[pt] Investigamos as propriedades ópticas de sistemas compostos por nanoilhas de ouro (Au) funcionalizadas com biotina, na superfície de monolitos de xerogéis de sílica (SiO2), visando desenvolvere uma plataforma para biossensores baseados na Ressonância de Plasmon de Superfície Localizada (LSPR), devido à sua elevada sensibilidade a alterações no ambiente químico próximo. Os xerogéis foram sintetizados por meio de um processo de sol-gel de catálise em duas etapas. Uma solução de água deionizada, etanol e tetraetil ortossilicato foi misturada sob agitação magnética a frio, com uma proporção molar de 8,5:3,5:1, e soluções de ácido clorídrico e hidróxido de amônio foram utilizadas como catalisadores para hidrólise e condensação, respectivamente, em moldes de polipropileno. Os géis foram envelhecidos nos moldes e convertidos em xerogéis por secagem a 600 graus C em um forno. Foi depositado um filme de Au de 20 nm na superfície dos xerogéis por sputtering. Em seguida, os xerogéis de sílica com filme de ouro (Au@SiO2) foram submetidos a um tratamento térmico em forno elétrico, para criar as nanoilhas de Au. Observou-se uma mudança de coloração, de azul para rosa, característica das AuNPs. Em seguida, os sistemas AuNP@SiO2 xerogéis foram funcionalizados em dois passos consecutivos com cisteamina (CA) e N-hidroxissuccinimidobiotina (NHSB) para a detecção de avidina em meio aquoso. O par biotina-avidina é um sistema amplamente conhecido para testar biossensores, devido à sua alta especificidade e sensibilidade muito baixa. O processo de funcionalização foi monitorado por absorbância óptica UV-vis para cada passo. Soluções aquosas com concentrações de avidina (10(-6) M, 10(-7) M, 10(-8) M, 10(-9) M e 10(-10M) foram utilizadas para testar a detecção e a sensibilidade. Observou-se um deslocamento médio de 52 nm na absorbância de todas as concentrações testadas, indicando que este sistema é promissor para aplicações em biossensores plasmônicos. / [en] In this study, the optical properties of systems composed of biotin-functionalized gold nanoislands on the surface of silica xerogel monoliths (SiO2) were investigated, aiming to develop an inorganic and inert solid platform for biosensors based on Localized Surface Plasmon Resonance (LSPR) due to their high sensitivity to changes in the nearby chemical environment. The silica xerogels were synthesized through a two-step catalytic sol-gel process, where a solution of deionized water, ethanol, and tetraethyl orthosilicate was mixed under cold magnetic stirring, with a molar ratio of 8.5:3.5:1, and solutions of hydrochloric acid and ammonium hydroxide were used as catalysts for hydrolysis and condensation, respectively, in polypropylene molds. Later, the gels were aged in molds and converted to xerogels by drying at 600 C degrees in an oven. To create the gold nanoislands, a thin film of 20 nm Au was deposited on one of the top surfaces of the xerogels by sputtering, and then the Au@SiO2 xerogels were subjected to heat treatment in an electric furnace. A color change of the samples from blue to pink was observed, characteristic of gold nanoparticles. Then, the AuNP@SiO2 xerogel systems were functionalized in two consecutive steps with cysteamine (CA) and N-hydroxysuccinimide-biotin (NHSB) for the detection of avidin in an aqueous solution. The biotin-avidin pair is a well-known system for testing biosensors, due to its high specificity and very low sensitivity. The functionalization process was monitored by UV-Vis optical absorbance for each step. Aqueous solutions with concentrations of avidin (10(-6) M, 10(-7) M, 10(-8) M, 10(-9) M and 10(-10) M) were used to test detection and sensitivity. An average shift of 52 nm was observed in the absorbance spectrum of all tested concentrations, indicating that this system is a promising structure for plasmonic biosensor applications.

