No description available.

No description available.

The introduction of lux genes, able to express bioluminescence, into terrestrial bacteria enabled the optimisation of a bioluminescence-based bioassay that was environmentally relevant. Individual assay parameters such as growth phase, cell washing, lyophilisation, pH tolerance and temporal response to a range of metal and xenobiotic pollutants were evaluated. The effects of a range of pollutants upon the metabolic response of the lux-marked organisms were assessed using declines in bioluminescence. The lux -based bioassay proved more sensitive to the sub-lethal effects of metal pollutants than tests relying on culturability. Uncontaminated soils were spiked with metal and xenobiotic solutions both as single pollutants and in combination with other contaminants. Relative toxicity of metal and xenobiotic pollutants in soil systems were investigated using ecotoxicity assays based upon lux-marked constructs of Rhizobium leguminosarum biovar trifolii (an important associative nitrogen fixer) and the respiration of the microbial community. The lux-marked bioassay proved to be more sensitive than the community microbial assay to the presence of multiple contaminants at sub-lethal concentrations. The relative toxicities of metal and organic xenobiotic compounds were shown to be time dependent and better represented using chronic assaying of lux-marked microorganisms. Following a field trial involving the application of paper mill sludge to land and subsequent crop failure a rapid diagnosis of soil pollutants was required. A suite of ecotoxicity assays including lux-based bioassays, respirometry and enzyme activity were used to assess the toxicity of paper mill sludge to the soil microbial biomass. The selected lux-marked soil bacteria showed potential for use as rapid, field-based screening techniques to provide early warning of the potential hazards of waste application.

The silicon based sensor is able to detect part per trillion ionic silver in 0.0098% hydrofluoric acid based on the open circuit potential (OCP) measurement. The OCP jump of 100 ppt ionic silver solution is up to 120 mV. The complex agent can effectively suppress the ionic silver concentration and suppress the OCP signal. The ability of complex agent to suppress the OCP signal depends on the formation constant of the complex with silver. The complex adsorbed on the sensor surface induces a second OCP jump, the height of the second jump depends on the formation constant of the complex. The MINEQL chemical equilibrium modeling program is used to calculate the ionic silver concentration when complex agent presents, a discrepancy is found between the MINEQL simulation result and the OCP signal of the silicon based sensor. The toxicity of ionic silver to C. dubia is studied parallel to the OCP signal of silicon based sensor. Less toxicity is found when the complex agent is present similar to the OCP signal. Another discrepancy is found between the MINEQL simulation and the toxicity test when MINEQL simulation is used to predict and control the ionic silver concentration. The data from both biosensor C. dubia and silicon based sensor support each other and both are not in agreement with MINEQL simulation prediction.

The recently proposed bioassay procedure that is based on the substrate induced quenching (SIQ) of an indicator fluorescence for the measurement of analyte concentrations is evaluated. In this type of assay a enzynatic reaction and a fluorescence quenching interaction are coupled together. Typically, an appropriate dehydrogenase enzyme reduces or oxidises the nicotinamide adenine dinucleotide cofactor. The change in the concentration of NADH results in variations in the excited fluorophore population as observed through fluorescence intensity. This latter aspect is used to monitor substrate (analyte) concentrations. Results on the investigation of the substrate induced quenching bioassay method and possibilities of using it as the basis of (i) a novel enzyme bioassay technique and (ii) a novel bioprobe format are presented. Ethanol was chosen as the model analyte, and a new assay procedure for its measurement was developed. A generic theoretical relation is discussed for the observed assay kinetics of substrate induced quenching (SIQ) and a model is described that includes the effects due to dynamic/static quenching of the fluorophore by either the enzyme substrate or product. The validity of the derived model is shown by comparison with experimental results for a SIQ based ethanol assay. The option of running the dehydrogenase reaction so as to consume NADH rather than generate it is also investigated. In order to demonstrate this approach acetaldehyde was chosen as the model analyte, and a assay procedure for its measurement was developed. The potential of the SIQ technique for incorporation into biosensor based upon a 'reservoir' format was demonstrated through the development of custom optical instrumentation and resevoir flowcell. Applicability of the SIQ technique to other biosensor formats such as flow-injection analysis and 'dry reagent' technology is discussed. The overall applicability of the SIQ technique is assessed through the generation of a number of SIQ assays on the following substrates: ethanol, glucose, glucose-6- phosphate, L-glutamic acid, isocitric acid, acetaldehyde, pyruvic acid, ot-ketoglutaric acid, and oxalacetic acid.

