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Title: Optical Responses of Biomolecules on Regular Metal Plasmonic Nanostructures Author: Martin Šubr Department: Institute of Physics of Charles University Supervisor of the doctoral thesis: prof. RNDr. Marek Procházka, Dr., Institute of Physics of Charles University Abstract: Adsorption of molecules on metal plasmonic nanostructures leads to significant enhancement of many optical processes, such as Raman scattering (surface-enhanced Raman scattering - SERS) or fluorescence (surface-enhanced fluorescence - SEF). Two groups of substrates were tested within this thesis: (i) Silver nanorods prepared by oblique angle vapor deposition, and (ii) silver and gold nanoislands growing on magnetron-sputtered polytetrafluoroethylene film. Step-by- step optimization process was performed on the nanoislands in order to obtain optimum SERS sensitivity and reproducibility. Detailed SERS intensity profiles were obtained using gradient nanostructures with the localized surface plasmon resonance (LSPR) condition varying across the sample and three different excitation wavelengths. It was also found that spectral position and height of the LSPR band can be controlled simultaneously using mixed gold/silver nanoislands. Detailed investigation of polarization- and angular- dependences of anisotropic silver nanorods was...

Nesta tese de doutorado foram desenvolvidas metodologias para obter espectros Raman intensificados pela superficie de adsorbatos orgânicos em eletrodos de Fe, Co e Ni, que são metais que apresentam baixo fator de intensificação SERS. Foram desenvolvidos procedimentos de ativação eletroquímica para eletrodos puros dos três metais. Os protocolos de ativação eletroquímica foram utilizados para obter espectros SERS de piridina, 1,10-fenantrolina e 2- e 4-aminopiridina. O fator de intensificação determinado para o três metais, da ordem de 102-103 vezes, depende fortemente do modo vibracional do adsorbato. Os espectros SERS da piridina nos metais de transição, quando comparados com os cálculos de espectros vibracionais de cluster da piridina com átomos metálicos por DFT mostram que a formação do radical α-piridil com a adsorção nos metais de transição, proposta na literatura, não ocorre. Os perfis de excitação SERS calculados pelo modelo de transferência de carga da piridina apresentaram boa correlação com os dados experimentais. Os resultados SERS para a 1,10-fenantrolina mostraram que a espécie que adsorve é a molécula livre, com a espécie adsorvida semelhante ao complexo sintetizado. A dependência das intensidades relativas com o potencial nos espectros SERS é diferente da observada para os espectros Raman ressonante dos complexos da phen com metais de transição, mostrando que o estado excitado monitorado pelas duas técnicas é diferente. O monitoramento da adsorção das 2-aminopiridinas nos metais de transição permitiu sugerir a adsorção pelo anel piridínico para potenciais menos negativos e através dos nitrogênios piridínico e amínico para potenciais mais negativos em eletrodos de Co e Ni, e para o Fe adsorve apenas pelo nitrogênio piridínico. Em solução eletrolítica 0,1 mol.L-1 KCl, a 4-aminopiridina passa de fracamente ligada para um complexo de superficie semelhante ao complexo sintetizado para potenciais mais negativos. Utilizando a solução eletrolítica 0,1 mol.L-1 KI, as duas espécies são observadas para um intervalo maior de potenciais. No eletrodo de Ni, observa-se 4-aminopiridina protonada para E = -0,7 V; para potenciais mais negativos um complexo de superficie semelhante ao complexo sintetizado é observado. Os filmes eletrodepositados de Co e Ni em eletrodos de Ag ativado eletroquimicamente permitiram a obtenção de espectros SERS da py com alto fator de intensificação. Os filmes com espessuras maiores que 2 monocamadas de Co ou Ni apresentaram bandas intensas da piridina adsorvida nestes metais, sem bandas da piridina adsorvida em Ag, indicando que os filmes não apresentam pinholes, com intensidade SERS 100 vezes maior do que os metais puros. As intensidades relativas dos espectros SERS são similares à obtidas nos espectros SERS da piridina nos metais puros para filmes finos mais espessos do que 7 monocamadas. Foram construídos substratos de Au SERS-ativos com alto desempenho e reprodutibilidade por eletrodeposição sobre uma máscara de microesferas de poliestireno. Os espectros SERS da 4-mercaptopiridina adsorvida nos substratos otimizados apresentaram intensidade 2 vezes maior do que o eletrodo de Au ativado eletroquimicamente. A reprodutibilidade do sinal SERS para estes substratos foi de ± 15 %, indicando que estes substratos podem ser utilizados como sensores para sistemas de interesse analítico. / Surface-enhanced Raman spectra of organic adsorbates on Fe, Co and Ni electrodes were acquired after the development of specific methodologies described in this PhD thesis. Electrochemical activation procedures were developed for the three bare metaIs electrodes. The electrochemical activation protocols were applied for the acquisition of SERS spectra of pyridine, 1,10-phenanthroline and 2- and 4-aminopyridine on Fe, Co e Ni electrodes. The total and relative intensities changes of SERS bands with the applied potentials were explained by the charge transfer mechanism, which had a large