Submitted by Etelvina Domingos (etelvina.domingos@ufpe.br) on 2015-03-12T17:33:14Z No. of bitstreams: 2 DISSERTAÇÃO Arnaldo César Dantas dos Santos Andrade.pdf: 7831380 bytes, checksum: 66d5438c268c796c6cdb3d1cd4e40f5a (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-12T17:33:14Z (GMT). No. of bitstreams: 2 DISSERTAÇÃO Arnaldo César Dantas dos Santos Andrade.pdf: 7831380 bytes, checksum: 66d5438c268c796c6cdb3d1cd4e40f5a (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2013 / Um dispositivo eletrônico para teste laboratorial remoto ou point-of-care testing (POCT) foi desenvolvido para detecção de glicose, sendo três as suas unidades estruturais: a fonte de luz, o sensor plasmônico e o transdutor de sinal. Após testes na primeira unidade estrutural do instrumento, optou-se por fonte de luz do tipo laser de ondas contínuas, trabalhando no comprimento de onda 780 nm. A segunda unidade estrutural é resultante de técnicas de engenharia molecular e síntese coloidal de um sensor plasmônico, estável, consumível em única dose. Funcionalizou-se a superfície dos nanobastões de ouro (NBAu), revestindo-os com polieletrólitos e em seguida conjugou-se com enzima glicose oxidase (GO) em camadas, pelo método layer-by-layer (LBL). As camadas foram caracterizadas por espectroscopia UVVis- NIR e obteve-se uma relação qualitativa entre estas e seus respectivos espectros de ressonância localizada de plasmon de superfície (LSPR). A LSPR possibilita uma ampla variedade de aplicações em dispositivos sensores baseados neste fenômeno. Os NBAu sintetizados neste trabalho apresentaram dois modos de absorção: (i) 550 nm o qual corresponde ao modo de oscilação transversal e (ii) 744 nm para o modo de oscilação longitudinal e sua morfologia foi obtida por microscopia eletrônica de transmissão (MET). Foi possível investigar a estabilidade de nanobastões funcionalizados com concentrações de poliestirenosulfonato de sódio (PSS). O sensor plasmônico NBAu-PSS-Poliacrilamida(PAM)- GO distinguiu absorções para soluções de concentrações distintas de glicose. Para a terceira unidade estrutural do instrumento foram selecionados transdutores de sinal e desenvolveu-se uma abordagem experimental que permitiu defini-los e programá-los a fim de reproduzir respostas correspondentes àquelas de analisadores convencionais. Um analisador de modulações LSPR foi programado no dispositivo eletrônico e ocorreu em conjunto com a síntese das nanoestruturas. A especificação do emissor de luz, a construção do sensor NBAu- PSS-PAM-GO e a definição do transdutor de sinal, permitiram elaborar uma instrumentação prática para a diagnóstico rápido. Este trabalho veio reforçar a importância da aplicação de nanoestruturas anisotrópicas para reconhecimento de macromoléculas. Uma estratégia semelhante foi contemplada neste mesmo dispositivo eletrônico, demonstrada em anticorpos conjugados aos nanobastões para reconhecimento da proteína troponina, como prova de conceito.

This thesis presents spectroscopic studies of metallic nanoparticle localized surface plasmons and plasmon enhanced fluorescence. We investigated the dielectric sensitivity of silver nanoprisms to an external electric field and gold nanorods to the formation of a self-assembled surface monolayer. Dark field microscopy was used to image plasmonic scattering from single nanoparticles, and a liquid crystal tunable filter was used to construct corresponding spectra. The plasmon resonances of silver nanoprisms displayed both reversible red shifts and irreversible blue shifts along with drastic intensity changes upon exposure to an applied bias. The plasmon resonances of gold nanorods showed sensitivity to the presence of alkanethiol molecules adhered to the particle surface by a moderate red shift. An increase in the effective external dielectric caused a shift toward longer wavelengths. We imaged plasmon enhanced fluorescence in order to optimize experimental parameters for a developing project that can characterize nanoparticle structure on sub-wavelength dimensions. Preliminary controls were performed to account for the effect of O₂ plasma treatment, solvent and alkanethiol monolayer formation on surface plasmon resonances. We found that O₂ plasma treatment for different time intervals did not result in a plasmon shift compared to untreated nanoparticles exposed to N₂; however when exposed to solvent the surface plasmons of the treated particles shifted five times as far toward the red. Interestingly, the solvent effect only resulted in a plasmon shift when the particles were N₂ dried after solvent incubation. Gold nanorods incubated in ethanol showed no wavelength maximum shift in pure solvent over time, but shifted moderately to the red after incubation in a solution of alkanethiol molecules. Conditions for the plasmon enhanced fluorescence study were optimized using a dye conjugate of the same alkanethiol molecule used previously by formation from solution in a monolayer on the gold nanorod surface. The appropriate synthesis for dye functionalization, molecular concentrations, solvents and optical settings were determined. / text

