Made available in DSpace on 2014-08-20T13:33:00Z (GMT). No. of bitstreams: 1 tese_marcelo_mendonca.PDF: 4204978 bytes, checksum: e41a9490cdb350a5e7add8e129afdd82 (MD5) Previous issue date: 2011-12-01 / The conventional methods used to detect the Listeria monocytogenes in foods are laborious and expensive, requiring several days for final identification. Monoclonal antibody (MAb) based immunoassays are highly specific and rapid to perform, especially when MAbs are raised to conserved virulence factors in the pathogen. Among diverse virulence factors of L. monocytogenes, the surface protein internalin A (InlA) is one of the most well-known and characterized protein, being an excellent target as it is highly exposed on the surface and exclusive of pathogenic species. In this work we report the production, characterization and use of a panel of MAbs against InlA (2D12, 3B7, 4E4), and a MAb (3F8) which specifically recognizes all bacteria belonging the genus Listeria. MAbs were produced by the immunization of BALB/c mice with a recombinant InlA together with heat killed L. monocytogenes. The MAbs produced showed excellent reativities by indirect ELISA, Western blot and immunofluorescence. A Cy5 conjugated anti-InlA MAb-2D12 was used as detection antibody for L. monocytogenes in a sandwich-like fiber optic immunoassay. Using MAb-2D12 as capture antibody on the waveguides, the limit of detection was ~3 x 102 CFU.mL-1, and when MAb-3F8 was used for capture the limit of detection was ~1 x 105 CFU.mL-1. Furthermore, MAbs 2D12 and 3F8 were used to coat paramagnetic beads and tested in the immunomagnetic separation (IMS) of L. monocytogenes from pure cultures, and artificially contaminated cheeses and hotdogs. After IMS capture, bacteria were released from the beads, used in the fiber optic assay or plated on agar for counting. In parallel, the capture of L. monocytogenes was confirmed by real-time qPCR and light-scattering technology (BARDOT). Using IMS to concentrate and separate L. monocytogenes, followed by a fiber optic platform, it was possible to detect in less than 22 h, approximately 40 CFU/g of L. monocytogenesi, even in the presence of L. innocua in cheese and hot dogs artificially contaminated. In addition, using mass spectrometry (MALDI-TOF-MS) the protein to which MAb-3F8 binds, was identified as fructose 1,6-bisphosphate aldolase (FBA). The results presented in this work indicate that using both systems together, the IMS and fiber optic immunosensor, were more reliable and faster, and could be applied in the routinely for detection of L. monocytogenes in food. Moreover, both MAbs have the potential to useful in others biosensor platforms, as well as in other detection and functionality immunoassays for InlA and FBA in Listeria. / Os métodos convencionais empregados para detecção de Listeria monocytogenes em alimentos são laboriosos e onerosos, requerendo vários dias para sua identificação final. A utilização de anticorpos monoclonais (MAbs) em imunoensaios para detecção rápida de bactérias tem como vantagem a alta especificidade e rapidez, principalmente quando direcionados para fatores de virulência conservados. Dentre os diversos fatores de virulência de Listeria, a proteína de membrana internalina A (InlA), é umas das mais bem caracterizadas, sendo um excelente alvo por ser altamente exposta na superfície e exclusiva de espécies patogênicas. Neste trabalho é relatado a produção, caracterização e utilização em métodos de diagnósticos de um painel de MAbs contra a InlA (2D12, 3B7, 4E4), e de um MAb (3F8) que reconhece especificamente todas as bactérias do gênero Listeria. Na produção dos MAbs, camundongos BALB/c foram imunizados com uma proteína recombinante InlA (rInlA) concomitantemente com L. monocytogenes inativadas por fervura. Os MAbs gerados demonstraram excelente reatividade por ELISA indireto, Western blot e imunofluorescência. O MAb anti-InlA 2D12 marcado com Cy5 foi usado como anticorpo de detecção de L. monocytogenes, no sistema tipo sanduíche de sensor de fibra óptica. Usando MAb-2D12 como anticorpo de captura nas fibras ópticas, obteve-se um limite de detecção de ~3 x 102 CFU.mL-1, e um limite de detecção de ~1 x 105 