Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-11-05T13:41:09Z No. of bitstreams: 1 deboraguimaraesdeoliveira.pdf: 3971933 bytes, checksum: 7b5a0ee6a3a033ca0149c8adc512aea2 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-11-23T11:16:07Z (GMT) No. of bitstreams: 1 deboraguimaraesdeoliveira.pdf: 3971933 bytes, checksum: 7b5a0ee6a3a033ca0149c8adc512aea2 (MD5) / Made available in DSpace on 2018-11-23T11:16:07Z (GMT). No. of bitstreams: 1 deboraguimaraesdeoliveira.pdf: 3971933 bytes, checksum: 7b5a0ee6a3a033ca0149c8adc512aea2 (MD5) Previous issue date: 2018-08-31 / A proteção superficial de nanoesferas de ouro (AuNEs) pode fornecer uma maior estabilidade coloidal, importante quando se trabalha com sistemas biológicos, por apresentarem situações diversas que podem levar à agregação das AuNEs. O biopolímero quitosana (Quit) é um material atóxico, apresenta baixo custo, e vem sendo muito utilizado em sistemas biológicos para diagnóstico de doenças. Sistemas sensíveis, como os biossensores utilizados na detecção de moléculas adsorvidas em AuNEs, podem ser monitorados por mudança na banda de ressonância de plasmon de superfície localizado (LSPR) e pela utilização da técnica de espalhamento Raman intensificado por superfície (SERS). Quando as moléculas são adsorvidas nas superfícies das AuNEs é possível observar a indução de agregação da suspensão coloidal pela mudança na banda LSPR. Devido à sensibilidade analítica, as superfícies das AuNEs são protegidas para evitar agregação e precipitação coloidal, e a obtenção de resultados falso-positivos. Esta tese teve como objetivo realizar vários estudos utilizando as AuNEs protegidas superficialmente por Quit para oferecer uma maior estabilidade e controlar a formação de hot spots na presença do analito. Para tal, sempre foram realizados experimentos comparando as AuNEs com e sem proteção superficial por Quit. Os estudos iniciais observaram a resistência à agregação das AuNEs com relação ao tempo de exposição em temperatura ambiente. Seguindo o raciocínio as AuNEs também foram expostas à diferentes concentrações dos agentes agregantes etanol (EtOH) e KCl. Os analitos utilizados como molécula prova para o SERS e também para observar possíveis mudanças na banda LSPR, foram os corantes: cristal violeta (CV), rodamina 6G (R6G) e IR-820, que apresentam cargas superficiais positiva ou negativa; a fim de identificar alguma preferência na adsorção por carga superficial do analito. Para os estudos em espectroscopia SERS, foram realizadas medidas variando concentração de Quit na presença dos corantes juntos ou separados, além de observar a interferência na ordem de adição do corante no sinal SERS. A Quit se mostrou promissora para aumentar a estabilidade das AuNEs e, portanto, experimentos para tentar modular e controlar a formação de hot spots foram realizados. O efeito SERS foi utilizado como uma ferramenta analítica em que foi estudada a relação direta do sinal SERS obtido dos corantes, com a sua concentração em solução. A partir destes resultados, foi possível construir isotermas de adsorção dos corantes estudados, que foram ajustadas ao modelo de Langmuir. / The surface protection of gold nanospheres (AuNEs) can provide greater colloidal stability, important when work with biological system, because the present diverse situations that can lead to the aggregation of AuNEs. The chitosan biopolymer (Quit) is a non-toxic material, presents low cost, and has been widely in biological systems for the diagnosis of diseases. Sensitive systems, such as biosensors used in detection of adsorbed molecules in AuNEs, can be monitored by changing in Localized Surface Plasmon Resonance´s band (LSPR) and by use of Surface-Enhanced Raman Spectroscopy technique (SERS). When the molecules are adsorbed on the AuNEs surfaces is possible to observe the induction of aggregation of the colloidal suspension by the change in the LSPR band. Due to the analytical sensitivity, the surfaces of the AuNES are