contribution in the SERS enhancement for these metaIs. The enhancement factor determined for the three metaIs, on the 102-103 times range, strongly depends on the adsorbate\' s vibrational modes. The SERS spectra of pyridine on the transition metals and vibrational spectra calculations of pyridine with metallic atoms showed that the formation of α-pyridil in the adsorption on transition metaIs, suggested in the literature, didn\'t occur. The calculated pyridine SERS excitation profiles present reasonable correlaton with the experimental data. The SERS results for 1,10-phenanthroline showed that the free molecule was the adsorbing species. The potential dependence of the SERS relative intensities was different from those of the resonance Raman spectra of 1,10-phenanthroline complexes with transition metal ions, indicating that different excited states were probed by the two techniques. 2-aminopyridine adsorbed through the pyridinic ring at less negative potentials and through both pyridinic and aminic nitrogens at more negative potentials on Co and Ni electrodes, but for Fe electrode it adsorbed exclusively through the pyridinic nitrogen. 4-aminopyridine adsorbed perpendicularly to the electrode. In 0,1 mol.L-1 KCl electrolytic solution, 4-aminopyridine changed from weak1y bound to a surface complex similar to the synthesized complex at more negative potentials. In 0,1 mol.L-1 KI electrolytic solution, both species were observed in a larger potential interval. On the Ni electrode, protonated 4aminopyridine was observed for V = -0.7 V, and for more negative potentials a surface complex, similar to the synthesized one, was observed. The electrodeposition of ultrathin film of Co and Ni on electrochemically-activated Ag electrodes allowed obtaining SERS spectra of pyridine with high enhancement factors. The SERS spectra of py for films thickness higher than 2 monolayers of Co or Ni presented intense bands of pyridine adsorbed on these metals, and no bands of pyridine adsorbed on Ag were observed, indicanting the absence of pinholes in the films. The relative intensities of SERS spectra on the thin films were similar to those obtained for the SERS of pyridine on the bare metaIs electrodes for films thicker than 7 monolayers, but with SERS intensity 100 times higher. The SERS activity and signal strength reproducibility of Au nanostructured substrates obtained by electrodeposition on a polystyrene masking were evaluated. The SERS spectra of 4-mercaptopyridine adsorbed on optimized electrodes presented intensities 2 times greater than those of the electrochemically activated Au electrode. The SERS intensity reproducibility for these substrates was ± 15%, indicating the potential use of such substrates as sensors.

The objectives of the work presented were to 1) fabricate reproducible nanorod array SERS substrates, 2) detection of bacteria using nanorod substrates, 3) detection of DNA hybridization using nanorod substrates and 4) critically evaluate the sensing method. Important findings from this work are as follows. A novel method for batch fabrication of substrates for surface enhanced Raman scattering (SERS) has been developed using a modified platen machined to fit in a commercial electron beam evaporator. The use of this holder enables simultaneous deposition of silver nanorod (AgNR) arrays onto six microscope slide substrates utilizing glancing angle deposition. In addition to multiple substrate fabrication, patterning of the AgNR substrates with 36 wells allows for physical isolation of low volume samples. The well-to-well, slide-to-slide, and batch-to-batch variability in both physical characteristics and SERS response of substrates prepared via this method was nominal. A critical issue in the continued development of AgNR substrates is their stability over time, and the potential impact on the SERS response. The thermal stability of the arrays was investigated and changes in surface morphology were evaluated using scanning electron microscopy and x-ray diffraction and correlated with changes in SERS enhancement. The findings suggest that the shelf-life of AgNR arrays is limited by migration of silver on the surface. Continued characterization of the AgNR arrays was carried out using fluorescent polystyrene microspheres of two different sizes. Theory suggests that enhancement between nanorods would be significantly greater than at the tops due to contributing electromagnetic fields from each nanostructure. In contrast to the theory, SERS response of microspheres confined to the tops of the AgNR array was significantly greater than that for beads located within the array. The location of the microspheres was established using optical fluorescence and scanning electron microscopy. The application of SERS to characterizing pathogens such as bacteria and viruses is an active area of investigation. AgNR array-based SERS substrates have enabled detection of pathogens present in biofluids. Specifically, several publications have focused on determining the spectral bands characteristic of bacteria from different species and cell lines. Studies were carried out on three strains of bacteria as well as the medium in which the bacteria were grown. The spectra of the bacteria and medium were surprisingly similar, so additional spectra were acquired for commonly