In this thesis, we focus on the structural identification of the interface using surface enhanced Raman spectroscopy (SERS) and inelastic electron tunnelling scattering (IETS). Two different molecular junctions, namely gold/ trans-1,2-bis (4-pyridyl) ethylene/gold junction and gold/4,4'-bipyridine/gold junctions in various conditions were studied and the corresponding configurations were determined. The enhancement in SERS was also studied by employing the time-dependent density functional theory. Furthermore, we studied some properties of the interface, such as the stability of the adsorbates and charge transfer properties of molecular junctions. The decrease in the stability of molecules was found when adsorbed on metallic surface and trapped in metallic junctions. Our studies explained several puzzles and by rational design, more stable molecular devices were obtained.

Biomedical sciences are in need of more versatile and more sensitive approaches for research and also for diagnostic purposes. In particular, intracellular detection and imaging of disease relevant proteins is a challenge. Although the state of the art method of intracellular imaging is fluorescence, it suffers from several drawbacks. Raman is an alternative imaging modality and this work investigates the use of different Raman techniques for detection and imaging of cellular constituents. In one aspect of the work, surface-enhanced Raman spectroscopy using gold nanoshells excitable at a wavelength of 780 nm was investigated. Initially the investigation of the uptake of the 150 nm diameter nanoparticles showed that NS are taken up voluntarily by a non-standard en- docytosis mechanism into mammalian fibroblast cells. Furthermore it was shown that internalized particles have no detrimental in uence on cell growth or cell viability. That these nanoparticles are non toxic was further confirmed by testing for markers of apoptosis and necrosis. Preliminary surface-enhanced Raman spectroscopy (SERS) studies produced spectra from intracellular compartments with an enhancement factor of 1010. To yield high specificity of the intracellular Raman protein sensor, two different approaches were studied. The first is based on the application of DNA aptamers which form a stacked G-quadruplex on target protein binding. A SERS sensor based on the well characterized Thrombin binding aptamer (TBA) yielded high reproducibility, high target specificity, and a limit of detection down to 0.1 fM. Further studies on a similar stacked G-quadruplex forming aptamer confirmed that observed detection signal is produced by the aptamer assuming its secondary structure but also showed that the stabilization and formation of the G-quadruplex secondary structure is strongly buffer dependent. A second sensing approach was based on a peptide (a3(IV)NC1) influential in Goodpasture's syndrome, an autoimmune disease. With the help of this peptide we found that an intracellular redoxpotential of -200 mV is necessary to make it accessible for the protease Cathepsin D. We found that SERS sensing has the ability to study the binding of Cathepsin D, its activity and with the help of a synthesized amino-acid SERS library the direct detection of the remaining peptide products. Finally this work concludes with imaging the changes of lipid droplet structure and distribution in fibroblast cells during the infection process of the murine cytomegalovirus (MCMV) in fixed and in living cells by coherent anti-Stokes Raman based on a Synchro-lock phase coupled setup. This showed that CARS imaging is able to non-invasively investigate the changes of lipid structures during different stages of the infection process and therefore promises to be a valuable tool in biological research.