protected to avoid aggregation and colloidal precipitation, and to obtain false positive results. This thesis aimed to perform several studies using the AuNEs surface protected by Quit to offer greater stability and to control the formation of hot spots in the presence of the analyte. For this, experiments were always done to compare the AuNEs with and without surface protection by Quit. The initial studies observed the resistance to AuNEs aggregation in relation to exposure time at room temperature. Following the reasoning, the AuNEs were also exposed to different concentrations of the aggregating agents ethanol (EtOH) and KCl. The analytes that were used as a test molecule for the SERS and also to observe possible changes in the LSPR band were the dyes: crystal violet (CV), rhodamine 6G (R6G) and IR-820, which have positive or negative surface charges; in order to identify some preference in surface adsorption of the analyte. For the SERS spectroscopy studies, measurements were performed varying the concentration of Quit in the presence of the dyes together or separated, besides observing the interference in the order of dye addition in the SERS signal. The Quit proved promising to increase the stability of AuNEs and, therefore, modification experiments, aiming at modulate and control the formation of hot spots, were performed. The SERS effect was used as an analytical tool in which the direct relationship of the SERS signal obtained from the dyes with their concentration in solution was studied. From these results, it was possible to construct adsorption isotherms of the studied dyes, which were adjusted to the Langmuir model.

Le travail de cette thèse porte sur la réalisation de substrats SERS (Surface Enhanced Raman Scattering) pour la détection d'espèce chimiques et biologiques. Il s'agit de mettre en place des méthodologies utilisant comme surface de départ celle d'une couche poreuse de silicium et un effet de Plasmon de surface induit par la présence d'un métal noble en général. Les spécificités des couches poreuses qui sont d'une part une rugosité nanométrique et d'autre part une porosité et donc une surface interne modulable ont une incidence directe sur la sensibilité de détection de molécules cibles. Nous avons développé dans ce manuscrit de thèse différentes méthodes d'élaboration de substrats SERS à base de couches poreuses de silicium. Nous avons envisagé deux voies, la première se base sur l'utilisation des nanoparticules d'or en solution colloïdale. La deuxième voie, plus originale, utilise le dépôt d'or par pulvérisation cathodique sur une couche poreuse préparée sur la face rugueuse de silicium. Des résultats SERS intéressants, concernant la détection de molécules chimique (Rhodamine 6G) et biologique (Albumine Sérum Bovin), ont été décrits et expliqués permettant d'envisager diverses solutions afin de les optimiser. / The work of this thesis focuses on the realization of SERS substrates (Surface Enhanced Raman Scattering) for the detection of chemical and biological species. This is to implement the methodologies using as the starting surface of a porous silicon layer and a surface Plasmon effect induced by the presence of a noble metal in general. The specificities of the porous layers which are firstly a nanometer roughness and secondly a porosity and therefore an inner surface have a direct impact on the target molecule detection sensitivity. We have developed in this PhD thesis different substrates development methodologies SERS-based on porous silicon layers. We considered two ways, the first is based on the use of gold nanoparticles in colloidal solution. The second route, more original, uses the deposition of gold by sputtering on a porous layer prepared on the rough side of a silicon wafer. Interesting SERS results regarding the detection of chemical (Rhodamine 6G) and biological (Bovin Serum Albumin) molecules, have been described and explained for considering various solutions to optimize them.