used bacterial growth media. In many instances, these spectra were similar to published spectra purportedly characteristic of specific bacterial species. In addition to bacterial samples, nucleic acid hybridization assays were investigated. Oligonucleotide pairs specifically designed to detect respiratory syncytial virus (RSV) in nasal fluids were prepared and evaluated. SERS spectra acquired on oligos, alone or in combination, contain the known spectral signatures of the nucleosides that comprise the oligo. However, spectra acquired on an oligo with a 5'- or 3' thiol were distinctly different from that acquired on the identical oligo without a thiol pendant group suggesting some control over the orientation of the oligo on the nanorod surface. The signal enhancement in SERS depends markedly upon the location of the probe relative to the substrate surface. By systematic placement of nucleotide markers along the oligo chain, the point at which the nucleotide disappears from the spectrum was identified. The overall findings for AgNR SERS substrates suggest that the applicability of SERS for detecting nucleic acid hybridization is limited. The strong distance dependence coupled with the lack of substrate stability at temperatures required for annealing oligos during hybridization suggest that AgNRs are not the platform to use for hybridization assays.

Nanoscience dominates virtually every field of science and technology in the 21st century. Nanoparticles are of fundamental interest since they possess unique size- dependent properties (optical, electrical, mechanical, chemical, magnetic etc.), which are quite different from the bulk and the atomic state. Bimetallic nanoparticles are of particular interest since they combine the advantages of the individual monometallic counterparts. The present study focuses on bimetallic nanoparticles containing gold as one of the constituents. Au-Pd, Au-Pt and Au-Ag bimetallic/alloy nanoparticles have been prepared by four different synthetic methods, and characterised by a variety of techniques, with an emphasis on Au-Ag alloy systems in the solution phase as well as in the form of nanostructured films on solid substrates. Au- Ag alloy nanoparticles have been used to demonstrate two different applications. The first is the use of Au-Ag monolayer protected alloy clusters in demonstrating single electron charging events in the solution phase as well as in the dry state. Single electron transfer events involving nanosized particles are being probed extensively due to their potential applications in the field of electronics. The second is an analytical application, involving the use of trisodium citrate capped Au-Ag alloy hydrosols as substrates for surface enhanced Raman and resonance Raman scattering [SE(R)RS] studies. The sols have been used for single molecule detection purposes. Various organic molecules such as quinones, phthalocyanines and methyl violet have been self- assembled in a stepwise manner on the nanoparticulate as well as bulk Au, Ag and Au-Ag surfaces, and characterised extensively by spectroscopic, electrochemical and spectroelectrochemical techniques.

Optical assays to detect cancer-associated molecular biomarkers in biological substrates are commonly performed with antibody-targeted organic dye contrast agents but the potential for precise quantification, long-term imaging, and multiplexed readouts is limited by chemical and optical instability, non-optimal spectral characteristics, and complicated synthetic chemistry of the dyes. This dissertation tested the hypothesis that a novel class of optical contrast agents termed polymer-protected Raman-encoded nanoparticle tags (PRENTs) provides practical advantages over existing optical technologies for molecular diagnostic applications. First, PRENTs were developed through a modular design utilizing gold-nanoparticle-Raman reporter complexes protected and functionalized by polyethylene glycol derivatives. PRENTs produced optical readouts through surface enhanced Raman scattering (SERS) that were brighter and more photostable than the fluorescence of semiconductor quantum dots under identical experimental conditions. Unique spectral signatures were produced with a broader class of Raman reporters than is possible with silica coated Raman tags. Spectral signatures and colloidal stability of PRENTs were unaffected by harsh chemical conditions that cause spectral changes and aggregation of dyes, quantum dots, and protein coated Raman tags. Antibody-targeted PRENTs specifically tagged cell surface cancer biomarkers on living cells at reasonable integration times. PRENTs were non-toxic to cells under conditions exceeding those required for sensitive molecular detection. Next, PRENTs were efficiently optimized for excitation with near-infrared light through inclusion of near-infrared chromophores as Raman reporters and exploitation of the size-dependent optical enhancement of gold nanoparticles. Third, the development of a slide-based Raman-linked immunosorbent assay using antibody-conjugated PRENTs enabled quantification of protein biomarkers with a dynamic range of 3 to 4 logs. In summary, this dissertation establishes PRENTs as novel optical tags with unique features useful for biomedical applications and provides insights for further assay development.