CFU.mL-1 foi visualizado com MAb-3F8 como captura. Os MAbs anti-InlA 2D12 e anti-Listeria 3F8 foram posteriormente utilizados para sensibilizar esferas paramagnéticas e testados na separação imunomagnética (IMS) de L. monocytogenes em culturas puras, e em queijo e salsichas tipo hotdog artificialmente contaminados. Após a captura por IMS, as bactérias foram liberadas, incubadas com a fibra óptica ou plaqueadas em agares para contagem. Em paralelo, a confirmação da captura de L. monocytogenes foi realizada por PCR quantitativo em tempo real e por light-scattering technology (BARDOT). Utilizando IMS para separar e concentrar L. monocytogenes, seguido da utilização em plataforma de fibra óptica, foi possível realizar a detecção em menos de 22 horas, de aproximadamente 40 UFC/g de L. monocytogenes em presença de L. innocua, em queijo e salsicha artificialmente contaminados. Além disso, a proteína alvo do MAb3F8 foi identificado como frutose 1,6-bifosfato aldolase através de espectrometria de massa (MALDI-TOF-MS). Os resultados obtidos nesse trabalho indicam que a utilização em conjunto dos sistemas de IMS e fibra óptica com os MAb-2D12 e MAb3F8, foram confiáveis e rápidos, e assim, podendo ser empregados em imunoensaios de rotina para detecção de L. monocytogenes em alimentos. Contudo, ambos MAbs possuem ainda grande potencial para serem mais explorados em outras plataformas de biossensores, assim como, em outros imunoensaios de detecção e funcionalidade de InlA e FBA em Listeria

In der medizinischen Diagnostik, Bioverfahrenstechnik und Umwelttechnik besteht ein steigender Bedarf an kompakten Analysegeräten für die schnelle Vor-Ort-Detektion spezifischer biochemischer Substanzen. Im Rahmen der Arbeit wurde daher ein neuartiger faseroptischer Sensor entwickelt, der in der Lage ist kleinste Brechzahländerungen, z.B. durch molekulare Bindungsprozesse, zu detektieren. Die hohe Empfindlichkeit an der vergoldeten Spitze der Sensorfaser beruht auf der Oberflächenplasmonenresonanz (SPR) einer einzelnen Mantelmode, die durch ein langperiodisches Fasergitter (LPG) ermöglicht wird. Die Übertragungsfunktion des Sensors wurde unter Verwendung eines Schichtwellenleitermodells schnell und präzise modelliert. Es konnte gezeigt werden, dass in einem wässrigen Umgebungsmedium die höchste Empfindlichkeit im Spektralbereich um 660 nm unter Annahme einer rund 35~nm dicken und 2~mm langen Goldbeschichtung erreicht wird. Weiterhin wurde nachgewiesen, dass mit einer intermediale Schicht aus Cadmiumsulfid die SPR der Mantelmode in einen höheren Spektralbereich verschoben und damit die Empfindlichkeit deutlich verbessert werden kann. Um eine geringe Polarisationsabhängigkeit des Sensors sicherzustellen, wurde ein nasschemisches Abscheidungsverfahren für die allseitige Goldbeschichtung der Sensorfaser entwickelt. Die spezifischen optischen Eigenschaften dieser Abscheidungen wurden mit Hilfe von LPGs untersucht, die durch eine spezielle UV-Belichtung hergestellt wurden. Die Experimente ergaben, dass die komplexe Permittivität nasschemischer Abscheidungen mit Schichtdicken oberhalb von 50~nm mit aufgedampften Goldschichten vergleichbar ist. Die Verluste der adressierten Mantelmoden wurden mit einer äquivalenten Sensoranordnung aus zwei identischen LPG untersucht. Dabei konnte ein Skalierfaktor abgeleitet werden, der die effiziente Berechnung der Mantelmodendämpfung erlaubt. Es wurde nachgewiesen, dass die Brechzahlauflösung etablierter volumenoptischer SPR-Sensoren mit einer einfachen Transmissionsmessung an einer geeigneten Wellenlänge erreicht werden kann. Die äußerst kompakte Sensorfläche des faseroptischen Sensors ermöglicht darüber hinaus die Untersuchung deutlich kleinerer Probenvolumina ohne ein zusätzliches mikrofluidisches System zu benötigen. Es wurde gezeigt, dass sekundäre Brechzahländerungen aufgrund von Temperaturschwankungen oder unspezifische Ablagerungen durch eine differentielle Auswertung zweier identischer Sensoren kompensiert werden können. Die verbleibende Querempfindlichkeit wird durch die Polarisationsabhängigkeit der Sensoren bestimmt. Die geringste Querempfindlichkeit konnte daher mit einer homogenen nasschemischen abgeschiedenen Sensorfläche