Nous avons étudié les comportements dynamiques d'auto-assemblage des tensioactifs cationiques non-chiral en présence du contre-anion chiral.Lorsque le nucléotide anionique chiral est ajouté à des vésicules cationiques, la transition morphologique se produit et transforme in situ des vésicules sphériques en hélices micrométriques.D'autres types de Gemini tensioactifs cationiques forment des rubans nanométriques hélicoïdaux, en présence de tartrate contre-anions. La forme et l'hélicité de ces rubans peuvent être contrôlés in situ par la variation de l'excès énantiomérique.En outre, les nanohélices organiques peuvent être transcrite en nanohélices 3D de silice via une polycondensation sol-gel.Ces nanohélices de silice fonctionnalisées avec des groupes aminés peuvent interagir fortement avec des nanoparticules d'or (GNPs; 1 ~ 20 nm). Le réseau 3D de -nanohélices GNPs/silice sont potentiellement utilisables pour des applications de capteurs basée sur les SERS comme ceux chimiques et biologiques ultra-sensibles en phase liquide. / We have studied the dynamic self-assembly behaviors of non-chiral cationic surfactants in thepresence of chiral counter-anion.When the chiral anionic nucleotides are added to cationic vesicles, morphology transitionoccurs and spherical vesicles transform in situ to micrometric helices.Other types of cationic surfactant, gemini surfactants form nanometric helical ribbons in thepresence of tartrate counter-anion. The shape and helicity of these self-assembled structurescan be controlled in situ by the variation of enantiomeric excess.In both cases, they form gels in water by creating extended networks of nanometric tomicrometric chiral fibers.Additionally, the organic nanohelices can be transcribed to 3D silica nanostructures via solgelpolycondensation. These silica nanohelices functionalized with amino group can interactstrongly with gold nanoparticles (1 ~ 20 nm). The 3D network of GNPs/silica-nanohelices canpotentially be used for SERS-based sensing applications such as ultra-sensitive chemical andbiological sensors in liquid phase.

In the first part of the Thesis, a new type of active system for SERS and SERRS of hydrophobic molecules, namely a 3-dimensional (3D) nanosponge aggregate with incorporated hydrophobic molecules has been developed, and tested by fullerene C60 and hydrophobic free- base tetraphenylporfine (H2TPP). The SERS and SERRS (surface enhanced /resonance/ Raman scattering) limits of detection (LODs) of C60 at four excitation wavelengths spanning the visible spectral region were found to be by one order of magnitude lower than in the reference system, which mimics the previously reported ways of utilization of Ag nanosponges as substrates for SERS and SERRS. The superiority of the newly developed sample is attributed to the efficient localization of the hydrophobic molecules into hot spots in 2D fractal aggregates of Ag nanoparticles (NPs). Diprotonation of H2TPP during the procedure using HCl as the preaggregation agent has been eliminated by employment of NaCl. On the other hand, investigation of the mechanism of H2TPP protonation during the former preparation procedure opened a possibility to employ Ag nanosponge aggregate as nanoreactor. In the second part of the Thesis, 2D assemblies of AgNPs were found to be better substrates for SERS of single layer graphene (SLG) than the 3D ones. In particular, the 2D...

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.

Lysosomen spielen entscheidende Rolle bei der zellulären Homöostase. Die Überwachung von Lysosomen, die Lipide ansammeln, ist eine erhebliche Herausforderung. Diese Arbeit konzentriert sich auf die Entwicklung der oberflächenverstärkten Raman-Streuung (SERS) als Methode zur Überwachung intakter Lysosomen, insbesondere hinsichtlich des Einflusses von Arzneimitteln, die den Lipidstoffwechsel stören. Um das Potenzial von SERS zur Untersuchung von Lysosomen in lebenden Zellen zu bewerten, wurden die Wechselwirkungen zwischen trizyklischen Antidepressiva und saurer Sphingomyelinase untersucht. Zunächst wurden Modellsysteme untersucht. Die Wechselwirkungen zwischen den Antidepressiva und Goldnanopartikeln wurden