Η χρήση των πολυμερών στον τομέα της ιατρικής βιομηχανίας κερδίζει ολοένα και μεγαλύτερο έδαφος τα τελευταία χρόνια έχοντας ήδη κάνει ισχυρή την παρουσία τους σε ένα ευρύ πεδίο κλάδων της βιοϊατρικής όπως στη μηχανική ιστών, στην εμφύτευση ιατρικών συσκευών και τεχνητών οργάνων, στην προσθετική και την οφθαλμολογία, στην οδοντιατρική και την αποκατάσταση οστών, στη χημειοθεραπεία και σε ποικιλία άλλων ιατρικών εφαρμογών. Με τη χρήση πολυμερικών συστημάτων μεταφοράς δραστικών ουσιών καθίσταται ικανή η ελεγχόμενη αργή αποδέσμευση φαρμάκων στο σώμα καθώς και η στοχευμένη απελευθέρωσή τους σε σημεία όπου υπάρχουν φλεγμονές ή όγκοι. Τοιουτοτρόπως, χημειοθεραπείες με χρήση βιοπολυμερών ως διαμεσολαβητές, προβάλλουν ως δυνητικές υποψήφιοι στην αντιμετώπιση του καρκίνου του εγκεφάλου με ενθαρρυντικά αποτελέσματα. Συγκρινόμενη με την τυπική συστημική χημειοθεραπεία, η ενδοογκική απελευθέρωση φαρμάκου με τη χρήση βιοπολυμερών θεωρητικώς παρουσιάζει αρκετά πλεονεκτήματα: τα βιοπολυμερή μπορούν να μεταφέρουν το φάρμακο απευθείας στον όγκο-στόχο αυξάνοντας τη συγκέντρωση τοπικά και παράλληλα μειώνοντας τη συστημική τοξικότητα· μπορούν έτσι να χρησιμοποιούνται στη θεραπεία ανοσοκατασταλμένων ασθενών που δεν μπορούν να υποβληθούν σε συστημική χημειοθεραπεία. Από τη στιγμή που είναι απαραίτητη η ποσοτικοποίηση των φαρμάκων για τον χαρακτηρισμό των συστημάτων αποδέσμευσης και για μελέτες φαρμακοκινητικής, θα πρέπει να επιλέγεται η καταλληλότερη μέθοδος ποσοτικοποίησης παρέχοντας υψηλή ευαισθησία και ακρίβεια, εξασφαλίζοντας μεγάλη ανιχνευτική ικανότητα ακόμη και για πολύ χαμηλές συγκεντρώσεις. Στην παρούσα εργασία δύο αναλυτικές τεχνικές, η απορρόφηση υπεριώδους-ορατού και η επιφανειακή ενίσχυση της σκέδασης Raman (Surface Enhanced Raman Scattering, SERS), χρησιμοποιήθηκαν για την ποσοτική εκτίμηση του αντινεοπλασματικού φαρμάκου Mitoxantrone και του αντιμυκητιακού παράγοντα Ambisome (Αμφοτερισίνη Β) που αποδεσμεύτηκαν από βιοσυμβατές πολυμερικές μήτρες συμπολυμερούς αιθυλενίου-οξικού βινυλεστέρα, συμπολυμερούς γλυκολικού-γαλακτικού οξέος και πολυπροπυλενίου. Το SERS είναι ένα νέο, εναλλακτικό, ταχύ και μη καταστροφικό εργαλείο που μπορεί να βρεί εφαρμογή και στην ποσοτική εκτίμηση ουσιών πάρα πολύ χαμηλών συγκεντρώσεων. Χάρις στην ενίσχυση που παρέχεται στο σήμα Raman από τα νανο-εκτραχυμένα υποστρώματα ευγενών μετάλλων ή τα νανο-συσσωματώματα κολλοειδών διαλυμάτων ευγενών μετάλλων, έχει αναφερθεί ακόμη και συλλογή φάσματος SERS από ένα μόνο μόριο. Συνεπώς, η εφαρμογή του SERS σε μελέτες ουσιών εξαιρετικά χαμηλών συγκεντρώσεων φαίνεται να είναι πολύ ενδιαφέρουσα. Κατασκευάστηκαν πολυμερικά υμένια με εγκλωβισμένες τις δραστικές ουσίες και η μελέτη αποδέσμευσης πραγματοποιήθηκε σε νερό. 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. / The application of polymeric materials for medical purposes is growing very fast. Polymers have found applications in such diverse biomedical fields as tissue engineering, implantation of medical devices and artificial organs, prosthesis, ophthalmology, dentistry, bone repair, chemotherapy and many other medical fields. Polymer-based delivery systems enable controlled slow release of drugs into the body and also they make possible targeting of drugs into sites of inflammation or tumors. Thus, biopolymer-mediated chemotherapy has shown promising results in the treatment of brain tumors. When compared to conventional systemic chemotherapy, intratumoral biopolymer-mediated drug delivery has several theoretical advantages: Biopolymers can deliver drugs into the tumor bed, thus maximizing local concentration while minimizing systemic toxicity. They may therefore be employed in the treatment of immunodepressed patients etc. Since drugs need to be quantified for drug delivery system characterization, intracellular distribution studies, free or vehicular, and for pharmacokinetic assays, the most suitable quantification method must be chosen. It should have a high sensitivity, specificity and reproducibility and should be capable of measuring at very low concentration range, as well. In the present study, two analytical techniques are utilized to quantitatively evaluate the antineoplastic drug Mitoxantrone and the antifungal agent Ambisome (Amphotericin b) released from active agents-loaded biocompatible polymer