Materials in contact with the biological milieu (biomaterials) spontaneously and nonspecifically adsorb constituent proteins which may lead to unwanted cell adhesion and responses or hinder device performance. These interactions and their related phenomena lead to complications in ~3% of implant surgeries. Thus, resistance to these nonspecific interactions is critical to the performance of many implanted biomaterials and biosensing surfaces. Further, these interactions have widespread importance to industrial materials in contact with biological environments such as food packaging, and agricultural and nautical surfaces. Thin film coatings of antifouling polymers are one of the leading methods for reducing nonspecific interactions. Both polymer composition (chemical composition and molecular weight) and polymer grafting density are the principal determinants of coating performance. For applications requiring specific bioactivity, such as selective ligand-analyte interactions for sensors, the polymer coating must remain antifouling and be amenable to functionalization with capture ligands. Tethered polymer coatings can be made by surface initiated polymerization (“graft-from”) which results in higher density coatings, but complex fabrication limits commercialization and capacity of functionalization with capture ligands. Simpler “graft-to” procedures, where pre synthesized polymers are immobilized to a surface, are more amenable to translation but suffer from inferior antifouling properties due to lower density coatings. New fabrication methods are therefore required to improve both graft-to and graft-from coatings. Herein, the effects of polymer density on material performance are explored and leveraged to produce novel functional surfaces using two classes of polymers, namely amphiphilic and thermoresponsive poly(oligo(ethylene glycol)) methyl ether methacrylate, and zwitterionic, functionalizable poly(carboxybetaine methacrylamide) (pCB), as well as copolymers thereof. Specifically, polymer grafting techniques which exploit grafting density effects on surfaces were developed, leading to surfaces: 1) that are both high-loading and antifouling due to two different grafting densities within bimodal architectures, and (2) with enhanced anti-fouling properties despite being prepared via a “grafting-to” method using shrinkable or expandable substrates. Interestingly, shrinking substrates with antifouling polymers resulted in a novel LSPR biosensor with high translation potential. Chapter 2 describes the pH controlled, one-pot production of two-layer brushes composed of an antifouling dense layer and a high-loading lower density layer where capture ligand immobilization was improved by 6 times compared to a single high density layer. Towards improving fouling and bioactivity of graft-to surfaces, Chapter 3 describes the first demonstration of Graft-then-Shrink where a stretched polystyrene (PS) substrate coated in a thin gold layer modified with thiol-terminated pCB was thermo-shrunk to one sixth in footprint to increase polymer surface coating content for enhanced antifouling properties and the production of micro/nano gold wrinkles to generate a localized surface plasmon resonance (LSPR) active surface. The low-cost sensors can vi detect biomolecular interactions by tracking changes in absorbance in the visible spectrum using ubiquitous plate readers. In Chapter 4, Graft-then-Shrink was extended to elastomeric materials, where thiol terminated polymers were grafted onto solvent swollen silicone via thiol-maleimide click chemistry, producing strongly antifouling materials. Taken together, these developments represent significant advances in the preparation and application of antifouling polymer coatings towards the improvement of antifouling surface properties of medical devices and resulted in the development of a novel, low-cost LSPR sensor without the need for specialized equipment. / Thesis / Doctor of Philosophy (PhD) / When a material, such as a medical implant or sensor, is placed in contact with tissues and biological fluids, biomolecules stick to the exposed surfaces through nonspecific interactions. It is important to minimize nonspecific interactions because they can lead to bacterial infections, inflammation, implant failure and loss of device performance. Coatings to minimize nonspecific interactions therefore remain an active area of research. In this thesis, we explored new methods to create biomolecule and cell repellent coatings of long, chainlike molecules known as polymers grafted onto surfaces. Specific types of polymers, known as antifouling, were particularly effective at reducing these interactions. Although it is important to block nonspecific interactions, many devices require bioactive surfaces through selective interactions. For example, sensors for analysis of blood products require the selective binding of the target ligand with minimal binding of non-target agents. To this end, functionalizable antifouling polymers are often modified with a capture or binding agent corresponding to the target ligand. Polymer coatings which are both antifouling and functionalizable for specific interactions, are called “romantic” because of their selective love of a single interaction. To synthesize these romantic polymer coatings, two main