nachgewiesen werden. / Compact analysis devices which facilitate the rapid detection of specific biochemical substances are in increasing demand in the fields of point-of-care medical diagnostics, bioprocess engineering and environmental engineering. The aim of this work was therefore to design a novel fiber-optic sensor able to detect small refractive index changes such as those caused by molecular binding processes. The high level of sensitivity at the gold-plated tip of the sensor fiber stems from the surface plasmon resonance (SPR) of a single cladding mode, which is the result of a long-period fiber grating (LPG). The transfer function of the sensor was calculated quickly and accurately using a slab waveguide model. It was observed that the highest level of sensitivity in an aqueous ambient medium is achieved at a wavelength of 660 nm assuming a gold coating of 35 nm in thickness and 2 mm in length. Furthermore, it was demonstrated that an intermedial cadmium sulfide layer shifts the SPR of the cladding mode towards higher wavelengths, thus leading to significantly enhanced sensitivity. An electroless plating process for the omnidirectional deposition of gold on the sensor fiber was developed in order to minimize the sensor\'s dependency on polarization. The specific optical properties of the gold layer deposited were investigated with the aid of LPGs fabricated using a special UV exposure method. The experiments showed the complex permittivity of electroless platings with a thickness of over 50 nm to be comparable with that of evaporated gold layers. The losses of the addressed cladding modes were investigated using an equivalent sensor setup consisting of two identical LPGs. This facilitated the determination of a scaling factor enabling the effcient calculation of cladding mode attenuation. It was demonstrated that it is possible to obtain the refractive index resolution of established volume optical SPR sensors with the aid of simple transmission measurements at a specific wavelength. Moreover, the extremely compact sensing area of the fiber-optic sensor enables the investigation of smaller sample volumes without the need for an additional microfluidic system. Secondary refractive index changes caused by temperature fluctuations or unspecific binding events can be compensated for by means of the differential interrogation of two identical fiber-optic sensors. The residual cross sensitivity is determined by the polarisation dependency of the sensor. The lowest cross sensitivity was therefore demonstrated in combination with a homogeneous electroless plated sensor surface.

The thesis deals with the possibility of using optical fiber as a sensor. The introduction of the thesis is devoted to the foundations of optics. In addition, the thesis deals with optical interferometry, polarization and dispersion in optical fiber. The main output of the thesis is the comparison of acoustic vibration measurements using interferometric and polarization methods and further measurements on the real route in Brno.

The diploma thesis deals with design of the converter card for the precise control of a laser diode. At first, there are described the principles of the laser diode, photodiode and basic functions of optical fiber sensors. Following chapter deals with description of developed converter card. In the diploma thesis are discussed all function blocks of the converter card and they are explained in detail. In the thesis are subsequently tested all problem parts of the converter card for the precise control of the laser diode, such as fast switching of the current flowing through the laser diode and design of the connection for evaluating of current pulse from the sensing photodiode. An integral part of thesis is a description of created printed circuit board and there are described all used layers and devices’ layout on the PCB. Final part of the diploma thesis deals with firmware for the microcontroller, which was made for testing of all function blocks of the converter card for the precise control of a laser diode. At the same time was developed software for PC, which communicates with the converter card via universal serial bus (USB).