matrices poly(propylene), poly(ethylene-co-vinyl acetate), poly(lactic-co-glycolic acid); the UV-Vis absorption and the Surface Enhance Raman Scattering (SERS). SERS is a new, versatile, fast and non destructive tool for the estimation of extremely small amounts of substances. Due to the enhancement provided to the Raman signal by the nano-rough noble-metal substrates or the nano-structured colloidal clusters of noble metals, even single molecule detection has been reported. Therefore, applying SERS to extremely low concentration measurements proves to be challenging. Drug loaded polymer specimens were prepared and the in vitro drug release was determined in water. Fast SERS quantitative measurements showed enhanced sensitivity compared to the UV-Vis absorption; SERS may enable low concentration quantitative assessment of controlled release of drugs from biopolymer-based delivery systems.

La diffusion Raman exaltée de surface (SERS) est utilisée pour la mise au point d’un biocapteur capable de détecter des pesticides dans l’eau, en se basant sur le suivi de l’activité enzymatique de l’Acétylcholinestérase (ACHE). Les nanoparticules d’or sont utilisées comme substrats SERS actifs. Le signal Raman exalté de l’analyte est optimisé en testant plusieurs types de nanoparticules.Le Raman SERS a permis la détection directe du Paraoxon (PO) et du carbaryl (CA) et la possibilité de suivi de l’activité de l’ACHE. En absence d'inhibiteurs, la molécule d’acétylcholine (ATC) est transformée en acide acétique et en choline par l’enzyme ACHE. La mesure de l’activité de l’ACHE repose sur le suivi des concentrations en ATC car sa transformation est inhibée en présence de pesticides. Il a été ainsi possible d’établir une relation linéaire entre la concentration de pesticides et l’exaltation du signal Raman de l’ATC non transformé. La méthode a permis la détection du PO et du CA, avec une limite de détection beaucoup plus faible que la détection directe. Ce biocapteur basé sur l’activité de l’ACHE a ensuite été utilisé pour l'évaluation d’autres polluants (inhibiteurs d’ACHE) comme les additifs contenus dans les plastiques notamment. Enfin, nous avons développé une seconde approche qui consistait à mesurer l’activité de l’ACHE en utilisant la diffusion dynamique de la lumière. En effet, nous avons montré que les paramètres physicochimiques (agrégation) des AuNPs en contact avec certaines molécules, sont fortement influencés par l’activité enzymatique de l’ACHE. C’est ce phénomène d’instabilité qui nous a permis de distinguer entre les deux cas : absence et présence de PO. / Surface-enhanced Raman scattering (SERS) was used to develop a biosensor for the detection of pesticides through the monitoring of the enzymatic activity of acetylcholinesterase (ACHE). Gold nanoparticles (AuNPs) were used as an active SERS substrate. The enhanced Raman signal of the analyte is optimized by testing several types of nanoparticles. Raman SERS allowed the direct detection of Paraoxon (PO) and carbaryl (CA) pesticides and the possibility of follow-up of the activity of the ACHE. In the absence of inhibitors, the acetylcholine (ATC) is transformed into acetic acid and choline by the enzyme ACHE. The measurement of ACHE activity is performed through the monitoring of ATC concentrations because its transformation is inhibited in the presence of pesticides. Results showed a linear correlation between the concentration of pesticides and the SERS signal of the untransformed ATC. The method was optimized for the quantification of paraoxon and carbaryl with a limit of quantification much lower than the one obtained with a direct detection. Their identification was also possible using chemometrics. This biosensors, based on the ACHE activities, was applied to the evaluation of emergent pollutants: additives of commercial polymers. Our results suggested that most of the tested polymers contained molecules that act as inhibitors of the ACHE. Finally, we propose another very simple approach to measure the ACHE activity using dynamic light scattering measurements. We found that the physicochemical parameters (aggregation) of AuNPs were strongly influenced by the enzymatic activity of ACHE when in contact with specified molecules, allowing to detect the presence of PO.