methods have been reported: 1) “grafting-from” where the polymer is grown from the surface, producing a very dense coating, and 2) “grafting-to” where the polymer is synthesized in solution, and then immobilized onto the material surface, which produces coatings of lower density. For antifouling polymer coatings to be as effective as possible, polymers should be tethered densely on the material surface, but to maximize the loading of capture agents, polymer density must be lower to allow for grafting within the layer. Further, the grafting-from method is typically more synthetically challenging hindering commercialization. To improve the selective bioactivity of graft-to and graft-from coatings as well as antifouling properties of graft-to coatings, we present two methods to improve the specific bioactivity of anti-fouling polymer coatings and the first description of Graft-then-Shrink, a method to enhance the antifouling properties of graft-to coatings for medical implants and label-free in vitro sensors. For graft-from coatings, we produced a hierarchical romantic surface that consists of two polymer layers, the lower of which is dense and antifouling, and the upper of which is low-density and can accommodate high-levels of bioactive agents, resulting in a best of both worlds; the density of the layers is controlled by a novel pH controlled polymerization procedure. A method to improve the less labor intensive “grafting-to” strategy was then devised, called “Graft-then-Shrink” where the antifouling polymers are grafted onto a shrinkable material, and then the material is shrunk, leading to an increase in grafted polymer content over grafting-to alone. This method was successfully applied to a heat shrinkable material and an elastomeric silicone material, a common material for medical devices, for improved antifouling properties. Finally, a method for combining the Graft-then-Shrink technique iv with a novel localized surface plasmon resonance (LSPR) biosensor was found, that provides a simple route to access romantic surfaces on high-sensitivity, easy to fabricate LSPR biosensors. Together, these fabrication methods will simplify and expedite the translation of antifouling and romantic surfaces for medical devices and sensors.

La fonctionnalisation chimique de surfaces hétérogènes (fonctionnalisation orthogonale) est une méthode efficace pour diriger l’ancrage de diverses cibles (biomolécules ou nano-objets) sur des zones précises prédéfinies sur un substrat. Ceci est particulièrement intéressant dans le domaine des biocapteurs à plasmons localisés (LSPR) où la transduction ne peut se faire que sur des nano- structures métalliques. L’enjeu est alors d’assurer que les molécules à détecter se fixent spécifiquement sur ces nanostructures et ne s’adsorbent pas sur la surface diélectrique environnante. Dans ce but, nous avons développé dans cette thèse des fonctionnalisations orthogonales de surfaces micro et nanostructurées d’or sur silice à l’aide de divers thiols et silanes. Par rapport à l’état de l’art dans ce domaine, nous avons notamment proposé un protocole en une seule étape et démontré la bonne orthogonalité de ces fonctionnalisations par différentes méthodes de caractérisation chimique de surface (notamment PM-IRRAS, XPS et ToF-SIMS). De plus, ces fonctionnalisations sélectives ont permis l’ancrage spécifique de diverses nanoparticules de latex sur des micro et nanostructures d’or entourées de silice, démontré par MEB. Actuellement, cette méthodologie est en cours d’application dans deux composants photoniques différents où l’on attend d’une part des effets d’exaltation de fluorescence par couplage de nano-antennes et nanobilles marquées et d’autre part un gain en sensibilité d’un biocapteur LSPR pour la détection de différentes biomolécules. / Orthogonal surface chemical functionalization is an efficient method for the selective trapping of different targets (biomolecules or nano-objects) onto predefined regions of a patterned substrate. This is specially interesting in the field of localized surface plasmon resonance (LSPR) biosensors, where transduction only occurs on metallic nanostructures. The aim is thus to ensure that the target molecules can be selectively anchored onto these nanostructures and not adsorbed on the surroun- ding dielectric surface. Thus, we have developped during this PhD different orthogonal functio- nalizations of micro and nanopatterned gold on silica surfaces with thiols and silanes. In regards to the state of the art in this topic, we have proposed a single-step protocol and demonstrated the good orthogonality of such functionalizations by extensive surface chemical characterization including PM-IRRAS, XPS and ToF-SIMS analysis. Furthermore, these functionalizations have been used for the selective anchoring of different latex nanoparticles onto micro and nanopatterns of gold surrounded by silica, as shown by SEM. At the moment, this methodology is being applied in two different photonic devices where we expect on the one hand a coupling between fluorescent nano- beads and plasmonic nano-antennas and, on the other hand, the increase in sensitivity of an LSPR biosensor for detecting different biomolecules.