Intracellular redox potential affects cellular function and its dysregulation is associated with disease. Current methods of monitoring intracellular redox potential are limited because they typically only report potentials of the redox buffer glutathione. Our group has developed redox-active probe molecules that change bond order depending on the probe oxidation state, and are instead sensitive to overall redox potential within the cell. Gold nanoshells coated with the probe form a novel intracellular redox nanosensor, and spectral discrimination of the oxidised and reduced states by Surface-Enhanced Raman Scattering (SERS) allows calculation of redox potential. Prior work by the group provided basic proof-of-principle for its use in measuring intracellular redox potential. The aim of this project, therefore, was to develop the tools and techniques to enable its application to meaningful biological questions, and extend the method into a pathologically relevant cell line. The initial stages of the project standardised the functionalisation of gold nanoshells with the NQ probe molecule and the application of the nanosensors to the A549 human lung cancer cell line. Toxicity tests confirmed the nanosensor was non-toxic. A protocol was then developed for rapidly obtaining SERS maps to enable localisation of nanosensors within the cell. This was successful, and the protocols can be applied to any combination of adherent cell type and nanosensor. A bespoke piece of software was created to determine redox potential and pH from SERS maps to produce a colourmap showing spatial variation of redox potential and pH with subcellular resolution. This software enables more rapid and precise calculation of redox potential or pH than manual processing. As a test case, changes in intracellular redox potential in response to treatment with toxic metal nanoparticles were studied and shown to correlate with other measures of oxidative stress. Hypoxia (abnormally low oxygen levels) is relevant in disease. Investigating redox potential in hypoxic cells requires precise control of the oxygen concentration during the acquisition of SERS spectra. To facilitate such experiments, a specialised imaging chamber was designed, constructed and tested. Such environmental control enables experiments to be carried out at various oxygen concentrations as well as under optimal cellular physiological conditions, enabling not only the response to alterations in oxygen levels to be studied but also extending the biological model system to more closely reflect animal physiology. Finally, a device was constructed that allowed the acquisition of SERS spectra from both intracellular and extracellular nanosensors in the same experiment, as the relationship between intracellular and extracellular redox potential is incompletely understood. The intracellular and extracellular nanosensors are spatially separated, allowing clear discrimination of the SERS spectra obtained simply by changing the orientation of the device. This device enables the effect of quantitative modification of extracellular redox potential on intracellular redox potential to be investigated. In summary, the work has greatly extended a method of measuring intracellular redox potential. It was taken from the proof-of-principle stage to being a robust method, capable of providing useful quantitative biological information. Improvements have been made in production and toxicity testing of the nanosensors, robustness of SERS data acquisition and analysis, environmental control during SERS data acquisition and application to disease-relevant cell culture models. The result is that we are now able to rapidly and reproducibly determine intracellular redox potential in single cells.

Submitted by Rafael Jesus Gonçalves Rubira null (rafael.gon.fis@gmail.com) on 2016-08-15T16:57:13Z No. of bitstreams: 2 Dissertação.pdf: 2967911 bytes, checksum: c00ff7d23b16f63e24d0d8ea23bcddd7 (MD5) Dissertação.pdf: 2967911 bytes, checksum: c00ff7d23b16f63e24d0d8ea23bcddd7 (MD5) / Rejected by Ana Paula Grisoto (grisotoana@reitoria.unesp.br), reason: Solicitamos que realize uma nova submissão seguindo as orientações abaixo: Foram submetidos 2 arquivos PDF’s, apenas 1 arquivo deve ser submetido. O arquivo PDF não deve estar protegido e a dissertação/tese deve estar em um único arquivo, inclusive os apêndices e anexos, se houver. Corrija estas informações e realize uma nova submissão contendo o arquivo correto. Agradecemos a compreensão. on 2016-08-16T14:54:28Z (GMT) / Submitted by Rafael Jesus Gonçalves Rubira null (rafael.gon.fis@gmail.com) on 2016-08-16T17:51:16Z No. of bitstreams: 1 Dissertação.pdf: 2967911 bytes, checksum: c00ff7d23b16f63e24d0d8ea23bcddd7 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-08-17T13:05:08Z (GMT) No. of bitstreams: 1 rubira_rjg_me_prud.pdf: 2967911 bytes, checksum: c00ff7d23b16f63e24d0d8ea23bcddd7 (MD5) / Made available in DSpace on 2016-08-17T13:05:08Z (GMT). No. of bitstreams: 1 rubira_rjg_me_prud.pdf: 2967911 bytes, checksum: c00ff7d23b16f63e24d0d8ea23bcddd7 (MD5) Previous issue date: 2016-07-22 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os herbicidas atrazina e prometrina pertencem à classe das s-triazinas, possuindo propriedades causadoras de malefícios em seres humanos, sendo classificados como agentes tóxicos. Esta dissertação para a defesa de Mestrado insere-se no projeto de mestrado cujo objetivo principal é investigar a detecção dos herbicidas atrazina e prometrina utilizando a técnica de espalhamento Raman amplificado em superfície (SERS, do inglês surface-enhaced Raman scattering), que possui alta sensibilidade e seletividade na detecção de analitos, sendo a aplicação sensorial o objetivo mais amplo deste projeto. Os herbicidas são dissolvidos em diferentes concentrações em colóides de prata (AgNPs: nanopartículas de prata) sintetizados a partir de nitrato de prata, tendo cloridrato de hidroxilamina como agente redutor. A atrazina foi detectada em concentrações da ordem de 10-12 mol/L em água ultrapura com boa resolução sinal ruído, e 10-11 mol/L em água deionizada. A prometrina também apresentou detecção em água ultrapura com concentrações da ordem de 10-12 mol/L e para sistemas com água deionizada e de torneira, a concentração foi da ordem de 10 9 mol/L. Tais valores estão abaixo dos limites de potabilidade de água permitidos pelas agências reguladoras (1,39 x 10 8 mol/L) para atrazina e 9,62 x 10-8 mol/L para a prometrina), limites estes que representam um desafio analítico por si só. Complementarmente, os espectros SERS foram classificados por técnicas computacionais de visualização da informação, possibilitando uma melhor análise dos dados. Em termos dos mecanismos de adsorção para as soluções em água ultrapura, a atrazina adsorve sobre as AgNPs via átomo de N entre o radical etil e o átomo de Cl e a prometrina têm o seu anel triazínico posicionado paralelo à superfície das AgNPs adsorvendo via átomos C-S. Tais mecanismos não puderam ser determinados para águas deionizada e de torneira. Complementarmente foram realizadas medidas de espectroscopia de impedância na detecção de ambos os herbicidas em filmes layer-by-layer (LbL) de PAH/AgNPs. / The atrazine and prometryne herbicides belong to the class of s-triazines, having properties that cause harm in humans and are classified as toxic agents. This thesis for the Master Qualification Examination General is part of the master project whose main objective is to investigate the detection of atrazine and Prometryn herbicides using surfaceenhanced Raman scattering (SERS) that has high sensitivity and selectivity in analyte detection, being sensory application the broader objective of this project. The herbicides are dissolved in various concentrations of silver colloids (AgNPs: silver nanoparticles). The atrazina was detected in concentration of order 10-12 mol/L in ultrapure water with good resolution signal noise and 10-11 mol/L in deionized water. The prometryn also presented detection in ultrapure water with concentrations of order 10-12 mol/L and for systems with deionized water and tap on the order of 10-9 mol/L. These values are below the limits of potable water allowed by regulatory agencies (1.39 x 10-8 mol/L atrazine and 9.62 x 10-8 mol/L for prometryn), these limits that represent an analytical challenge by itself. In addition, the SERS spectra were classified by computer techniques of information visualization, enabling better data analysis. In terms of adsorption mechanisms for solutions ultrapure water, atrazine adsorbs on AgNPs via C atom of the radical ethyl and Cl and prometryn have their triazínico ring positioned parallel to the surface of AgNPs adsorbing via C-S atoms. Such mechanisms could not be determined for deionized and tap water. In addition were carried impedance spectroscopy measurements at the detection of both herbicides in LbL PAH/AgNPs films. / FAPESP: 2014/00244-0