Nesta tese de doutorado foram desenvolvidas metodologias para obter espectros Raman intensificados pela superficie de adsorbatos orgânicos em eletrodos de Fe, Co e Ni, que são metais que apresentam baixo fator de intensificação SERS. Foram desenvolvidos procedimentos de ativação eletroquímica para eletrodos puros dos três metais. Os protocolos de ativação eletroquímica foram utilizados para obter espectros SERS de piridina, 1,10-fenantrolina e 2- e 4-aminopiridina. O fator de intensificação determinado para o três metais, da ordem de 102-103 vezes, depende fortemente do modo vibracional do adsorbato. Os espectros SERS da piridina nos metais de transição, quando comparados com os cálculos de espectros vibracionais de cluster da piridina com átomos metálicos por DFT mostram que a formação do radical α-piridil com a adsorção nos metais de transição, proposta na literatura, não ocorre. Os perfis de excitação SERS calculados pelo modelo de transferência de carga da piridina apresentaram boa correlação com os dados experimentais. Os resultados SERS para a 1,10-fenantrolina mostraram que a espécie que adsorve é a molécula livre, com a espécie adsorvida semelhante ao complexo sintetizado. A dependência das intensidades relativas com o potencial nos espectros SERS é diferente da observada para os espectros Raman ressonante dos complexos da phen com metais de transição, mostrando que o estado excitado monitorado pelas duas técnicas é diferente. O monitoramento da adsorção das 2-aminopiridinas nos metais de transição permitiu sugerir a adsorção pelo anel piridínico para potenciais menos negativos e através dos nitrogênios piridínico e amínico para potenciais mais negativos em eletrodos de Co e Ni, e para o Fe adsorve apenas pelo nitrogênio piridínico. Em solução eletrolítica 0,1 mol.L-1 KCl, a 4-aminopiridina passa de fracamente ligada para um complexo de superficie semelhante ao complexo sintetizado para potenciais mais negativos. Utilizando a solução eletrolítica 0,1 mol.L-1 KI, as duas espécies são observadas para um intervalo maior de potenciais. No eletrodo de Ni, observa-se 4-aminopiridina protonada para E = -0,7 V; para potenciais mais negativos um complexo de superficie semelhante ao complexo sintetizado é observado. Os filmes eletrodepositados de Co e Ni em eletrodos de Ag ativado eletroquimicamente permitiram a obtenção de espectros SERS da py com alto fator de intensificação. Os filmes com espessuras maiores que 2 monocamadas de Co ou Ni apresentaram bandas intensas da piridina adsorvida nestes metais, sem bandas da piridina adsorvida em Ag, indicando que os filmes não apresentam pinholes, com intensidade SERS 100 vezes maior do que os metais puros. As intensidades relativas dos espectros SERS são similares à obtidas nos espectros SERS da piridina nos metais puros para filmes finos mais espessos do que 7 monocamadas. Foram construídos substratos de Au SERS-ativos com alto desempenho e reprodutibilidade por eletrodeposição sobre uma máscara de microesferas de poliestireno. Os espectros SERS da 4-mercaptopiridina adsorvida nos substratos otimizados apresentaram intensidade 2 vezes maior do que o eletrodo de Au ativado eletroquimicamente. A reprodutibilidade do sinal SERS para estes substratos foi de ± 15 %, indicando que estes substratos podem ser utilizados como sensores para sistemas de interesse