In der medizinischen Diagnostik, Bioverfahrenstechnik und Umwelttechnik besteht ein steigender Bedarf an kompakten Analysegeräten für die schnelle Vor-Ort-Detektion spezifischer biochemischer Substanzen. Im Rahmen der Arbeit wurde daher ein neuartiger faseroptischer Sensor entwickelt, der in der Lage ist kleinste Brechzahländerungen, z.B. durch molekulare Bindungsprozesse, zu detektieren. Die hohe Empfindlichkeit an der vergoldeten Spitze der Sensorfaser beruht auf der Oberflächenplasmonenresonanz (SPR) einer einzelnen Mantelmode, die durch ein langperiodisches Fasergitter (LPG) ermöglicht wird. Die Übertragungsfunktion des Sensors wurde unter Verwendung eines Schichtwellenleitermodells schnell und präzise modelliert. Es konnte gezeigt werden, dass in einem wässrigen Umgebungsmedium die höchste Empfindlichkeit im Spektralbereich um 660 nm unter Annahme einer rund 35~nm dicken und 2~mm langen Goldbeschichtung erreicht wird. Weiterhin wurde nachgewiesen, dass mit einer intermediale Schicht aus Cadmiumsulfid die SPR der Mantelmode in einen höheren Spektralbereich verschoben und damit die Empfindlichkeit deutlich verbessert werden kann. Um eine geringe Polarisationsabhängigkeit des Sensors sicherzustellen, wurde ein nasschemisches Abscheidungsverfahren für die allseitige Goldbeschichtung der Sensorfaser entwickelt. Die spezifischen optischen Eigenschaften dieser Abscheidungen wurden mit Hilfe von LPGs untersucht, die durch eine spezielle UV-Belichtung hergestellt wurden. Die Experimente ergaben, dass die komplexe Permittivität nasschemischer Abscheidungen mit Schichtdicken oberhalb von 50~nm mit aufgedampften Goldschichten vergleichbar ist. Die Verluste der adressierten Mantelmoden wurden mit einer äquivalenten Sensoranordnung aus zwei identischen LPG untersucht. Dabei konnte ein Skalierfaktor abgeleitet werden, der die effiziente Berechnung der Mantelmodendämpfung erlaubt. Es wurde nachgewiesen, dass die Brechzahlauflösung etablierter volumenoptischer SPR-Sensoren mit einer einfachen Transmissionsmessung an einer geeigneten Wellenlänge erreicht werden kann. Die äußerst kompakte Sensorfläche des faseroptischen Sensors ermöglicht darüber hinaus die Untersuchung deutlich kleinerer Probenvolumina ohne ein zusätzliches mikrofluidisches System zu benötigen. Es wurde gezeigt, dass sekundäre Brechzahländerungen aufgrund von Temperaturschwankungen oder unspezifische Ablagerungen durch eine differentielle Auswertung zweier identischer Sensoren kompensiert werden können. Die verbleibende Querempfindlichkeit wird durch die Polarisationsabhängigkeit der Sensoren bestimmt. Die geringste Querempfindlichkeit konnte daher mit einer homogenen nasschemischen abgeschiedenen Sensorfläche nachgewiesen werden. / Compact analysis devices which facilitate the rapid detection of specific biochemical substances are in increasing demand in the fields of point-of-care medical diagnostics, bioprocess engineering and environmental engineering. The aim of this work was therefore to design a novel fiber-optic sensor able to detect small refractive index changes such as those caused by molecular binding processes. The high level of sensitivity at the gold-plated tip of the sensor fiber stems from the surface plasmon resonance (SPR) of a single cladding mode, which is the result of a long-period fiber grating (LPG). The transfer function of the sensor was calculated quickly and accurately using a slab waveguide model. It was observed that the highest level of sensitivity in an aqueous ambient medium is achieved at a wavelength of 660 nm assuming a gold coating of 35 nm in thickness and 2 mm in length. Furthermore, it was demonstrated that an intermedial cadmium sulfide layer shifts the SPR of the cladding mode towards higher wavelengths, thus leading to significantly enhanced sensitivity. An electroless plating process for the omnidirectional deposition of gold on the sensor fiber was developed in order to minimize the sensor\'s dependency on polarization. The specific optical properties of the gold layer deposited were investigated with the aid of LPGs fabricated using a special UV exposure method. The experiments showed the complex permittivity of electroless platings with a thickness of over 50 nm to be comparable with that of evaporated gold layers. The losses of the addressed cladding modes were investigated using an equivalent sensor setup consisting of two identical LPGs. This facilitated the determination of a scaling factor enabling the effcient calculation of cladding mode attenuation. It was demonstrated that it is possible to obtain the refractive index resolution of established volume optical SPR sensors with the aid of simple transmission measurements at a specific wavelength. Moreover, the extremely compact sensing area of the fiber-optic sensor enables the investigation of smaller sample volumes without the need for an additional microfluidic system. Secondary refractive index changes caused by temperature fluctuations or unspecific binding events can be compensated for by means of the differential interrogation of two identical fiber-optic sensors. The residual cross sensitivity is determined by the polarisation dependency of the sensor. The lowest cross sensitivity was therefore demonstrated in combination with a homogeneous electroless plated sensor surface.