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第17583号 / 工博第3742号 / 新制||工||1570(附属図書館) / 30349 / 京都大学大学院工学研究科材料化学専攻 / (主査)教授 平尾 一之, 教授 田中 勝久, 教授 三浦 清貴 / 学位規則第4条第1項該当

The increased incidence of food pathogen outbreaks placed a new emphasis on the requirement of a rapid, sensitive, and reliable detection method for pathogens in food samples. Surface-enhanced Raman spectroscopy (SERS) is a technique that tremendously enhances the weak Raman scattering of an analyte by using a metallic nano-substrate. Herein, we developed an innovative SERS sandwich assay platform which is based on 3-mercaptophenylboronic acid (3-MPBA) or aptamer as a capturer, and 3-MPBA and silver nanoparticles (AgNPs) as the reporter for non-selective and selective detection of bacteria. Both optical and chemical (SERS mapping) imaging were used as mechanisms for bacterial detection and quantification. Using Salmonella enterica and Listeria monocytogenes as the model bacteria, we have identified a unique bacterial SERS signal upon the interaction between the captured bacteria, 3-MBPA and AgNPs, which was used as the base for reliable detection of bacteria using SERS mapping. The non-specific assay also possesses unique optical properties allowing for the enhanced visualization of bacteria at low microscope magnifications (10 and 20x objective lenses). Using 3-MBPA owe achieved sensitive detection and quantification of as low as 102 CFU/mL and a capture efficiency of 92.1% for nonselective detection of Salmonella. The capability of the assay method to detect specific bacteria using an aptamer was also demonstrated. Besides the SERS applications of this assay, it was discovered that the 3-MPBA coated gold chip developed for this assay enhances the visualization of bacteria under a light microscope allowing for facile and rapid detection and quantification. In anticipation for industrial applications, sample preparation methods and strategies were developed for simple and carbohydrate food matrices.