analítico. / Surface-enhanced Raman spectra of organic adsorbates on Fe, Co and Ni electrodes were acquired after the development of specific methodologies described in this PhD thesis. Electrochemical activation procedures were developed for the three bare metaIs electrodes. The electrochemical activation protocols were applied for the acquisition of SERS spectra of pyridine, 1,10-phenanthroline and 2- and 4-aminopyridine on Fe, Co e Ni electrodes. The total and relative intensities changes of SERS bands with the applied potentials were explained by the charge transfer mechanism, which had a large contribution in the SERS enhancement for these metaIs. The enhancement factor determined for the three metaIs, on the 102-103 times range, strongly depends on the adsorbate\' s vibrational modes. The SERS spectra of pyridine on the transition metals and vibrational spectra calculations of pyridine with metallic atoms showed that the formation of α-pyridil in the adsorption on transition metaIs, suggested in the literature, didn\'t occur. The calculated pyridine SERS excitation profiles present reasonable correlaton with the experimental data. The SERS results for 1,10-phenanthroline showed that the free molecule was the adsorbing species. The potential dependence of the SERS relative intensities was different from those of the resonance Raman spectra of 1,10-phenanthroline complexes with transition metal ions, indicating that different excited states were probed by the two techniques. 2-aminopyridine adsorbed through the pyridinic ring at less negative potentials and through both pyridinic and aminic nitrogens at more negative potentials on Co and Ni electrodes, but for Fe electrode it adsorbed exclusively through the pyridinic nitrogen. 4-aminopyridine adsorbed perpendicularly to the electrode. In 0,1 mol.L-1 KCl electrolytic solution, 4-aminopyridine changed from weak1y bound to a surface complex similar to the synthesized complex at more negative potentials. In 0,1 mol.L-1 KI electrolytic solution, both species were observed in a larger potential interval. On the Ni electrode, protonated 4aminopyridine was observed for V = -0.7 V, and for more negative potentials a surface complex, similar to the synthesized one, was observed. The electrodeposition of ultrathin film of Co and Ni on electrochemically-activated Ag electrodes allowed obtaining SERS spectra of pyridine with high enhancement factors. The SERS spectra of py for films thickness higher than 2 monolayers of Co or Ni presented intense bands of pyridine adsorbed on these metals, and no bands of pyridine adsorbed on Ag were observed, indicanting the absence of pinholes in the films. The relative intensities of SERS spectra on the thin films were similar to those obtained for the SERS of pyridine on the bare metaIs electrodes for films thicker than 7 monolayers, but with SERS intensity 100 times higher. The SERS activity and signal strength reproducibility of Au nanostructured substrates obtained by electrodeposition on a polystyrene masking were evaluated. The SERS spectra of 4-mercaptopyridine adsorbed on optimized electrodes presented intensities 2 times greater than those of the electrochemically activated Au electrode. The SERS intensity reproducibility for these substrates was ± 15%, indicating the potential use of such substrates as sensors.