This research proposes a novel polymer-based optical waveguide made with Polydimethylsiloxane (PDMS) for optical detection applications. Alternative to other fiber-based sensor, the proposed optical sensor uses PDMS waveguide as the main sensing component. PDMS has excellent optical properties which is essential for bio-photonic detection, including highly optical transparency, good flexibility and high bio-compatibility. Uncured PDMS polymer is cast in a Teflon tubing to form the PDMS rod. Since the reflective index of PDMS is as high as 1.43, that the bare PDMS can be an optical waveguide while the reflective index of the surrounding media is smaller than 1.43. The cast PDMS waveguide is then connect with plastic optical fibers to form the proposed optical waveguide system. In order to improve the optical performance of the PDMS waveguide, a surface coating process is used to reduce the surface roughness of the PDMS waveguide. The measured insertion loss with and without performing the surface coating procedure is 1.14 and 1.71dB/cm, respectively. Once the PDMS waveguide is formed, Au nanoparticles (Au-Nps) were coated on the PDMS surface with the assistance of a positive charge polymer of PDDA to form an optical waveguide capable of localized SPR detection. In addition, an atmospheric plasma treating process is used to enhance the coating ratio and speed of Au-Nps. UV-VIS spectrum and the SEM observation of the Au-particle coated PDMS waveguide confirm that the plasma treatment process significantly improves the coating results of Au-Nps. Liquid samples with different refractive index were used to demonstrate the LSPR sensing ability of the fabricated optical waveguide. The label free DNA detection was demonstrated by the system. The thiolated single strand DNA was modify on the PDMS optical waveguide as a DNA probe and bound with target DNA by DNA hybridization. The detection limit is as low as 10 pM. This research provides a simple and fast fabrication method to fabricate waveguide-based LSPR sensors.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-04-27T14:25:47Z No. of bitstreams: 1 dhienifferferreiradecarvalho.pdf: 5689053 bytes, checksum: fec45b3ef7e9df5f96e4b20d31f2177e (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-13T13:02:17Z (GMT) No. of bitstreams: 1 dhienifferferreiradecarvalho.pdf: 5689053 bytes, checksum: fec45b3ef7e9df5f96e4b20d31f2177e (MD5) / Made available in DSpace on 2017-05-13T13:02:17Z (GMT). No. of bitstreams: 1 dhienifferferreiradecarvalho.pdf: 5689053 bytes, checksum: fec45b3ef7e9df5f96e4b20d31f2177e (MD5) Previous issue date: 2011-12-14 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Nesta dissertação foi utilizada a espectroscopia Raman intensificado por superfície (Surface Enhanced Raman Scattering – SERS) para monitorar a adsorção de moléculas de interesse biológico em superfície metálica de ouro ou cobre. Foram sintetizadas diferentes nanopartículas metálicas de ouro, variando-se a concentração, a ordem de adição dos reagentes e a temperatura em que as sínteses foram feitas. Através dos espectros de extinção dos coloides metálicos foi possível estimar a distribuição de tamanhos obtida em cada uma das sínteses, e as amostras foram também analisadas por medidas de microscopia eletrônica de varredura e espalhamento dinâmico de luz. As nanopartículas de cobre foram obtidas em meio aquoso, sofrendo oxidação da superfície com formação de óxido de cobre (II), cuja cinética foi acompanhada por espectros de extinção. Quando um adsorbato com caráter redutor foi adicionado à superfície metálica, houve redução do óxido de cobre, presente na superfície da nanopartícula, permitindo a obtenção do espectro SERS da forma oxidada da espécie adsorvida. A molécula que possibilitou o estudo deste mecanismo redox foi a p fenilenodiamina, sendo obtido o espectro SERS da espécie oxidada radical cátion. Serotonina, adrenalina, L-carnosina, melatonina, rifampicina e albumina sérica bovina foram os adsorbatos utilizados para a obtenção dos espectros SERS em coloide de ouro. Estas moléculas apresentam anéis aromáticos, que contribuem para uma elevada polarizabilidade molecular e por isso se espera elevado sinal SERS. Estas espécies também possuem átomos de nitrogênio e oxigênio, os quais estão disponíveis como sítios de coordenação para a interação com a superfície metálica. Para obtenção dos