Orientador: Ronei Jesus Poppi / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-08T23:09:23Z (GMT). No. of bitstreams: 1 Rocha_WericksonFortunatodeCarvalho_M.pdf: 1597999 bytes, checksum: b9442a7ba3c5fb6665bc87f6c0abf34f (MD5) Previous issue date: 2007 / Resumo: Este trabalho teve como objetivo realizar a validação de modelos de calibração multivariada através do cálculo de figuras de mérito e de cartas de controle multivariadas do medicamento Nimesulida por meio do infravermelho próximo. Foram preparadas 69 amostras sintéticas contendo o princípio ativo (Nimesulida) na faixa 10,38-39,47% (m/m) em excipiente (lactose, povidona-KV29- 32, celulose 200, lauril sulfato de sódio, croscarmelose sódica e estereato de magnésio). Destas, 49 amostras foram utilizadas para a calibração e 20 para a validação as quais foram separadas através do algoritmo de Kennard-Stone.O tratamento utilizado nos espectros foi a correção do espalhamento multiplicativo. Na sequência, foram obtidos os seguintes valores para as figuras de mérito: limite de detecção (0,61), limite de quantificação (2,03), exatidão (RMESC 0,66 %(m/m), RMESCV 0,92 %(m/m), RMESP 1,05 %(m/m) de Nimesulida), seletividade média (0,0056), sensibilidade (0,0036), inverso da sensibilidade analítica (0,20 %(m/m)-1 de Nimesulida) e razão sinal ruído (181,11). Na segunda parte do trabalho foram utilizadas 113 amostras sintéticas para construção das cartas de controle multivariadas. Foram desenvolvidas três cartas de controle e calculado os limites de controle para cada carta. Os valores encontrados foram: - carta NAS (Superior = 6,54x10; Inferior = 5,25x10), carta interferente (7,45) e carta resíduo (2,38x10). Através dessas cartas de controle multivariadas foi possível identificar as amostras que estavam dentro e fora de controle. Obteve-se 64 amostras fora de controle e 20 amostras dentro de controle de acordo com o planejamento experimental realizado. Sendo assim, foi possível identificar, de forma qualitativa, as amostras de Nimesulida que estavam dentro e fora de controle. Portanto, a dissertação desenvolvida sugere um novo método analítico que pode ser usado para controle de qualidade em fármacos e para validação de modelos de calibração multivariada, pois os resultados obtidos, indicam que o modelo desenvolvido pode ser utilizado na indústria farmacêutica como uma alternativa ao método-padrão / Abstract: This study was mainly intended to elaborate the validation of multivariate calibration models based on the determination of figures of merit and multivariate control charts constructed with data from Nimesulide tablets upon near-infrared spectroscopy use. A total of 69 synthetic samples were prepared containing the active principle (Nimesulide) in the range of 10,38-39, 47% (m/m) in excipients (Cellulose,Sodium Lauryl Sulphate, Magnesium Stearate, Carmellose Sodium,Povidone and Lactose). It was used 49 samples for the calibration and 20 for the validation that were separated by the Kennard-Stone algorithm. It was used the multiplicative scatter correction on the spectra set. Then, the following values for figures of merit were calculated: limit of detection (0,61) , limit quantification (2,03) , accuracy (RMESC 0,66 %(m/m), RMESCV 0,92%(m/m), RMESP 1,05%(m/m) of Nimesulide), mean selectivity (0,0056), sensitivity (0,0036), inverse analytical sensitivity (0,20 %(m/m)- 1 of the Nimesulide) and signal-to-noise ration (181, 11). In the second application, 113 synthetic samples were used for the construction of multivariate control charts. Three control charts were designed and the control limits for each chart were calculated. The values found were: - chart NAS (upper = 6,54x10; below = 5,25x10), chart interferent (7,45) and chart residual (2,38x10). From these charts it was possible to identify the samples that were in and out of control. It was obtained 64 samples out of control and 20 samples in control, according to the experimental design. Therefore, it was possible to identify, in a qualitative way, the Nimisulide samples that were in control and the ones that were out of control. Hence it follows that, this research project suggests a new analytical method that can be used for quality control in drugs and also for the validation of multivariate calibration models. Thus, the results obtained indicate that this model can be used in pharmaceutical industries as an alternative to the standard procedure / Mestrado / Quimica Analitica / Mestre em Química