Le besoin croissant de biocapteurs optiques compacts, sélectifs, ultrasensibles, rapides et bas coût dans les domaines médical et environnemental a engendré une émergence de solutions technologiques, notamment les capteurs à bases de microrésonateurs optiques. Ces types de biocapteurs sont capables de fournir une détection sélective de très faibles concentrations de biomolécules si leurs surfaces sont fonctionnalisées. En revanche, les deux méthodes optiques d'interrogation actuelles, balayage spectral et variation de l’intensité, ne peuvent ni fournir la sensibilité de la phase du signal optique propagé dans le capteur, ni les paramètres opto-géométriques (perte par propagation, l'indice effectif, coefficient de couplage, etc) nécessaires pour une modélisation de la réponse du capteur. Pour accéder à ces informations, nous avons proposé d’utiliser l'interféromètre optique à faible cohérence sensible à la phase comme une technique alternative d’interrogation et de caractérisation de microrésonateurs. La première partie des travaux de cette thèse est consacrée à l’étude de conception et de réalisation de microrésonateurs monomodes possédant un facteur de qualité supérieur à 20000 dans l'eau. Cette étude a été validée par la réalisation technologique, à l'aide des procédés de photolithographie classique et de gravure sèche au plasma d'oxygène, de microrésonateurs polymères possédant des facteurs de qualité allant jusqu'à 38 200. La deuxième partie des travaux de thèse est dédiée à l'adaptation du dispositif PS-OLCI, initialement développé au Laboratoire National de Métrologie et d'Essais (LNE) pour interroger les composants des télécommunications optiques, pour la caractérisation de microrésonateurs optiques. Les résultats obtenus en évaluant les performances spatiales et spectrales de différents microrésonateurs ont montré que le dispositif PS-OLCI n'est pas seulement un outil d'interrogation et de caractérisation mais aussi un véritable outil d’aide à la conception de microrésonateurs optiques. Une modélisation, validée par l'ajustement des mesures expérimentales, de la réponse PS-OLCI d'un microrésonateur, met en évidence la relation existant entre l'interférogramme et les intégrales de Fresnel. La dernière partie de nos travaux concerne l'association du dispositif PS-OLCI et d’un composant optofluidique, constitué de microrésonateurs et d’un circuit microfluidique en polymères, pour la détection d’espèces biologiques. A cet effet, la molécule de glucose a été choisie pour démontrer la détection homogène ou volumique en solution aqueuse en obtenant respectivement les limites de détection de l'ordre de 50 µg/ml et de 2 µg/ml en exploitant l’intensité ou la phase des mesures PS-OLCI. Ces performances démontrent la capacité de notre capteur à déceler des biomolécules en faible concentration ainsi que la pertinence de la mesure de la phase, d'où l'intérêt du dispositif PS-OLCI. Pour remédier au problème de sélectivité du capteur en détection homogène, la méthode de détection surfacique est utilisée. La problématique de chimie de surface des polymères, c'est à dire la fonctionnalisation des surfaces des guides polymères en vue d’une détection surfacique, a d'abord été effectuée. Les limites de détection obtenues en détection surfacique sont ensuite évaluées à leur tour pour différents types de molécules particulièrement les protéines telles que la streptavidine ou la biotine. Les performances de détection de streptavidine obtenues sont au moins 10 fois meilleures que celles obtenues à l’aide de la technique de Résonance de Plasmons de Surface considérée à ce jour comme la technique de référence en biodétection sans marqueur. Ces premiers résultats, présentant des marges importantes d’amélioration, contribuent à démontrer que les capteurs à base de microrésonateurs optiques sont des candidats potentiels très prometteurs pour la détection de très faibles concentrations de biomolécules pour l’analyse biochimique. / The increasing need for compact, selective, ultrasensitive, fast and affordable optical biosensors in the medical and environmental sectors gave rise to new technological solutions, especially regarding sensors based on optical microresonators. If their surfaces are functionalized, these biosensors can provide a selective detection of low concentrations of biomolecules. However, two common optical interrogation methods – spectral scanning and intensity variation – cannot provide the same sensitivity as the method using phase detection of the guided modes nor the opto-geometrical parameters (propagation loss, effective refractive index, coupling coefficient), needed for the modeling of the sensor response. To get this information, we proposed to use the Phase Sensitive-Optical Low Coherence Interferometer (PS-OLCI) as a new alternative technical solution for interrogation and characterization of microresonators. The first part of this thesis is dedicated to the conception and fabrication of single mode microresonators with a quality factor higher than 20 000 in water. This work was validated by the manufacture of polymer microresonators with a quality factor up to 38 200, using UV photolithography and Reactive Ion Etching (RIE) processes. The second part of this work covers the adaptation of PS-OLCI setup, initially developed at Laboratoire national de métrologie et d'essais (LNE), to interrogate optical telecommunication devices, for the characterization of optical microresonators. The results, obtained through the analysis of spatial and spectral performances of various microresonators, showed that the PS-OLCI setup is not only an interrogation and characterization tool but also a real support tool for designing optical microresonators. The performed modelling of the PS-OLCI and microresonator association response, validated by the fitting of the experimental data, demonstrated the relation between PS-OLCI measurements and Fresnel integrals. The last part of this work is dedicated to label free biosensing experiments using PS-OLCI setup associated to an optofluidic component, made of polymer optical microresonators and polymer microfluidic circuit, to detect biological species. The glucose molecule was chosen to demonstrate the homogeneous sensing experiments in aqueous solution. The obtained detection limits are around 50 µg/ml when we exploited intensity and around 2 µg/ml when we exploited the PS-OLCI measurements phase. These results demonstrate the high sensitivity of the proposed biosensor as well as the value of the optical phase measurement, hence the interest of PS-OLCI set up. To address the problem of sensor selectivity in homogeneous sensing method, surface sensing experiences were performed. The first step of this method was the functionalization of the sensor surface, by binding adequate recognition molecular sites to the sensor surface in order to immobilize target molecules. Proteins were then chosen to perform this very same type of experiences. The preliminary results in the 0.02 pg/mm2 range clearly show that our sensors is ten times more sensitive than Surface Plasmon Resonance, which is actually considered among the most successful label free sensing methods. These first results, which can be improved, demonstrate that the sensors based on optical microresonators are promising candidates for the detection of low concentrations of biomolecules for biochemical investigation.

Orientador: Sidney José Lima Ribeiro / Banca: Marcelo Nalin / Banca: Antonio Aparecido Pupim Ferreira / Banca: Gustavo Fernandes Souza Andrade / Banca: Airton Abrahan Martin / Resumo: A espectroscopia de Raman intensificada por superfície (SERS, termo em inglês Surfaceenhanced Raman Spectroscopy) é uma técnica promissora que mostra a sensibilidade para a detecção da interação de biomoléculas que são importantes para detecção precoce de doenças. O vírus da imunodeficiência humana (HIV) têm sido um grande problema por várias décadas. Existem vários métodos de deteção baseados na interação específica de anticorpos, tais como, o ELISA e os testes rápidos (TR's). No entanto, novas estratégias têm sido desenvolvidas para rápido diagnóstico do vírus HIV, e uma prova de conceito de detecção do peptídeo p17-1 foi descrito neste trabalho. A proteina matriz p17 é uma essencial proteína no ciclo de replicação do vírus HIV. As fases iniciais da replicação do vírus envolve a pré integração do complexo do DNA no núcleo do p17 desempenhando um papel na ligação de RNA viral e transporte para a membrana. Neste trabalho foram descritos duas plataformas SERS para a detecção do vírus HIV baseado no peptide p17 -1 (sequência LSGGELDRWEKIRLPGG). O anticorpo foi imobilizado em um substrato de ouro usando duas diferentes camadas automontadas (SAM). A primeira SAM, os substratos