Electrolyte interactions with colloidal nanoparticles (NPs) in aqueous solutions have been implicated in a wide range of research and applications. Existing studies on electrolyte interactions with NPs are primarily based on the electrical double layer (EDL) theory. However, the EDL model provides very limited information on how electrolytes directly bind to NPs, electrolyte impact on charge distribution on NPs, and NP morphological modification upon electrolyte binding. Furthermore, the previous reports have mainly focused on either cations or anions binding onto NPs, while the potential cation and anion coadsorption onto NPs and NPacilitated cation-anion interactions remain largely uncharted. Filling these knowledge gaps are critical to enhance the fundamental understanding of interfacial interactions of electrolytes with NPs. Experimental characterization of cations and anions at the solid/liquid interface is a challenging analytical task. In the first study, we demonstrated the first direct experimental evidence of ion pairing on gold nanoparticles (AuNPs) in water by using surface enhanced Raman spectroscopy (SERS) in combination with electrolyte washing. Unlike ion pairing in aqueous solutions where the oppositely charged ions are either in direct contact or separated by a solvation shell, the ion pairing on AuNPs refers to cation and anion coadsorption onto the same NP surface regardless of separation distance. Ion pairing reduces the electrolyte threshold concentration in inducing AuNP aggregation and enhances the competitiveness of electrolyte over neutral molecules in binding to AuNPs. In the second study, we demonstrated that binding, structure, and properties of an ionic species on AuNPs are significantly dependent on the counterion adsorbed on AuNPs. These counterion effects include electrolyte-induced AuNP aggregation and fusion, quantitative cation and anion coadsorption on AuNPs, and SERS spectral distortion induced by the ionic species on AuNP surfaces. In the final study, we proposed that ion pairing as the main mechanism for reducing electrostatic repulsion among organothiolates self-assembled on AuNPs in water by using a series of experimental and computational studies. The work described in this dissertation provides a series of new insights into electrolyte interfacial interactions with AuNPs.

This dissertation focuses on four research topics: self-assembly of colloidal nanoparticles, surface modifications of the properties of ionically self-assembled multilayer films, surface enhanced Raman spectroscopy of functionalized gold nanoparticles, and two photon uncaging in gel. Those techniques are used for development of novel nanofabrication methods for top-down and bottom-up assembly of nanostructures, by modifying the properties of nanopatterned surfaces with photoactive ligands, and other technologies. First I describe the development of an improved method for nanosphere lithography, a variation of the convective self-assembly technique. The method exhibited high reproducibility and yielded high quality monolayer crystals by withdrawing a meniscus of liquid polystyrene spheres solution and subsequent evaporation of the solvent. The monolayer crystal was used as an evaporation mask to create surface arrays of gold nanotriangular particles. Metal nanoparticles, with sharp features or narrow gaps, exhibit strong plasmonic properties. I took advantage of those properties to attempt to create patchy modifications of the surface functionalization of gold nanotriangular particles treated with photosensitive molecules. Two molecules denoted, P3-DTC, and LIP3, were used as functional molecules attached to the gold nanoparticles. After interaction with 356nm UV light, part of those molecules cleaves off the surface of the nanoparticles rendering the surface modified with a new functional group. The modification takes place only at the plasmonic hot spots of those nanoparticles, resulting in a patchy modification of the properties of the nanoparticles. I built polymer Ionically Self-assembled Multilayer (ISAM) films using a Layer-by-Layer deposition technique and treated them to alter their surface adhesion properties. Poly (allylamine hydrochloride) (PAH), and poly (styrene sulfonate) (PSS) are a very well-studied system of polyelectrolytes for LbL deposition. ISAM films built from those polyelectrolytes are rich in amine groups to which nanoparticles, cells, tissue cultures, ligands can be made to adhere. In my work I developed a method for selective modification of the surface adhesiveness, by neutralizing the amine groups trough acetylation with acetic anhydride. With resolution from a few microns to a few hundred nanometers, I selectively passivated some areas of the ISAM film while others I left unaltered. I tested the effect of the acetic anhydride passivation by performing Horse Radish Peroxidase (HRP) test which quantifies the amount of free amines on the surface of the film. I also demonstrated the patchy modification of surface adhesiveness by introducing gold nanospheres which attached only to the amine active areas of the modified ISAM film. Photoactivatable fluorophores, i.e. compounds and other entities that may transform into a fluorescent form on absorption of a photon can be employed in multidimetional volume patterning. I studied the photoactivation of aryl azides in gelatin matrix. Specifically, I used Azidocoumarin 151 as a test molecule to undergo two-photon activation, and then measured the resulting photoluminescence. The activation of the Azidocoumarin 151 can be used to create arbitrary 3D patterns of modified functionality inside the gel. The activated molecules can be used as sites for further modification of the patterning inside the volume of the gel. Possible modifications include attaching biomolecules, nanoparticles, or individual cells. / Ph. D.