Free analyte is measured to be able to understand the pharmacological effects of a drug in the body, the binding to its ligand, and the effective drug level among other things. Thereby, it is important with correct measurements of free analyte, although it is not that easy to achieve since overestimations can occur. In this project, several immunoassays were developed for the bioanalytical methods Gyrolab and ELISA to measure free analyte, where the biotherapeutics Avastin® and Lucentis®, and the ligand VEGF were used as analytes. One difference between the methods is the short contact time of just a few seconds for Gyrolab compared to the sample incubation time of a couple of hours for ELISA. One difference between the antibodies is that Lucentis is an affinity-matured Fab region, and therefore, has a stronger affinity to VEGF compared to Avastin. When free Avastin was measured, the difference was significant, with ELISA estimating higher concentrations compared to Gyrolab. However, this was not the case for all assays. In some cases, this was probably due to differences between the methods that could not be seen. Otherwise, the results with no difference between the methods, when measuring free analyte with Lucentis as the drug, were expected due to the stronger affinity and longer halftime of dissociation. However, the difference with the longer sample incubation time for ELISA compared to the short contact time for Gyrolab seems to influence the measurement of free analyte, depending on the affinity of the components being measured.

Résumé : Malgré le nombre croissant de capteurs dans les domaines de la chimie et la biologie, il reste encore à étudier en profondeur la complexité des interactions entre les différentes molécules présentes lors d’une détection à l’interface solide-liquide. Dans ce cadre, il est de tout intérêt de croiser différentes méthodes de détection afin d’obtenir des informations complémentaires. Le principal objectif de cette étude est de dimensionner, fabriquer et caractériser un détecteur optique intégré sur verre basé sur la résonance plasmonique de surface, destiné à terme à être combiné avec d’autres techniques de détection, dont un microcalorimètre. La résonance plasmonique de surface est une technique reconnue pour sa sensibilité adaptée à la détection de surface, qui a l’avantage d’être sans marquage et permet de fournir un suivi en temps réel de la cinétique d’une réaction. L’avantage principal de ce capteur est qu’il a été dimensionné pour une large gamme d’indice de réfraction de l’analyte, allant de 1,33 à 1,48. Ces valeurs correspondent à la plupart des entités biologiques associées à leurs couches d’accroche dont les matrices de polymères, présentés dans ce travail. Étant donné que beaucoup d’études biologiques nécessitent la comparaison de la mesure à une référence ou à une autre mesure, le second objectif du projet est d’étudier le potentiel du système SPR intégré sur verre pour la détection multi-analyte. Les trois premiers chapitres se concentrent sur l’objectif principal du projet. Le dimensionnement du dispositif est ainsi présenté, basé sur deux modélisations différentes, associées à plusieurs outils de calcul analytique et numérique. La première modélisation, basée sur l’approximation des interactions faibles, permet d’obtenir la plupart des informations nécessaires au dimensionnement du dispositif. La seconde modélisation, sans approximation, permet de valider le premier modèle approché et de compléter et affiner le dimensionnement. Le procédé de fabrication de la puce optique sur verre est ensuite décrit, ainsi que les instruments et protocoles de caractérisation. Un dispositif est obtenu présentant des sensibilités volumiques entre 1000 nm/RIU et 6000 nm/RIU suivant l’indice de réfraction de l’analyte. L’intégration 3D du guide grâce à son enterrage sélectif dans le verre confère au dispositif une grande compacité, le rendant adapté à la cointégration avec un microcalorimètre en particulier. Le dernier chapitre de la thèse présente l’étude de plusieurs techniques de multiplexage spectral adaptées à un système SPR intégré, exploitant en particulier la technologie sur verre. L’objectif est de fournir au moins deux détections simultanées. Dans ce cadre, plusieurs solutions sont proposées et les dispositifs associés sont dimensionnés, fabriqués et testés. / Abstract : In spite of the growing number of available biosensors, many biochemical reactions and biological components have not yet been studied in detail. Among them, some require the combination of several detection techniques in order to retrieve enough information to characterize them fully. An unknown reaction based, for example, on DNA hybridization could be characterized with an electrochemical sensor, a mechanical sensor and an optical sensor, each giving a different type of information. The main objective of the work presented here is to design, fabricate and characterize a flexible integrated optical biosensor based on surface plasmon resonance, intended to be then combined with other detection techniques, and in particular, a microcalorimeter. Surface Plasmon Resonance (SPR) is well known to be a sensitive technique for surface-based biochemical detection. It has the advantage to be an unlabeled method and provides real time information on the kinetics of a reaction. The flexibility of the proposed SPR biosensor comes from the fact that it is designed for a large range of analyte refractive indices, from 1.33 to 1.48. These values are suitable for most biological entities and their ligand layers, and especially for hydrophilic polymer matrices used to trap DNA or protein entities and introduced in this work. As several biochemical studies require the simultaneous comparison of measurements to a reference or to another measurement, the second objective of this project is to study the potential of multi-analyte detection in an integrated SPR device on glass. The first three chapters of the thesis are focused on the main objective. The design based on two different models is presented, at the same time as the related simulation tools. The first model is based on the weak coupling approximation and permits to obtain most of the information for the device’s design. The second model, having no approximation, is used to validate the first model and complete and refine the design. The fabrication process of the glass chip is then introduced, as well as the characterization instruments and protocols. A device is obtained, with a volumetric sensitivity between 1000 nm/RIU and 6000 nm/RIU depending on the analyte refractive index. The 3D integration of the waveguide within the glass substrate makes the device extremely compact and adapted to the integration with the microcalorimeter in particular. The last chapter describes the study of several spectral multiplexing techniques adapted to an integrated SPR system using the glass technology. The goal is to provide at least two simultaneous measurements. Several detection techniques are examined and the related devices are designed, fabricated and characterized.