espectros SERS foram utilizadas diferentes radiações excitantes, na busca da ressonância com as transições do plasmon de superfície localizado das nanopartículas de ouro. Foi ainda realizado o cálculo teórico vibracional para cada um dos adsorbatos estudados, isolados ou coordenados a um ou mais átomos de ouro ou cobre, através dos quais foi realizada a atribuição dos espectros Raman e SERS dos sólidos e dos complexos de superfície formados, respectivamente. Através desta atribuição e das regras de seleção de superfície foi possível determinar a geometria de adsorção da biomolécula à superfície da nanopartícula metálica. / In this dissertation the Surface Enhanced Raman Scattering (SERS) spectroscopy was used to monitor molecules with biological interest adsorbed on gold or copper metallic surfaces. Different gold metallic nanoparticles were synthesized varing the concentration, the addition order of the reagents and the temperature in which the syntheses were made. The size distributions were estimated for each colloid synthesis by the extinction spectra of the suspensions, and the samples were also analyzed by scanning electronic microscopy and dynamic light scattering techniques. Copper nanoparticles were obtained in aqueous media, undergoing surface oxidation with the formation of Cu(II) oxide, whose kinetic was followed by extinction spectra. When a reducer adsorbate was added to the nanoparticle suspension, the copper oxide, present in the metallic surfaces, undergoing reduction, allowing the achievement of SERS spectrum of the adsorbate in the oxidized form. The molecule which allowed the study of this redox mechanism was p-phenylenediamine, and the SERS spectrum was obtained from the radical cation species. Serotonin, adrenaline, L-carnosine, melatonin, rifampicin and bovine serum albumin were the adsorbates selected to obtain the SERS spectra in gold colloids. These molecules exhibit aromatic rings, whose is expected high contribution for the molecular polarizability, and in consequence of this a great SERS signal. Such species also have nitrogen and oxygen atoms, which are available coordination sites for interaction with the metallic surface. Different exciting radiations were used for searching of the resonance with the localized surface plasmon transitions of the gold nanoparticles. It was made the vibrational theoretical calculations for each studied adsorbate, isolated and coordinated to one or more gold or copper atoms, that were used for the assignment of Raman and SERS spectra of the solid and the surface complex, respectively. Through such assignment and the surface selection rules it was possible to determinate the adsorption geometry of biomolecule on the metallic surface.

Indiana University-Purdue University Indianapolis (IUPUI) / Designing and developing a diagnostic technology that is capable of highly sensitive and specific, multiplexed, high-throughput, and quantitative biomarker assays for disease diagnosis and progression is of the upmost importance in modern medicine and patient care. Current diagnostic assays capable of multiplexed and high-throughput analysis include mass spectrometry, electrochemistry, polymerase chain reaction (PCR), and fluorescence-based techniques, however, these techniques suffer from a lack in sensitivity, false responses, or extensive sample processing that are detrimental to clinical diagnostics. To overcome these sensitivity challenges, the field of nanoplasmonics has become utilized when developing diagnostic assays. Plasmonic-based diagnostic tests utilize the unique optical, chemical, and physical property of nanoparticles to increase the sensitivity of the assay. In this dissertation, novel diagnostic platforms that utilize nanoparticles and their localized surface plasmon resonance (LSPR) property will be introduced. LSPR is an optical property in noble metallic nanoparticles that is referred to as the collective oscillation of free electrons upon light irradiation. It is highly dependent on the shape, size, and dielectric constant (refractive index) of the surrounding medium of the nanoparticle and LSPR sensing is based on a change in these properties. In this dissertation, the LSPR property is utilized to fabricate nanoplasmonic-based diagnostic platforms that are capable of multiplexed and high-throughput biomarker assays, with high sensitivity and