durch SERS charakterisiert. Die Daten zeigten, dass Moleküle mit den Nanopartikeln interagieren. Als Modellsystem der lipidreichen Umgebung wurden Komposite aus Liposomen und Goldnanopartikeln von SERS und Cryo-EM untersucht. Die SERS-Spektren sind charakteristisch für die Lipidzusammensetzung der Vesikel. Die Wechselwirkungen zwischen den Antidepressiva und den Lysosomen wurden in der Fibroblastenzelllinie 3T3 durch SERS und komplementäre Methoden untersucht. In Übereinstimmung mit den SERS-Spektren von Modellsystemen zeigen die SERS-Spektren lebender Zellen Signaturen sowohl der Antidepressiva als auch der Lipide. Um die Unterschiede in den Lysosomen zwischen behandelten und nicht behandelten Zellen aufzudecken, wurde ein zufälliger Waldansatz verwendet. Darüber hinaus wurde SERS verwendet, um die Lipidverteilung in Leishmania-infizierten Makrophagen zu untersuchen, von denen bekannt ist, dass sie Lipide akkumulieren. Die Ergebnisse zeigen, dass SERS verwendet werden kann, um die Lipidzusammensetzung in lebenden Zellen verschiedener Zelltypen zu untersuchen. Als neue methodische Entwicklung zeigt die Random-Forest-Analyse von SERS-Daten, dass Ansätze des maschinellen Lernens für ein besseres Verständnis von Daten aus biologischen Systemen nützlich sein können. / Lysosomes play a crucial role in cellular homeostasis. Monitoring lysosomes that accumulate lipids represents a considerable challenge. This thesis focuses on the development of surface-enhanced Raman scattering (SERS) as a method to monitor intact lysosomes, in particular regarding the influence of drugs that interfere with lipid metabolism. To evaluate the potential of SERS for studying lysosomes in live cells, the interactions between tricyclic antidepressants and acid sphingomyelinase were studied. First, model systems were investigated. The interactions between the antidepressants and gold nanoparticles were characterized by SERS. The data showed that molecules interact with the nanoparticles. As a model system of the lipid-rich environment, composites of liposome and gold nanoparticles were studied by SERS and cryo-EM. The SERS spectra are characteristic of the vesicles’ lipid composition. The interactions between the antidepressants and the lysosomes were studied in the fibroblast cell line 3T3 by SERS and complementary methods. In agreement with the SERS spectra of model systems, the SERS spectra of live cells show signatures of both, the antidepressants and the lipids. To reveal the differences in the lysosomes between treated and non-treated cells, a random forest approach was used. Moreover, SERS was used to study the lipid distribution in Leishmania-infected macrophages known to accumulate lipids. The results show that SERS can be used to investigate lipid composition in live cells of different cell types. As a new methodological development, the random forest analysis of SERS data shows that machine learning approaches can be useful for a better understanding of data from biological systems.

We have investigated some of the many photonic and plasmonic properties of the diatom Coscinodiscus wailesii. We start by showing that when diatom frustules are converted to high-index magnesium silicide while maintaining their structure, they exhibit a broad (1μm - 2μm) photonic bandgap that varies in wavelength according to the position and angle of the incident light on the frustule. We then demonstrate the use of the micro and nanostructured silica diatom frustule as a low-cost, easily prepared substrate for surface-enhanced Raman spectroscopy by coating the frustule in 25 nm of silver and a monolayer of thiophenol. Some potential applications of diatoms to water quality measurements are suggested, and steps are taken to image a diatom frustule and chloroplasts simultaneously in vivo using rhodamine 19 dye and fluorescence microscopy. We propose future experiments that could ascertain whether there is any biological effect of the light filtering properties of the diatom frustule, and put forth some suggestions as to how to influence the morphology and photonic properties of the frustule via chemical contaminants in the diatom seawater growth medium.