de ouro foram imersos em uma solução aquosa de 11 mercaptoundecanóico (MUA). Na segunda SAM, os substratos foram imersos em uma mistura aquosa de politietileno glicol (SHPEG- COOH e SH-PEG-CH3). Aqui serão chamados de SAM-MUA e SAM-PEG, respectivamente. Ambas as SAM's foram imersas emu ma solução de anticorpo (anti-p17) e foram descritas como plataforma d captura MUA e PEG. Ambas plataformas foram funcionalizadas com o peptídeo p17-1. Sondas SERS foram preparadas com nanopartículas de ouro e revestidas com uma molécula Raman reporter (azul de Nilo A) e funcionalizadas com um anticorpo anti-p17. Estas estruturas (sonda SERS e plataformas de captura) formam um ensaio sanduíche... / Abstract: Surface-enhanced Raman Scattering (SERS) technique offers great promises for simplified and sensitive detection of biomolecular interactions that are relevant for early disease diagnostics. Human immunodeficiency virus (HIV) has been a problem for decades. There are several methods of diagnostics based on antibodies specific reactions, such as enzyme-linked immunosorbent assays (ELISAs) and rapid test (RT). However, new strategies have been developed for rapid HIV diagnostics and, as a proof-of-concept, peptide p17-1 was considered here. The matrix protein p17 is a structural protein that is essential in the life cycle of the retrovirus The early stages of the virus replication involve the pre integration of the DNA complex into the nucleus P17 plays a role in RNA viral binding and transport to the membrane. Here were describe two new SERS platform for HIV detection based on peptide p17-1 (sequence LSGGELDRWEKIRLPGG). The antibody anti-p17 was immobilized in a planar gold surface using two differents self-assembled (SAM) techniques. First SAM, were obtained by immersion of the surface into ethanolic solution of 11-Mercaptoundecanoic acid (MUA). Second SAM were obtained by immersion in aqueous solution aquous mixtures of (SH-PEG-COOH/SH-PEG-CH3) and polyethylene glycol (PEG,). Here were describe the two platforms as SAM-MUA and SAMPEG, respectively. Both SAM's were immersed in a solution containing the anti-p17. Samples at this step were called capture platform-MUA and capture platform-PEG. Both capture platforms were funcionalizated with the peptide p17-1. SERS probes were prepared with gold nanoparticles coated with a Raman reporter molecule (Nile Blue A) and, functionalized with an anti-p17. These structures (SERS probe and capture platforms) allow for a sandwich assay, a strategy regularly used for high-sensitivity detection. The light blue color in the SERS mapping represents peptide strong... / Doutor

No campo da eletroanalítica existe uma intensa atividade de pesquisa sobre eletrodos de pasta de carbono. As principais vantagens da pasta de carbono são a rapidez e a simplicidade do processo de preparação, e a possibilidade de renovação da superfície do eletrodo a cada nova medida. Isso é possível devido à modificação interna do material eletródico, em que o modificador escolhido está diretamente relacionado com o analito de interesse a ser determinado. Os biossensores de pasta de carbono são muito utilizados na determinação de pesticidas da classe dos organofosforados e carbamatos pelo processo de inibição enzimática. Um dos principais problemas na determinação de substâncias utilizando eletrodos de pasta de carbono e biossensores de pasta de carbono é o seu alto limite de detecção, que dificulta a análises traços de substâncias. Uma opção que há décadas vem sendo adotada é a utilização dos eletrodos de nanotubos de carbono, funcionalizados ou não, na elaboração da pasta. Estes sensores, juntamente com os biossensores de pasta de nanotubos de carbono, possuem propriedades catalíticas e estruturais que fornecem um menor limite de detecção e quantificação na determinação de várias substâncias associadas. Estas características são singulares e inerentes à conformação dos nanotubos de carbono, sendo a elevada área ativa e poder eletrocatalítico umas das propriedades destes nanomateriais mais relatadas na literatura. Diante da falta de trabalhos que descrevam, paralelamente, o desempenho destes biossensores na determinação de carbamatos, neste trabalho objetivou-se explorar comparativamente o comportamento eletroquímico de biossensores de pasta de carbono e de pasta de nanotubos de carbono modificados com a enzima acetilcolinesterase para