“Lab on a Chip” technologies have revolutionized the field of bio-chemical analytics. The crucial role of optical techniques in this revolution resulted in the emergence of a field by itself, which is popularly termed as “optofluidics”. The miniaturization and integration of the optical parts in the majority of optofluidic devices however still remains a technical challenge. The works described in this thesis focuses on developing integration methods to combine various optical techniques with microfluidics in an alignment-free geometry, which could lead to the development of portable analytical devices, suitable for field applications. The integration approach was applied to implement an alignment-free optofluidic chip for optical chromatography; a passive optical fractionation technique fractionation for cells or colloids. This system was realized by embedding large mode area photonic crystal fiber into a microfluidic chip to achieve on-chip laser beam delivery. Another study on passive sorting envisages an optofluidic device for passive sorting of cells using an optical potential energy landscape, generated using an acousto-optic deflector based optical trapping system. On the analytical side, an optofluidic chip with fiber based microfluidic Raman spectroscopy was realized for bio-chemical analysis. A completely alignment-free optofluidic device was realized for rapid bio-chemical analysis in the first generation by embedding a novel split Raman probe into a microfluidic chip. The second generation development of this approach enabled further miniaturization into true microfluidic dimensions through a technique, termed Waveguide Confined Raman Spectroscopy (WCRS). The abilities of WCRS for online process monitoring in a microreactor and for probing microdroplets were explored. Further enhanced detection sensitivity of WCRS with the implementation of wavelength modulation based fluorescent suppression technique was demonstrated. WCRS based microfluidic devices can be an optofluidic analogue to fiber Raman probes when it comes to bio-chemical analysis. This allows faster chemical analysis with reduced required sample volume, without any special sample preparation stage which was demonstrated by analyzing and classifying various brands of Scotch whiskies using this device. The results from this study also show that, along with Raman spectroscopic information, WCRS picks up the fluorescence information as well, which might enhance the classification efficiency. A novel microfabrication method for fabricating polymer microlensed fibers is also discussed. The microlensed fiber, fabricated with this technique, was combined with a microfluidic gene delivery system to achieve an integrated system for optical transfection with localized gene delivery.

No description available.