Solid oxide fuel cells (SOFCs) represent a next generation energy source with high energy conversion efficiency, low pollutant emission, good flexibility with a wide variety of fuels, and excellent modularity suitable for distributed power generation. As an electrochemical energy conversion device, SOFC’s performance and reliability depend sensitively on the catalytic activity and stability of the electrode materials. To date, however, the development of electrode materials and microstructures is still based largely on trial-and-error methods because of inadequate understanding of the mechanisms of the electrode processes. Identifying key descriptors/properties of electrode materials or functional heterogeneous interfaces, especially under in situ conditions, may provide guidance to the design of electrode materials and microstructures. This thesis aims to gain insight into the electrochemical and catalytic processes occurring on the electrode surfaces using unique characterization tools with superior sensitivity, high spatial resolution, and excellent surface specificity applicable under in situ/operando conditions. Carbon deposition on nickel-based anodes is investigated with in situ Raman spectroscopy and SERS. Analysis shows a rapid nucleation of carbon deposition upon exposure to small amount of propane. Such nucleation process is sensitive to the presence of surface coating (e.g., GDC) and the concentration of steam. In particular, operando analysis of the Ni-YSZ boundary indicates special function of the interface for coking initiation and reformation. The coking-resistant catalysts (BaO, BZY, and BZCYYb) are systematically studied using in situ Raman spectroscopy, SERS, and EFM. In particular, time-resolved Raman analysis of the surface functional groups (-OH, -CO3, and adsorbed carbon) upon exposure to different gas atmospheres provides insight into the mechanisms related to carbon removal. The morphology and distribution of early stage carbon deposition are investigated with EFM, and the impact of BaO surface modification is evaluated. The surface species formed as a result of sulfur poisoning on nickel-based anode are examined with SERS. To identify the key factors responsible for sulfur tolerance, model cells with welldefined electrode-electrolyte interfaces are systematically studied. The Ni-BZCYYb interface exhibits superior sulfur tolerance. The oxygen reduction kinetics on LSCF, a typical cathode material of SOFC, is studied using model cells with patterned electrodes. The polarization behaviors of these micro- electrodes, as probed using a micro-probe impedance spectroscopy system, were correlated with the systematically varied geometries of the electrodes to identify the dominant paths for oxygen reduction under different electrode configurations. Effects of different catalyst modifications are also evaluated to gain insight into the mechanisms that enhance oxygen reduction activity. The causes of performance degradation of LSCF cathodes over long term operation are investigated using SERS. Spectral features are correlated with the formation of surface contamination upon the exposure to air containing Cr vapor, H2O, and CO2. Degradation in cathode performance occurs under normal operating conditions due to the poisoning effect of Cr from the interconnect between cells and the high operating temperature. The surface-modified LSCF cathode resists surface reactions with Cr vapor that impairs electrode performance, suggesting promising ways to mitigate performance degradation.