a determinação dos pesticidas carbaril e metomil. Estes nanotubos foram purificados em meio ácido e caracterizados por microscopia eletrônica de varredura e por voltametria cíclica. Parâmetros cinéticos da enzima acetilcolinesterase como a constante de Michaelis-Menten e velocidade máxima de hidrólise do substrato acetiltiocolina foram obtidos e analisados pelo método espectrofométrico. Os biossensores confeccionados tiveram seus parâmetros eletródicos de operação otimizados e foram utilizados na determinação do pesticida carbaril em amostra fluvial. Os biossensores de pasta de carbono e de pasta de nanotubos de carbono modificados com a enzima acetilcolinesterase apresentaram uma repetibilidade com desvio padrão relativo de 2,44% e 1,77%, respectivamente. A reprodutibilidade dos biossensores elaborados apresentou um desvio padrão relativo de 6,60% e 4,13%, respectivamente. Na determinação do pesticida carbaril, os biossensores de pasta de carbono e de pasta de nanotubos de carbono modificados com a enzima acetilcolinesterase apresentaram um limite de detecção de 0,0389 e 0,0163 µmol L-1, respectivamente. Na determinação do pesticida metomil, os biossensores de pasta de carbono e de pasta de nanotubos de carbono modificados com a enzima acetilcolinesterase apresentaram um limite de detecção de 0,0398 e 0,0083 µmol L-1, respectivamente. Em ambas as determinações, o biossensor elaborado com os nanotubos funcionalizados apresentou maior sensibilidade. Quando comparado com o biossensor de pasta de carbono frente à determinação do carbaril, o biossensor de pasta de nanotubos apresentou um limite de detecção aproximadamente 2,4 vezes menor e, quando comparado com o biossensor de pasta de carbono na determinação do pesticida metomil, o biossensor de pasta de nanotubos apresentou um limite de detecção aproximadamente 5 vezes menor. Os limites de quantificação dos biossensores de pasta de carbono e de pasta de nanotubos de carbono modificados com a enzima acetilcolinesterase, para os pesticidas carbaril e o metomil, foram de 0,1297 e 0,0542 µmol L-1 e de 0,1323 e 0,0277 µmol L-1, respectivamente. O biossensor de pasta de nanotubos de carbono modificado com a enzima acetilcolinesterase foi aplicado na determinação de carbaril em amostra de água de rio e ácido húmico, fornecendo um LD de 0,0722 e 0,0704 µmol L-1, respectivamente. Comparado com a curva original realizada em tampão fosfato, este biossensor apresentou um limite de detecção e quantificação de aproximadamente 4,32 e 4,43 vezes maior no procedimento utilizando ácido húmico e água fluvial, respectivamente. / There in the field of electroanalytical an intense research activity on carbon pastes electrodes. The main advantages of carbon paste are the speed and simplicity of the preparation process, and the possibility of renewing the electrode surface with each new measure. This is possible due to the internal modification of the electrode material, in which the modifier selected is directly related to the analytes to be determined. The carbon paste biosensors are widely used in determining the class of pesticides organophosfates and carbamates by enzyme inhibition process. A major obstacle to the determination of substances with a carbon paste electrode and carbon nanotubes paste biosensors is its high detection limit, which complicates the analysis of trace substances. One option that has for decades been adopted is the use of electrodes of carbon nanotubes, functionalized or otherwise, in elaboration of the paste. These sensors, together with the carbon paste nanotubes biosensors, have structural and catalytic properties that provide a lower limit of detection and quantitation in the determination of several related substances. These characteristics are natural and inherent in the arrangement of carbon nanotubes, the high electrocatalytic active area and catalytic power properties of these nanomaterials are most often reported in the literature. Given the lack of studies that describe, in parallel, the performance of these biosensors in the determination of carbamates, this study aimed, comparatively, to explore the electrochemical behavior of carbon paste and carbon nanotubes paste biosensors modified with enzyme acetylcholinesterase to determine pesticide carbaryl and methomyl in distillated and river water. These nanotubes were purified in acidic medium and characterized