Cavity enhanced spectroscopies (CES) are currently amongst the most sensitive spectroscopic techniques available for probing gas-phase samples, however their application to the liquid-phase has been more limited. Sensitive analysis of submicrolitre liquid samples is highly desirable, as miniaturisation allows for the reaction and analysis of scarce or expensive reagents, produces less waste, and can increase the speed of separations and reactions, whilst having a small footprint and high throughput. Absorption spectroscopy is a particularly desirable technique due to its universal, label-free nature, however its application to small volume liquid samples is hampered by the associated short absorption pathlengths, which limit sensitivity. CES improve sensitivity by trapping light within a confined region, increasing the effective pathlength through the sample. Three distinct types of optical cavity were constructed and evaluated for the purposes of making optical absorption measurements on liquid samples. The first incorporated a high optical quality flow cell into a "macrocavity" formed from two dielectric mirrors separated by 51.3 cm. Cavity losses were minimised by positioning the flow cell at Brewster's angle to the optical axis, and the setup was used to perform a single-wavelength cavity ringdown spectroscopy experiment to detect and quantify nitrite within aqueous samples. The detection limit was determined to be 8.83 nM nitrite in an illuminated volume of only 74.6 nL. Scattering and reflective losses from the flow cell surfaces were found to be the largest barrier to increased sensitivity, leading us to focus on the integration of cavity mirrors within a microfluidic flow system in the work that followed. In the second set of experiments, cavity enhanced absorption spectroscopy (CEAS) measurements were performed on Thymol Blue using custom-made microfluidic chips with integrated cavity mirrors. Unfortunately, due to the plane-parallel configuration of the mirrors and the corresponding difficulty in sustaining stable cavity modes, the results were underwhelming, with a maximum cavity enhancement factor (CEF) of only 2.68. At this point, attention was focussed toward a more well-defined cavity geometry: open-access plano-concave microcavities. The microcavities consist of an array of micron-scale concave mirrors opposed by a planar mirror, with a pathlength that is tunable to sub-nanometer precision using piezoelectric actuators. In contrast to the other experimental setups described, themicrocavities allow for optical measurements to be performed in which we monitor the change of wavelength and/or amplitude of a single well-defined cavity mode in response to a liquid sample introduced between the mirrors. In the first microcavity experiment, we used 10 μm diameter mirrors with cavity lengths from 2.238 μm to 10.318 μm to demonstrate refractive index sensing in glucose solutions with a limit of detection of 3.5 x 10^-4 RIU. The total volume of detection in our setup was 54 fL. Thus, at the limit of detection, the setup can detect the change of refractive index that results from the introduction of 900 zeptomoles (500,000 molecules) of glucose into the device. The microcavity sensor was then adapted to enable broadband absorption measurements of methylene blue via CEAS. By recording data simultaneously from multiple cavities of differing lengths, absorption data is obtained at a number of wavelengths. Using 10 μm diameter mirrors with cavity pathlengths from 476 nm to 728 nm, a limit of detection, expressed as minimum detectable absorption per unit pathlength, of 1.71 cm^-1 was achieved within a volume of 580 attolitres, corresponding to less than 2000 molecules within the mode volume of the cavity. Finally, a new prototype was developed with improved cavity finesse, a much more intense and stable light source, and improved flow design. Using a single plano-concave microcavity within the array with a cavity pathlength of 839.7 nm, and 4 μm radius of curvature mirror, absorption measurements were performed on Methylene Blue. Analysis of this data indicated a CEF of around 9270, and a limit of detection based on the measured signal-to-noise ratio of 0.0146 cm^-1. This corresponds to a minimum detectable concentration of 104 nM Methylene Blue, which given the mode volume of 219 aL, suggests a theoretical minimum detectable number of molecules of 14.

Malgré le nombre croissant de capteurs dans les domaines de la chimie et la biologie, de nombreuses réactions n’ont pas encore été correctement identifiées et étudiées. C’est entre autres le cas des interactions intermoléculaires à l’interface liquide/solide trouvées dans les chimies de surface utilisées pour les méthodes de diagnostics médicaux et l’identification de divers processus biologiques. Afin de correctement comprendre les mécanismes en jeux, il est important de pouvoir croiser différentes méthodes de détection pour obtenir des informations complémentaires.MuLe principal objectif de cette étude est de dimensionner, fabriquer et caractériser un détecteur optique intégré sur verre basé sur la résonance plasmonique de surface, destiné à terme à être combiné avec d’autres techniques de détection. La résonance plasmonique de surface est une technique reconnue pour sa sensibilité adaptée à la détection de surface, qui a l’avantage d’être sans marquage et permet de fournir un suivi en temps réel de la cinétique d’une réaction. L’avantage principal de ce capteur est qu’il a été dimensionné pour une large gamme d’indice de réfraction de l’analyte, allant de 1,33 à 1,48. Ces valeurs correspondent à la plupart des entités biologiques associées à leurs couches d’accroche, particulièrement les matrices de polymères. Ces matrices sont de plus en plus utilisées non seulement pour leur capacité à augmenter la densité d’analytes présents à la surface du capteur, mais aussi pour leurs propriétés favorisant l’adsorption spécifique et leur utilisation comme élément actif de reconnaissance biologique.Étant donné que beaucoup d’études biologiques nécessitent la comparaison de la mesure à une référence ou à une autre mesure, le second objectif du projet est d’étudier le potentiel du système SPR intégré sur verre pour la détection multianalyte.MuLes trois premiers chapitres se concentrent sur l’objectif principal du projet. Le dimensionnement du dispositif suivant un cahier des charges préétabli est présenté, ainsi que les outils de simulation. Le procédé de fabrication de la puce optique sur verre est ensuite décrit, ainsi que les instruments et protocoles de caractérisation. Une comparaison est faite entre les simulations et les résultats expérimentaux, et les performances des outils numériques ainsi que celles du dispositif sont évaluées.Le dernier chapitre de la thèse présente l’étude de plusieurs techniques de multiplexage spectral adaptées à un système SPR intégré, exploitant en particulier la technologie sur verre. L’objectif est de fournir au moins deux détections simultanées. Dans ce cadre, plusieurs solutions sont proposées et les dispositifs associés sont dimensionnés, fabriqués et testés. / In spite of the growing number of available biosensors, many biochemical reactions and biological components have not yet been studied in detail. Among them, some require the combination of several detection techniques in order to retrieve enough information to characterize them fully. An unknown reaction based, for example, on DNA hybridization could be characterized with an electrochemical sensor, a mechanical sensor and an optical sensor, each giving a different type of information.MuThe main objective of the work presented here is to design, fabricate and characterize a flexible integrated optical biosensor based on surface plasmon resonance, intended to be then combined with other detection techniques. Surface Plasmon Resonance (SPR) is well known to be a sensitive technique for surface-based biochemical detection. It has the advantage to be an unlabeled method and provides real time information on the kinetics of a reaction. The use of an integrated technology enables us to integrate several sensors on the same chip for the same sample, making them compact and low-cost. The flexibility of the proposed SPR biosensor comes from the fact that it is designed for a large range of analyte refractive indices, from 1.33 to 1.48 in the 600 nm-1000 nm wavelength range. These values are suitable for most biological entities and their ligand layers, and especially for hydrophilic polymer matrices used to trap DNA or protein entities. These biochemical matrices are used more and more for their ability to trap high densities of analyte, provide a strong binding and serve as an active detection medium with good anti-fouling properties.MuAs several biochemical studies require the simultaneous comparison of measurements to a reference or to another measurement, the second objective of this project is to study the potential of multianalyte detection in an integrated SPR device on glass.The first three chapters of the thesis are focused on the main objective. The design according to predefined specifications is presented, at the same time as the simulation tools. The fabrication process of the glass chip is introduced, as well as the characterization instruments and protocols. Simulation and experimental results are then compared, and the device performance is assessed.The last chapter describes the study of several spectral multiplexing techniques adapted to an integrated SPR system using the glass technology. The goal is to provide at least two simultaneous measurements. Several detection techniques are examined and the related devices are designed, fabricated and characterized.

