seconde partie, un réseau ordonné de sondes opto-électrochimiques est développé sur la face distale d’un faisceau de fibres optiques qui a été attaquée sélectivement par voie humide. Une structure macroporeuse est fabriquée en utilisant un cristal colloïdal comme « template ». L’or est ensuite déposé dans les interstices avant de dissoudre les nanoparticules de latex formant le cristal colloïdal. Ce réseau de microcavités macroporeuses a été testé avec succès comme substrat pour des mesures de Raman exalté de surface (SERS). / In this thesis, arrays of nano- and microelectrodes are developed to obtain electrochemical and optoelectrochemical sensors, by using the template synthesis as a microfabrication technique. In the first part, ensembles of nanoelectrodes (NEEs), obtained using a track-etched polycarbonate membrane as template, are functionalised in order to obtain electrochemical immunosensors. A biorecognition chain, antigen-antibody, is immobilized on the wide polycarbonate membrane letting uncovered the gold nanodisk electrodes. A label redox enzyme, linked to the biorecognition chain, is recognized and quantified electrochemically. Two different detection schemes are developed and low protein detection limits are achieved. In the second part, a macroporous micrometer sized opto-electrochemical sensor is developed on the distal face of an imaging fiber (coherent optical fiber bundle). A microwell array is obtained by controlled chemical etching, by exploiting the different chemical composition between cores and clads. Colloidal templates are created inside the microcavities, using polystirene beads of 280 nm. Gold is deposited inside the cavities, filling the void in the colloidal template, exploiting electroless and electrochemical deposition techniques. The gold macroporous structure inside the wells is successfully tested as SERS substrate.

L' objet d'étude de cette thèse est l' origami d' ADN. Le nombre important d'applications utilisant cette technique, et leurs diversités, sont des preuves que ces structures présentées en 2007 sont une avancée importante pour la technologie ADN. Dans cette thèse nous présentons dans un premier temps les résultats de simulations sur les propriétés mécaniques de ces nouvelles structures. Les résultats d'expériences et de simulations sur le processus de formations pour de petites structures d'ADN et sur des origamis d'ADN sont présentés dans une deuxième partie. Finalement une application de ces structures à la détection SERS est proposée, et les résultats expérimentaux concernant la réalisation de cette structure sont discutés. / DNA origami are new nanostructures (2006) whose physical properties are still to be understood. In this work we were first interested in their mechanical properties. The first approach of this study was through the use of polymer physics, as it is the classical way to study DNA. We then used computer simulations to model the system in a more detailed manner and to extract general rules on the mechanical behaviour of DNA constructs. The other aspect that we studied is the process of formation of DNA origamis. We first realised an experimental study of the process of formation of the simplest origami that we could envision. This study was intended to investigate basic principles on the process of formation of DNA structures. A coarse grain model is then developed to have a first insight onto the formation process. Then an experimental study on large origamis follows with a modeling of the annealing and melting curves based on the principles determined from the study of the simplest origami. We also worked on the development of a SERS platform.

Nesta tese foram utilizadas as espectroscopias de espalhamento Raman intensificado pela superficie(Surface-EnhancedRaman Scattering- SERS) e Raman ressonante para monitorar a adsorção e o comportamento faradáico do ácido esquárico e seus derivados, além de um sal de transferência de carga de esquarato e tetratiofulvaleno e dois copolímeros de esquarato e pirróis. Outro tema desenvolvido neste estudo foi a construção de substratos SERS-ativos de elevado desempenho. A técnica SERS tem sido campo de grande interesse desde a detecção do espectro aman de uma única molécula, em 1997.O desenvolvimento de substratos SERS-ativos de elevado desempenho depende da apropriada manipulação de superficies metálicas nanoestruturadas, o que nos levou a adquirir conhecimento na síntese destes substratos. Colóides e filmes de Ag e Au foram preparados e seu desempenho SERS comparado com a superficie de eletrodos ativados por ciclos de oxidação-redução. A intensificação obtida para esses filmes foi comparávelà observada em eletrodos. O ânion esquarato, produto da dupla desprotonação do ácido esquárico, apresenta substancial delocalização de carga, sendo amplamente usado na síntese de materiais orgânicos condutores. Foram realizadas as caracterizações vibracionais do ácido esquárico, hidrogeno-esquarato e esquarato, além de seu radical, através da técnica SERS. Baseado nos resultados experimentais, um mecanismo de adsorção destas espécies sobre eletrodos de Au e filmes de ilhas de Ag ou Au foi proposto. Os resultados SERS também mostram que o ácido esquárico adsorvido sobre Au é decomposto em um processo catalisado pela superficie metálica, emboraestas espécies sejam muito estáveis em solução. Baseado nos resultados SERS do tetratiofulvaleno e de suas espécies oxidadas, foi eletroquimicamente, e formado pelo radical-cátion tetratiofulvaleno e o radical-ânion esquarato. Os elevados fatores de intensificação de Raman ressonante e SERS do tetratiofulvaleno impediram a detecção do esquarato no sal de transferência de carga. Duas poliesquaraínas polí(1-metilpirrol-co-ácido esquárico) e poli(1-dodecilpirrol-coácido esquárico) foram sintetizadas e caracterizadas pelas espectroscopias Raman ressonante, SERS e ressonância paramagnética de spin (EPR). Estes resultados nos levaram a propor uma estrutura polimérica diferente da apresentada pela literatura. Nossos resultados mostraram a presença de um radical orgânico delocalizado, do ânion esquarato protonadoe de dicátions similares aos presentes no polipirrol. / In this Thesis Surface-Enhanced Raman Scattering (SERS) and resonance Raman spectroscopy were used for monitoring the adsorption and faradaic behavior of squaric acid and its derived species. In addition, the charge transfer salt of squarate and tetrathiofulvalene and the copolymers of squarate and pyrroles were also studied. Another theme developed in this study was the manufacturing of. high performance SERS-active substrates. SERS technique has been a field of great interest since the detection of a single molecule Raman spectrum in 1997. The development of high perforrnance SERS-active substrates depends on the proper manipulation of nanostructured metal surfaces, and order to acquire know-how in the synthesis of such substrates. Ag and Au island films as well as colloid substrates were prepared and compared with electrode surfaces SERS activated by oxidation-reduction cycles. The enhancement factor obtained for such films is comparable to those observed in electrodes. The squarate anion, product of the double deprotonation of squaric acid, shows substantial charge delocalization, being largely used in the synthesis of conducting organic materiaIs. The vibrational characterization of squaric acid, hydrogen-squarate, squarate as well as its radical was carried out from the SERS data. Based on the experimental data an adsorption mechanism of such species on Au electrodes and Au or Ag islands was proposed. SERS results also show that squaric acid adsorbed on Au is decomposed in a process catalyzed by the metal surface, although in solution it proves to be a very stable specles. The vibrational characterization of an electrochemically forrned charge transfer salt between tetrathiofulvalene radical-cation and squarate radical-anion was done based on the SERS data of tetrathiofulvalene and its oxidation species. The large resonance Raman and SERS enhancement factors of tetrathiofulvalene preclude the detection of the squarate species in the charge transfer salt. Two polysquaraines: poly(1-methylpyrrole-co-squaric acid) and poly(1-dodecylpyrrole-co-squaric acid) were synthesized and characterized by resonance Raman, SERS and Electron Paramagnetic Resonance Spectroscopy (EPR) techniques. The results lead us to propose a polymeric structure different from that present in the literature. Our data showed a delocalized organic radical in the polymeric matrices together with dications similar to those present in polypirrole and protonated squarate anion.