Η παρούσα διατριβή εξειδίκευσης αποτελεί το προοίμιο μιας μακρόπνοης εμπλοκής του εργαστηρίου υλοποίησής της στη μελέτη ενδεχόμενης μετανάστευσης ουσιών που χρησιμοποιούνται ως ενισχυτικά φραγής ή/και ως χημικοί αισθητήρες σε βιοπολυμερικές συσκευασίες τροφίμων και αποδέσμευσής τους σε προσομοιωτές τροφίμων. Στο πλαίσιο αυτό, η εργασία αυτή αποτελεί μια προσπάθεια ανάδειξης της μεθόδου επιφανειακής ενίσχυσης της σκέδασης Raman (Surafce Enhanced Raman Scattering) ως κατάλληλης για τον ποσοτικό προσδιορισμό μικρού μοριακού βάρους ενώσεων που ενδεχομένως αποδεσμεύονται σε υδατικά διαλύματα ή/και συγκεκριμένους προσομοιωτές τροφίμων. Η μελέτη εστιάστηκε στην ελεγχόμενη αποδέσμευση φαρμακευτικών ουσιών από μια βιοπολυμερική μήτρα κυρίως κατά το πρώιμο στάδιο της μελέτης και τις παραμέτρους που επηρεάζουν το φαινόμενο αυτό. Βασικός στόχος της μελέτης ήταν η κατά το δυνατό μείωση του ορίου ανίχνευσης με SERS της αποδεσμευόμενης ουσίας με την εμβάπτιση της βιοπολυμερικής μήτρας που την εμπεριέχει σε πρότυπα υδατικά διαλύματα. Πραγματοποιήθηκε μια διεξοδική μελέτη των υποστρωμάτων που χρησιμοποιούνται στο SERS και συγκεκριμένα του νανοκολλοειδούς αργύρου (Ag). Για τον σκοπό αυτό, πραγματοποιήθηκαν πειράματα τα οποία έδειξαν την εξάρτηση της έντασης SERS από τη συσσωμάτωση των υποστρωμάτων Ag συναρτήσει του χρόνου και του παράγοντα συσσωμάτωσης, NaCl. Η εφαρμογή του SERS σε μελέτες ουσιών εξαιρετικά χαμηλών συγκεντρώσεων αναδεικνύεται ως ένα πολύ ενδιαφέρον πεδίο έρευνας. Επίσης, ως πρότυπο πείραμα, παρουσιάζεται μεθοδολογία μελέτης με την τεχνική SERS της αποδέσμευσης του αντικαρκινικού φαρμάκου Μitoxantrone (ΜΤΧ) από εμπορικά ράμματα Maxon. Για το σκοπό αυτό, παρασκευάστηκαν πολυμερικά υμένια με εγκλωβισμένη τη δραστική ουσία και η μελέτη της αποδέσμευσης της σε νερό και PBS (phosphate buffered saline) πραγματοποιήθηκε με SERS και UV-Vis, αντίστοιχα. Η φασματοσκοπία UV-Vis χρησιμοποιήθηκε συμπληρωματικά. Στηριζόμενοι στη μεθοδολογία που αναπτύξαμε εξάγαμε ποσοτικά αποτελέσματα από τρία διαφορετικά εργαστηριακά δείγματα, τα οποία προήλθαν από ανάμιξη εμπορικών ραμμάτων Maxon με 1% κ.β. MTX: (α) στην άμορφη φάση έπειτα από ταχεία ψύξη του τήγματος, (β) στην ημικρυσταλλική φάση με σχετικά χαμηλό ποσοστό κρυσταλλικότητας, που λάβαμε έπειτα από ανόπτηση της άμορφης φάσης για περιορισμένο χρόνο στη θερμοκρασία κρυστάλλωσης και (γ) σε μια επίσης ημικρυσταλλική φάση με αρκετά μεγάλο ποσοστό κρυστάλλωσης (όσης και τα εμπορικά ράμματα). Τα αποτελέσματα δείχνουν πως υπάρχει συσχέτιση μεταξύ κρυσταλλικότητας και αποδέσμευσης του φαρμάκου, με τα μικρότερα ποσά αποδέσμευσης στην περίπτωση του άμορφου δείγματος. Αυτό που παρατηρήθηκε στα πρώιμα στάδια της αποδέσμευσης από τις μετρήσεις SERS φαίνεται να επαληθεύεται από αντίστοιχα αποτελέσματα σε μεταγενέστερα στάδια αποδέσμευσης που λάβαμε με εφαρμογή της συμβατικής τεχνικής απορρόφησης ορατού – υπεριώδους (UV-Vis). Ωστόσο, οι ποσοτικές μετρήσεις με τη χρήση του SERS σε πολύ μικρές συγκεντρώσεις έδειξαν μεγαλύτερη ανιχνευτική ευαισθησία σε σχέση με αυτές που πραγματοποιήθηκαν με την απορρόφηση UV-Vis. Συμπερασματικά, το SERS δείχνει ικανό στον ποσοτικό προσδιορισμό ενεργών ουσιών που αποδεσμεύονται από βιοσυμβατά πολυμερικά συστήματα μεταφοράς δραστικών ουσιών σε πολύ μικρές συγκεντρώσεις. / This thesis of specialization is the precursor of a long-term involvement of the laboratory of Applied Molecular Spectroscopy of FORTH/ICE-HT in the implementation of the study of the migration of substances used as barrier and/or as chemical sensors in biopolymer based food packaging and their release into food simulants. In this context, this work attempts to highlight the method of surface enhanced Raman scattering (SERS) as appropriate for quantifying low molecular weight compounds that may be released in aqueous solutions and/or specific food simulants. The study focused on the controlled release of pharmaceuticals from a biopolymeric matrix mainly during the early stage of the study and the parameters affecting this phenomenon. The main objective of the study was to reduce SERS detection limit of the released substance by emerging the substance-incorporated biopolymeric matrix in standard aqueous solutions. In this context, we developed methods to maximize SERS enhancement and consequently reduce the limit of detection of an active substance, Mitoxantrone (MTX). This was achieved by a thorough study of the substrates used in SERS, namely