Detection and quantification of small peptides, such as yeast pheromones, are often challenging. We developed a highly sensitive and robust affinity-assay for the quantification of the α-factor pheromone of Saccharomyces cerevisiae based on recombinant hydrophobins. These small, amphipathic proteins self-assemble into highly stable monolayers at hydrophilic-hydrophobic interfaces. Upon functionalization of solid supports with a combination of hydrophobins either lacking or exposing the α-factor, pheromone-specific antibodies were bound to the surface. Increasing concentrations of the pheromone competitively detached the antibodies, thus allowing for quantification of the pheromone. By adjusting the percentage of pheromone-exposing hydrophobins, the sensitivity of the assay could be precisely predefined. The assay proved to be highly robust against changes in sample matrix composition. Due to the high stability of hydrophobin layers, the functionalized surfaces could be repeatedly used without affecting the sensitivity. Furthermore, by using an inverse setup, the sensitivity was increased by three orders of magnitude, yielding a novel kind of biosensor for the yeast pheromone with the lowest limit of detection reported so far. This assay was applied to study the pheromone secretion of diverse yeast strains including a whole-cell biosensor strain of Schizosaccharomyces pombe modulating α-factor secretion in response to an environmental signal.

Detection and quantification of small peptides, such as yeast pheromones, are often challenging. We developed a highly sensitive and robust affinity-assay for the quantification of the α-factor pheromone of Saccharomyces cerevisiae based on recombinant hydrophobins. These small, amphipathic proteins self-assemble into highly stable monolayers at hydrophilic-hydrophobic interfaces. Upon functionalization of solid supports with a combination of hydrophobins either lacking or exposing the α-factor, pheromone-specific antibodies were bound to the surface. Increasing concentrations of the pheromone competitively detached the antibodies, thus allowing for quantification of the pheromone. By adjusting the percentage of pheromone-exposing hydrophobins, the sensitivity of the assay could be precisely predefined. The assay proved to be highly robust against changes in sample matrix composition. Due to the high stability of hydrophobin layers, the functionalized surfaces could be repeatedly used without affecting the sensitivity. Furthermore, by using an inverse setup, the sensitivity was increased by three orders of magnitude, yielding a novel kind of biosensor for the yeast pheromone with the lowest limit of detection reported so far. This assay was applied to study the pheromone secretion of diverse yeast strains including a whole-cell biosensor strain of Schizosaccharomyces pombe modulating α-factor secretion in response to an environmental signal.

No description available.