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No. of bitstreams: 1 alinelucianofilgueiras.pdf: 4846834 bytes, checksum: f046903a257bca51b49b36d371931270 (MD5) Previous issue date: 2017-03-24 / Nesta tese investigou-se as interações químicas dos antimicrobianos tinidazol, metronidazol, aztreonam, rifampicina e tetraciclina (TC) adsorvidos sobre superfícies metálicas de nanopartículas de ouro (AuNP) ou prata (AgNP) através das técnicas espectroscópicas de espalhamento Raman intensificado por superfície (SERS), de espalhamento Raman ressonante intensificado por superfície (SERRS) e de absorção no ultravioleta, visível e infravermelho próximo (UV-VIS-NIR). Também foram investigadas, a citotoxidade das AgNP, quitosana (QUIT), TC e das combinações AgNP com quitosana (AgNP+QUIT), e AgNP com QUIT e TC (AgNP+QUIT+TC) frente a células de fibroblastos bovinos. Foram obtidas as imagens de microscopia eletrônica de transmissão (TEM) das bactérias K.pneumoniae e S. aureus na ausência e presença das AgNP. Diferentes rotas sintéticas de AuNP e AgNP foram propostas, com o objetivo de se aprimorar as possíveis aplicações dessas sínteses. As AgNP devem ser pequenas para aplicações em experimentos biológicos e devem estar em ressonância com as radiações excitantes de comprimento de onda em 532, 633 ou 1064 nm quando forem utilizadas nos experimentos SERS. Os espectros SERS dos adsorbatos estudados foram obtidos na ausência e na presença dos modificadores de superfície 2-mercaptoetanol, polivinil álcool (PVA) ou íons cloreto. A presença destes modificadores superficiais permitiu monitorar os mecanismos de adsorção, que levaram a padrões espectrais SERS distintos. A análise dos espectros SERS da rifampicina foi baseada nas modificações observadas na estrutura eletrônica do grupo cromóforo, enquanto que para o tinidazol, metronidazol e aztreonam as diferenças nos padrões espectrais foram devidas a proximidade do sítio de adsorção do analito em relação à superfície metálica. A atribuição vibracional dos espectros Raman e SERS foi baseada em cálculos teóricos obtidos da teoria do funcional de densidade (DFT) das moléculas isoladas ou interagindo com átomos de metal. Este trabalho foi realizado em parceria com o Prof. Dr. Diego Paschoal da Universidade Federal do Rio de Janeiro e com o Prof. Dr. Hélio Ferreira dos Santos da Universidade Federal de Juiz de Fora. As imagens TEM das bactérias K. pneumoniae e S.aureus obtidas na ausência e presença das AgNP+QUIT foram realizadas em colaboração com o Dr. Celso Sant’Ana e o aluno Mateus Eugênio do Instituto Nacional de Metrologia, Qualidade e Tecnologia. Os ensaios de citotoxicidade das diferentes combinações de AgNP frente as células de fibroblastos bovinos foram realizados através do ensaio colorimétrico com brometo de 3, (-4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium (MTT). Este trabalho foi realizado em colaboração com a Profa. Dra. Michele Munk da Universidade Federal de Juiz de Fora. / In this thesis were investigated the chemical interaction of the antimicrobians tinidazole, metronidazole, aztreonam, rifampicin and tetracycline adsorbed on metallic surfaces of gold (AuNP) or silver nanoparticles (AgNP) by using the spectroscopic techniques surface enhanced Raman scattering (SERS), surface enhanced resonance Raman scattering (SERRS) and absorption in ultraviolet, visible and near infrared regions (UV-VIS-NIR). The cytotoxic effects of silver nanoparticles, chitosan (QUIT), tetracycline (TC) and its combinations AgNP with chitosan (AgNP+QUIT), and AgNP with QUIT and TC (AgNP+QUIT+TC) against bovine fibroblast cells were also investigated. The images using transmission electron microscopy (TEM) were obtained from the bacteria K.pneumoniae and S. aureus in the absence and presence of AgNP +QUIT. Different AuNP and AgNP were synthesized, with the aim of improving the possible applications of these syntheses. These have to be small in the biological experiments and should be in resonance with laser lines at 532, 633 or 1064 nm when used in the SERS experiments. The SERS spectra of the studied adsorbates were obtained in the absence and presence of the surfaces modifiers 2-mercaptoethanol, chloride ions or polyvinyl alcohol (PVA). The presence of these surfaces modifiers allowed monitoring adsorption mechanisms, which led to distinct SERS spectral patterns. The analyses of the SERS spectra of rifampicin were based on the observed changes in the electronic structure of the chromophore group, while for the tinidazole, metronidazole and aztreonam the differences in the spectral patterns were due to the proximity of anchor site of the analyte in relation to the metallic surface. The vibrational assignments of the Raman and SERS spectra were based on theoretical calculations obtained from density functional theory (DFT) of the isolated molecules or in interaction with metallic atoms. The DFT studies were made in collaboration with the Prof.Dr.Diego Paschoal from Universidade Federal do Rio de Janeiro and Prof.Dr Hélio Ferreira dos Santos from Universidade Federal de Juiz de Fora. The TEM images of K. pneumoniae and S.aureus bacteria obtained in the absence and in the presence of AgNP+QUIT were made in collaboration with the researcher Celso Sant’Ana and its student Mateus Eugênio from Instituto Nacional de Metrologia, Qualidade e Tecnologia. The cytotoxicity assays of the different combination of AgNP and chitosan and tetracycline against bovine fibroblast cells were made though colorimetric assay using MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The biological assay were made in collaboration with the Prof.Dr. Michele Munk and its students Leonara Fayer and Rafaella Zanetti from Universidade Federal de Juiz de Fora.