by scanning electron microscopy and cyclic voltammetry. The kinetic parameters of acetylcholinesterase as the Michaelis-Menten constant and Maximum Velocity of hydrolysis of acetylthiocholine were obtained and analyzed by spectrophotometric method. Biosensors fabricated electrodes had their parameters optimized operation and were used in the determination of carbaryl pesticide in a sample fluvial. Carbon paste and carbon nanotubes paste biosensors modified with enzyme acetylcholinesterase showed a repeatability wih relative standard deviation of 2.44% and 1.77%, respectively. The reproducibility of the biosensor developed showed a relative standard deviation of 6.60% and 4.13%, respectively. In determining the pesticide carbaryl, the carbon paste and carbon nanotubes paste biosensors modified with enzyme acetylcholinesterase showed a detection limit of 0.0389 and 0.0163 µmol L-1, respectively. In determining the pesticide methomyl, the carbon paste and carbon nanotubes paste biosensors modified with enzyme acetylcholinesterase showed a detection limit of 0.0398 and 0.0083 µmol L-1, respectively. In both determinations, the biosensor prepared with functionalized nanotubes showed higher sensitivity. Compared with the carbon paste biosensor in the determination of carbaryl, the nanotubes paste biosensor showed a detection limit of approximately 2.4 times lower and, when compared with the carbon paste biosensor in the determination of the pesticide methomyl, the biosensor elaborated with nanotubes showed a detection limit of approximately five times lower. The limits of quantification of carbon paste and carbon nanotubes paste biosensors modified with enzyme acetylcholinesterase, in the determination of carbaryl and methomyl pesticides, were 0.1297 and 0.0542 µmol L-1, and 0.1323 and 0.0277 µmol L-1, respectively. The carbon nanotubes biosensor modified with the enzyme acetylcholinesterase was applied to the determination of carbaryl in the sample of river water and humic acid, giving a detection limit of 0.0722 and 0.0704 mol L-1, respectively. Compared with the original curve performed in phosphate buffer, the biosensor showed a detection an quantification limit of approximately 4.32 and 4.43 times higher in the procedure using humic acid and fluvial water, respectively.

Durant la dernière décennie, la résonance de plasmon de surface (SPR) est devenue très populaire pour effectuer des analyses au cours d’un greffage chimique (ou biochimique) et étudier ainsi des réactions chimiques. Ce travail de thèse avait pour but de développer une méthode lithographique alternative, la nanoimpression assistée UV dite «douce», qui permet de fabriquer des réseaux de nanomotifs sur de très grandes surfaces (voir chapitre 1 - état de l’art) pour générer des nanostructures métalliques SPR intégrables. Les chapitres 2 et 3 étudient les paramètres expérimentaux de la nanoimpression pour obtenir des nanostructures hautement résolues et avec un minimum de défaut. Notre étude optique a été menée ensuite sur des réseaux de nanotrous imprimés dans des films d’or (chapitre 4). Le mécanisme physique du phénomène de transmission assistée par les plasmon est étudié de manière systématique d’après l’évolution de la position du pic de transmission avec les paramètres structuraux. Des mesures réalisées dans un système fluidique ont ensuite montré une réponse à un faible changement de l’indice de réfraction à la surface du réseau. Enfin, le dernier chapitre (chapitre 5) présente une nouvelle géométrie de biocapteurs optique basé sur une structure tri-couche dans une géométrie de type «nanocavité» à plasmon localisé (LSPR). Ces capteurs LSPR à nanocavités permettent d’améliorer le facteur de mérite d’un ordre de grandeur par rapport aux LSPR classiques. Leurs propriétés de résonance sont discutées à l’aide d’outils de simulation numérique. Enfin, nous démontrons qu’un tel capteur possède une grande sensibilité à la détection de biomolécules et serait donc adapté à l’étude d’interactions immunochimiques. / During the last decade, surface plasmon resonance (SPR) has become widely used to characterize a biological surface and to characterize binding events in the fields of chemistry and biochemistry. Research in this field has been favoured by the tremendous growth in nanofabrication methods among which soft lithographies are alternatively emerging. The purpose of