AABBSSTTRRAACCTT Dimers and small aggregates as well as compact aggregates of Ag nanoparticles (NPs) were assembled and chemically anchored to supporting surfaces. The supporting surfaces were either glass slides or SiO2 - coated Cu or Au grids for TEM, both chemically functionalized by 3-aminopropyltrimethoxysilane (APTMS). Compact aggregates of Ag NPs incorporating protoporphyrin IX (PPIX) molecules were prepared by adsorption of chlorides in the presence of PPIX. Dimers and small aggregates of Ag NPs were assembled by selected molecular linkers: 4,4'-diaminoazobenzene (DAAB), 4,4'-diaminoterphenyl (DATP) and 5,10,15,20-tetrakis(4-aminophenyl)porphine (TAPP). The most efficient strategy of dimers and small aggregates preparation has been their assembling by a three - step procedure involving (i) attachment of isolated Ag NPs to the NH2 groups of APTMS functionalized TEM grid, (ii) attachment of molecular linker (with two functional NH2 groups in para position) to Ag NPs by a one terminal NH2 group, and (iii) attachment of Ag NPs to the second, free terminal NH2 group of the linker. In this procedure, the control over the perpendicular orientation of the bifunctional linker and its attachment by one terminal group to Ag NP surface has been accomplished by functionalization of Ag NPs by adsorbed citrate...

Cette thèse s’intéresse à l’exaltation du signal Raman sur des nanomatériaux cuivrés. Des couches minces d’épaisseur de cuivre variable ont été préparées et étudiées avant et après oxydation dans l’air à des températures inférieures à 200°C. Leur microstructure a été caractérisée par microscopies MEB et AFM. L’épaisseur des couches de cuivre et d’oxyde cuivreux a été mesurée localement par ces techniques, et comparée aux résultats d’études spectroscopiques par ellipsométrie et absorption UV-visible. Une modélisation des spectres d’absorption UV-visible, basée sur des calculs d’interférences à partir des équations de Fresnel, permet de déterminer à la fois les épaisseurs des couches et leurs indices de réfraction. L’étude Raman de ces échantillons permet de discuter et de quantifier le phénomène d’exaltation Raman par interférences (IERS). D’autres échantillons nanostructurés à base de cuivre, recouverts de graphène monofeuillet, ont été étudiés. Les variations d’intensité Raman du graphène sont discutées en termes d’IERS. La dernière partie du manuscrit est consacrée à l’étude du signal SERS de molécules déposées sur des substrats commerciaux nanostructurés d’or, et à leur évolution après avoir recouvert ces substrats d’une couche mince de cuivre. / This thesis studies the enhanced Raman signatures on copper based materials. Thin copper films were prepared and studied before and after thermal oxidation in air, under 200 °C. Their microstructure has been characterized by SEM and AFM. The thickness of the copper and cuprous oxide films have been characterized locally by those techniques, and by ellipsometry and UV-visible absorption spectroscopic techniques. A modeling of the UV-visible spectra has been performed based on interference calculations using Fresnel equations, allowing the determination of both the thicknesses and the refractive indices of the films. Raman study of these samples allows a quantification of the interference enhanced Raman phenomenon (IERS). Other copper nanostructured samples covered with single layer graphene (SLG) have been studied, and The Raman intensity of SLG discussed in terms of IERS. The last part of the manuscript is dedicated to SERS studies of molecules deposited on nanostructured golden commercial substrates and to the evolution of the Raman the signal after covering these substrates with a thin copper layer.