nanocolloidal silver (Ag) suspensions. For this purpose, we contacted experiments which show the dependence of the SERS intensity on the aggregation of Ag substrates as a function of both time and the aggregating agent, NaCl. Also, as a standard experiment, present SERS methodology was applied in the study of the release of the anticancer drug Mitoxantrone (MTX) from commercially available sutures, Maxon. For this purpose, polymeric films prepared with the encapsulated active substance were immersed either in water or/and in PBS (phosphate buffered saline) and the release of MTX was probed by both SERS and UV-Vis. Based on the developed methodology we obtained quantitative results from three different laboratory samples produced by mixing commercial Maxon sutures with 1 wt% MTX: (a) an almost completely amorphous mixture produced by quenching from the melt, (b) a semi-crystalline one possessing low crystallinity that was produced by annealing the amorphous sample at the temperature close to the crystallization one and (c) a semi-crystalline one possessing high crystallinity similar to the commercial product. The results indicate a correlation between crystallinity and drug release rate; the more amorphous the sample is the less quantity of the drug is released. SERS was able to probe the active agent at the early state of release; UV-Vis has supported these results at a later state of the release process. In conclusion, SERS may enable low concentration quantitative assessment of controlled release of drugs from biopolymer-based delivery systems.

This thesis is focused on understanding the interactions between molecules and surface-enhanced Raman scattering (SERS) substrates that are typically unresolved due to the diffraction limit of light. Towards this end, we have developed and tested several different sub-diffraction-limited imaging techniques in order to observe these interactions. First, we utilize an isotope-edited bianalyte approach combined with super-resolution imaging via Gaussian point-spread function fitting to elucidate the role of Raman reporter molecules on the location of the SERS emission centroids. By using low concentrations of two different analyte molecules, we find that the location of the SERS emission centroid depends on the number and positions of the molecules present on the SERS substrate. It is also known that SERS enhancement partially results from the molecule coupling its emission into the far-field through the plasmonic nanostructure. This results in a particle-dictated, dipole-like emission pattern, which cannot be accurately modeled as a Gaussian, so we tested the applicability of super-resolution imaging using a dipole-emission fitting model to this data. To test this model, we first fit gold nanorod (AuNR) luminescence images, as AuNR luminescence is primarily coupled out through the longitudinal dipole plasmon mode. This study showed that a three-dimensional dipole model is necessary to fit the AuNR emission, with the model providing accurate orientation and emission wavelength parameters for the nanostructure, as confirmed using correlated AFM and spectroscopy. The dipole fitting technique was next applied to single- and multiple-molecule SERS emission from silver nanoparticle dimers. We again found that a three-dimensional dipole PSF was necessary to accurately model the emission and orientation parameters of the dimer, but that at the single molecule level, the movement of the molecule causes increased uncertainty in the orientation parameters determined by the fit. Finally, we describe progress towards using a combined atomic force/optical microscope system in order to position a carbon nanotube analyte at known locations on the nanoparticle substrate. This would allow for the simultaneous mapping of nanoparticle topography and exact locations of plasmonic enhancement around the nanostructure, but consistently low signal-to-noise kept this technique from being viable. / text