this thesis work was to develop soft UV nanoimprint lithography, an emerging flexible technology allowing patterning on large area of subwavelength photonic nanostructures. The main advantages offered by soft UV nanoimprint lithography concern the simple patterning procedure and the low cost of the experimental setup (see state-of-art presented in chapter 1). Chapters 2 and 3 present the fabrication of master stamps, the study of nanoimprinting parameters coupled with the optimization of the etching process in order to get metallic nanostructures with limited pattern defects. The physical mechanisms of the transmission phenomenon exalted by surface plasmons were studied based on arrays of imprinted gold nanoholes (chapter 4). Extraordinary light transmission has been experimentally demonstrated. The geometrical effects on the position transmission peak were systematically analyzed. Proof-of-concept measurements performed in simple fluidic device indicate a response to small changes in refractive index in the surface vicinity. Finally, chapter 5 proposes a novel design for the optical sensor which is based on “nanocavities” exhibiting coupled localized plasmons. This LSPR sensor offers an improvement of one order of magnitude of the Figure of Merit compared to classical LSPR sensors. The resonance properties of these innovative nanocavities have been studied from numerical simulations and discussed based on their geometrical dependence. Since this system has demonstrated higher sensitivity for detection of biomolecules, it is thus fully adapted to study immunochemical binding interactions.

Genetic mutations perturbing inactivation of human cardiac voltage-gated sodium channels (VGSCs), specifically Nav1.5, can cause long QT syndrome type 3 (LQT3). LQT3 is a cardiac disorder in which patients experience syncope and ventricular tachyarrhythmia, and are thus predisposed to sudden cardiac death. Deeper understanding of the structural dynamics of VGSC inactivation is needed to inform treatment of and drug design for potentially life-threatening arrhythmias. A well supported hypothesis is that the VGSC inactivated state is stabilized by hydrophobic interactions between the inactivation gate and an unknown binding site potentially involving the underside of the channel pore, C-terminus (C-T), and auxiliary proteins. Despite advances in biophysical and structural characterization of VGSCs, the specific molecular components and timing of their interactions within the inactivation complex remain unclear. Fluorescence imaging approaches that connect conformational change with channel function in mammalian cells could provide much needed mechanistic insight on the structural dynamics of the VGSC inactivation complex. This thesis describes the development of a site-specific fluorescent unnatural amino acid (UAA) labeling and spectral imaging methodology to probe the cardiac VGSC, Nav1.5, inactivation complex in live mammalian cells. First, UAA mutagenesis experiments were performed to validate orthogonal synthetase-tRNA (aaRS-tRNA) technology for fluorescent labeling of intracellular and membrane proteins in mammalian cells. Next, towards investigating conformational dynamics and intramolecular interactions related to inactivation, the Nav1.5 inactivation gate was labeled with a single environmentally sensitive fluorescent UAA L-anap. While the function of L-anap labeled channels was altered, their function remained within pathophysiological range. Then, imaging of L-anap labeled Nav1.5 in mammalian cells afforded characterization of unique L-anap spectra at different sites in the inactivation gate. Finally, using potassium-depolarization (K-depolarization) as rough means of voltage control, L-anap spectral shifts demonstrated conformational changes between the closed and open-inactivated states, which depended on the presence of the distal C-T (DCT). Site-specific L-anap labeling of the inactivation gate combined with spectral imaging and K-depolarization affords a general imaging assay to directly monitor conformational rearrangements of the Nav1.5 inactivation gate in channels expressed in live mammalian cells. While interactions with the DCT are specifically probed, this general assay provides an opportunity to bring necessary unification of ideas about VGSC inactivation, as well as insight on outstanding questions of VGSC regulation.