specificity. The work presented in this dissertation is presented as six chapters, (1) Introduction. (2) Methods, (3) Fabrication of a LSPR-based multiplexed and high-throughput biosensor platform and its application in performing microRNA assays for the diagnosis of bladder cancer. In this chapter, the advancement of single-plex solid state LSPR-based biosensors into a multiplexed and high-throughput diagnostic biosensor platform is reported for the first time. The diagnostic biosensor platform is first fabricated utilizing different gold nanoparticles (spherical nanoparticles, nanorods, and triangular nanoprisms), and then with the gold triangular nanoprisms as the nanoparticle of choice, microRNA assays were performed. The developed biosensor platform is capable of assaying five different types of microRNAs simultaneously at an attomolar limit of detection. Additionally, five microRNA were assayed in 20-bladder cancer patient plasma samples. (4) Development/optimization of the biosensor platform presented in Chapter 3 for the detection of COVID-19 biomarkers. In this chapter, the biosensor platform utilized in Chapter 3 was designed to assay 10 different COVID-19 specific biomarkers from three classes (six viral nucleic acid gene sequences, two spike protein subunits, and two antibodies) with limit of detections in the attomolar range and with high specificity. The high-throughput capability of the biosensor platform was advanced, with the platform performing analysis of a single biomarker in 92 patient samples simultaneously. Additionally, the biomarker platform was utilized to assay all 10 biomarkers in a total of 80 COVID-19 patient samples. (5) Further optimization of the biosensor platform for the development of a highly specific antibody detection test for COVID-19. During the COVID-19 pandemic, knowledge was gained on the specificity of antibodies produced against COVID-19. In this chapter, that knowledge was applied towards the optimization of the biosensor platform presented in Chapter 4 in order to assay SARS-CoV-2 neutralizing antibody IgG. The optimization of the biosensor platform included the size of the gold triangular nanoprisms and the receptor molecule of choice. The biosensor platform assayed this highly specific COVID-19 IgG antibody with a limit of detection as low as 30.0 attomolar with high specificity and no cross reactivity. Additionally, as a proof of concept, the biosensor platform was utilized in a high-throughput format to assay SARS-CoV-2 IgG in a large cohort of 121 COVID-19 patient samples simultaneously. (6) Advancement of the biosensor platform from a 96-well plate to a 384-well plate and its application in assaying microRNA for early diagnosis of pancreatic cancer. In this chapter, the high-throughput capabilities of the biosensor platform presented in Chapters 3-5 was expanded by increasing the sensor amount in one platform from 92 to 359. The 384-well plate biosensor platform was designed, optimized, and utilized to perform microRNA assays for early-stage pancreatic cancer diagnosis. The optimization of the biosensor platform included the manipulation of LSPR-based parameters and the -ssDNA receptor molecule in order to obtain low limit of detections (high sensitivity). Additionally, the biosensor platform assayed two microRNA in a large cohort (n=110) of pancreatic cancer and chronic pancreatitis patient samples.

Silver-gold nanocages have attracted considerable research interest recently due to their excellent performance in the fields of biomedicine and photocatalysis. These applications oftentimes manipulate at elevated temperatures and therefore impose demands on the thermal stability of the cage structures. To better understand this subject, in this work, we systematically evaluated the thermal stability of two nanocages with different wall thicknesses of 3.8 nm and 13 nm, both in the solution-phase (diethylene glycol) and solid-phase (in-situ STEM). The results revealed that the nanocages with thicker walls exhibited better thermal stabilities in both phases. By monitoring and analyzing the morphology changes of the nanocages, we determined that the nanocages with thin and thick walls undergo deformation processes differently. Nevertheless, they both deformed into more thermodynamically stable structures eventually. The plasmonic properties of the nanocages were also examined.