A fatigue test has been performed on 7075-T651 aluminum specimens which were bonded with polyimide coated optical fibers with discrete Bragg gratings. These fibers were bonded with AE-10 strain gage adhesive. The results indicate that lower strain amplitudes do not produce cause for concern, but that larger strain amplitudes (on the order of 3500 μ) may cause some sensors to become unreliable. The strain response of acrylate coated optical fiber strain sensors bonded to aluminum specimens with AE-10 and M-Bond 200 strain gage adhesives was investigated with both axial and cantilever beam tests. These results were compared to both the strain response of conventional strain gages and to model predictions. The results indicate that only about 82.6% of the strain in the specimen was transferred through the glue line and fiber coating into the fiber. Thus, multiplying by a strain transfer factor of approximately 1.21 was sufficient to correct the optical fiber strain output. This effect was found to be independent of the adhesive used and independent of the three-dimensional profile of the glue line used to attach the fiber. Finally, this effect did not depend on whether the fiber had a polyimide or an acrylate coating. Further investigation was conducted on the feasibility of using optical fiber strain sensors for monitoring subcritical damage (such as matrix cracks) in fiber reinforced composite materials. These results indicate that an array of optical fibers which monitor the strain profile on both sides of a composite panel may be sufficient for these purposes / Master of Science

Améliorer la sécurité routière passe par une meilleure compréhension des causes d'accidents. Il est donc nécessaire de développer des observatoires discrets pour étudier la manière de conduire de tous les automobilistes. Une partie de cette analyse implique l'utilisation de capteurs mesurant les trajectoires des véhicules sur une portion de route. Deux capteurs innovants ont été crées pour pallier au manque de capteurs suffisamment précis pour ces travaux de recherche : le premier est un capteur à fibres optiques présentant une succession de réseaux de Bragg et le second, protégé par un brevet, est fondé sur une technologie résistive. Le premier repère la déformation locale de fibres optiques noyées à moins d'un centimètre sous la surface de la chaussée. Il utilise la variation de longueurs d'onde engendré par l'extension de la fibre à la zone de contact roue - sol. En utilisant un algorithme adéquat, il est insensible à la température. Le second est constitué de deux conducteurs dont un est résistif. Le poids du véhicule engendre un contact électrique entre les deux conducteurs, transformant la résistance électrique de l'ensemble. Les modèles développés, électrique ainsi que de variation thermique, permettent sa meilleure utilisation. Ils ont été soumis à une expérimentation sur une route départementale. Le capteur optique s'avère plus performant mais coûteux. Le deuxième n'est pas assez robuste mais présente des perspectives intéressantes. / This action stake is not technology for itself. It is a great help the development of new safety functions, e.g. the estimation of driver’s behaviour based uponthe vehicle’s trajectory. This trajectory is determined via two sensors we developed. This system must not disturb the driver and should therefore remain invisible to him. The first one is a fiber Bragg grating (FBG) sensor. It detects local strain due to the vehicle weight. The fiber is embedded in the road thanks to resin used in other traffic sensors. The vehicle location is spotted according to the variations of Bragg wavelengths. The fiber extension located under the ground - wheel contact zone changes the step of the Bragg grating. The second one is based upon two conductors. One of them has a grater electrical resistance. The vehicle’s weight creates a link between the two conductors. The resulting electrical resistance provides a lateral position estimation of the vehicle. Electrical and thermic models and simulation even increase the sensor reliability. A caveat is lodged. Both of them were tested on a secondary road. To put in a nutshell, the FBG sensor gives better results but is very expensive (sensors and interrogator too). The resistance sensor is not much raw nevertheless it has interesting perspectives.