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No. of bitstreams: 1 linuspaulingdefariapeixoto.pdf: 3833578 bytes, checksum: 7044b2daa711546991179b32265a99dc (MD5) Previous issue date: 2015-08-21 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O presente trabalho visou a síntese e caracterização de nanopartículas de ouro (AuNPs) e sua aplicação na construção de biossensores plasmônicos utilizando para a detecção as técnicas de LSPR (ressonância de plasmon de superfície localizado) e SERS/SEF (espalhamento Raman intensificado por superfície / fluorescência intensificada por superfície). As AuNPs foram sintetizadas na forma de nanobastões (AuNRs) e nanobipirâmides (AuNBs) utilizando o método de crescimento por partículas precursoras (seed) e utilizando surfactantes para direcionamento do crescimento das mesmas. Devido ao melhor rendimento obtido para AuNRs, avaliou-se apenas sua sensibilidade em suspensão ou imobilizadas em lâminas de vidro. Para a imobilização em vidro, o 3-mercaptopropiltrimetoxisilano foi utilizado como molécula ligante entre o vidro e as AuNPs e posteriormente os substratos modificados foram caracterizados por espectroscopia UV-VIS, microscopia eletrônica de varredura e voltametria cíclica. A superfície dos AuNRs foi modificada para a detecção de duas biomoléculas-prova: (1) estreptavidina, realizada pelo mecanismo de interação por bioafinidade com a biotina, utilizando cisteamina como ligante entre as AuNPs e a biotina; (2) anti-BSA, através do mecanismo de interação antígeno-anticorpo, ativando a superfície das AuNPs (previamente modificada com ácido mercaptoundecanóico) com N-(3-dimetilaminopropil)-N′-etilcarbodiimida e N-hidroxisuccinimida. A sensibilidade LSPR foi monitorada pelo deslocamento do máximo da banda do plasmon longitudinal dos AuNRs frente ao aumento do índice de refração local devido às modificações na superfície dos AuNRs, alcançando 297 nm RIU-1. A detecção por SERS foi realizada através do modo extrínseco, utilizando um corante (IR-820) como molécula marcadora sobre a superfície dos AuNRs. Os biossensores construídos tiveram desempenho satisfatório na detecção de moléculas provas, além de boa sensibilidade frente a modificações no índice de refração do meio; isso foi observado tanto para AuNPs em suspensão ou imobilizadas em lâminas de vidro. Os procedimentos reportados são simples, rápidos e eficientes para a aplicação em biossensores. Adicionalmente, a integração das AuNPs em suspensão (modificadas com moléculas marcadoras) com as AuNPs imobilizadas nas lâminas de vidro, se mostrou um método interessante para a detecção de biomoléculas utilizando SERS/SEF. / This work was focused on the synthesis and characterization of gold nanoparticles (AuNPs) for applying as LSPR (localized surface plasmon resonance) and SERS/SEF (surface enhanced Raman spectroscopy / surface enhanced fluorescence) based biosensors. AuNPs were synthesized in two different forms, nanorods (AuNRs) and nanobipyramids (AuNBs), controlling the growth of seeds by using surfactants. A better yield was obtained to AuNRs and therefore, only the sensitivity of these nanoparticles was evaluated either using the AuNRs in suspension or immobilized on glass slides. A monolayer of 3-mercaptopropylmetoxisilane was used as a linker between AuNPs and glass surface, and after the modified glass slides were characterized with UV-VIS spectroscopy, scanning electronic microscopy and cyclic voltammetry. AuNRs surface was modified aiming the detection of two probe molecules: (1) streptavidin allowed by the bioafinity for biotin, using cysteamine as monolayer between AuNPs and biotin; (2) anti-BSA allowed by an antigen-antibody interaction activating the surface (previously modified with mercaptoundecanoic acid) with N-(3-dimetilaminopropil)-N′-etilcarbodiimide and N-hidroxisuccinimide. The LSPR sensitivity was evaluated by monitoring shifts in the longitudinal plasmon mode of AuNRs with changes in local refractive index due to surface binding events, reaching 297 nm RIU-1. SERS sensitivity was carried out in an extrinsic mode using a dye (IR-820) as SERS label on AuNRs surface. The biosensors developed in this work (AuNPs in suspension and adsorbed on glass slides) are efficient as biosensor as they presented good sensitivity for change in refractive index and for surface binding with probe molecules. The main advantages of these biosensors is the simple methodology summed to the short time of analysis. In addition, coupling the labeled AuNPs in suspension with the AuNPs adsorbed on glass slides is an interesting methodology for SERS/SEF detection of biomolecules.