Carbon dioxide sequestration is one of many mitigation tools available to help reduce carbon dioxide emissions while other disposal/repurposing methods are being investigated. Geologic sequestration is the most stable option for long-term storage of carbon dioxide (CO2), with significant CO2 trapping occurring through mineralization within the first 20-50 years. A fiber optic based monitoring system has been proposed to provide real time concentrations of CO2 at various points throughout the geologic formation. The proposed sensor is sensitive to the refractive index (RI) of substances in direct contact with the sensing component. As RI is a measurement of light propagating through a bulk medium relative to light propagating through a vacuum, the extraction of the effects of any specific component of that medium to the RI remains very difficult. Therefore, a requirement for a selective barrier to be able to prevent confounding substances from being in contact with the sensor and specifically isolate CO2 is necessary. As such a method to evaluate the performance of the selective element of the sensor was investigated. Polybenzimidazole (PBI) and VTEC polyimide (PI) 1388 are high performance polymers with good selectivity for CO2 used in high temperature gas separations. These polymers were spin coated onto a glass substrate and cured to form ultra-thin (>10 μm) membranes for gas separation. At a range of pressures (0.14 –0.41 MPa) and a set temperature of 24.2±0.8 °C, intrinsic permeabilities to CO2 and nitrogen (N2) were investigated as they are the gases of highest prevalence in underground aquifers. Preliminary RI testing for proof of concept has yielded promising results when the sensor is exposed exclusively to CO2 or N2. However, the use of both PBI and VTEC PI in these trials resulted in CO2 selectivities of 0.72 to 0.87 and 0.33 to 0.63 respectively, for corresponding feed pressures of 0.14 to 0.41 MPa. This indicates that both of the polymers are more selective for N2 and should not be used in CO2 sensing applications as confounding gas permeants, specifically N2, will interfere with the sensing element. / Graduate / 0428 / 0495 / 0542 / ben.t.davies@gmail.com

First two chapters of this paper deals with the division of optical fiber sensors, digital signal processing and includes price comparison of four experimental sensors. In chapter three analysis, description and evaluation of measurment has been demonstrated on Mach - Zehnder interferometer. Last chapter is about application for signal analysis from vibration sensors. Description contains snippets from source code and graphical user interface. This paper includes three fields - fiber optics, digital signal processing and creation of application - and thanks to them it provides solid basis for study of optic fiber sensors.

The thesis deals with integral and distributed optical fiber sensors of electric current. In the area of integral sensors the analysis is performed by Jones matrix calculus and the simultaneous influence of induced circular and undesired linear birefringence using an ortho-conjugation retroreflector. Furthermore, the proposed conjugation loop variant using half-wave phase retarders is analyzed. The theoretical conclusions are confirmed by simulation and experimental measurement. In the field of distributed fiber optic sensors, analysis and simulation for the detection of plasma currents in thermonuclear fusion reactors, based on the POTDR technique, is performed. An improvement procedure is proposed for circular-shaped vacuum vessel reactors. Plasma current detection procedure in divertor-type reactors have been newly designed and simulated to investigate the effect of the OTDR detector noise on plasma current detection accuracy.