Dissertations / Theses on the topic 'Optical Molecular Sensors'

In this Doctorate Thesis project, a small library of four structurally related melamine-bridge bis(porphyrin-ZnII) receptors, was synthesized The coordination properties of these porphyrin derivatives has been investigated by UV-vis spectroscopy for a series of aliphatic diamines of general formula H2N (CH2)n NH2 (n = 4-8). A marked colour variation occours due to a favourable host-guest ditopic interaction. The binding constants, higher than 10e7 M-1, make these receptors excellent candidates for the development of sensor devices for continuous flow analyses, in which the porphyrin derivatives are covalently supported onto appropriate solid materials. These dimeric metalloporphyrin hosts (molecular tweezers), have also been successfully exploited as chirality probes for determination of the absolute configuration (AC) for a wide variety of chiral molecules (diamines, amino esters, amino amides, amino alcohols and secondary monoalcohols) by using circular dichroism spectroscopy, in collaboration with Prof. Nina Berova of Columbia University. The deposition of porphyrin derivatives on different matrices was also investigated. Characterizations of substrates were carried out by scanning electron microscopy (SEM), atomic force microscopy (AFM) and scanning tunneling microscopy. These results show promising potential in the synthesis of highly ordered networks of surface-supported functional materials for sensing and solar energy applications.
Il progetto di ricerca di questa Tesi di Dottorato ha riguardato la sintesi, la caratterizzazione e lo studio delle proprietà di ricognizione molecolare di nuovi derivati bis-porfirinici contenenti un ponte triazinico. Studi di spettroscopia UV-vis hanno permesso di determinare l’affinità di tali recettori nei confronti delle diammine lineari di formula generale H2N(CH2)nNH2, con n = 4-8. Le costanti di formazione dei complessi host-guest sono molto grandi, fino a 10e7 M-1, grazie all’effetto ditopico realizzato dai due centri porfirinici. La coordinazione delle diammine al dimero porfirinico è associata ad una variazione marcata del colore e questo fatto ha favorito l’impiego di tali derivati in ambito sensoristico. A tal proposito, è stata messa a punto una procedura per supportare i dimeri porfirinici su materiali polimerici per la costruzione di sensori da utilizzare per l’analisi in flusso continuo. Questi derivati sono stati anche utilizzati come pinze molecolari (tweezers) per la determinazione della configurazione assoluta di molecole chirali (diammine, ammino esteri, ammino ammidi e ammino alcoli, monoalcoli secondari) mediante l’impiego della spettroscopia di dicroismo circolare (CD), in collaborazione con la Prof. Berova della Columbia University. Parallelamente, sono stati realizzati studi di deposizione di derivati porfirinici su superfici in vista di applicazioni di tipo sensoristico e in campo energetico (fotovoltaico). La caratterizzazione dei substrati è stata condotta mediante misure di microscopia elettronica a scansione (SEM), microscopia a forza atomica (AFM) e microscopia a scansione ad effetto tunnel (STM). Questi studi di deposizione hanno dimostrato come, scegliendo le opportune condizioni di deposizione, sia possibile costruire dei sistemi ordinati a lungo raggio, su superfici di diversa natura, rendendo questi sistemi candidati ideali per lo sviluppo di nuovi materiali ad alto contenuto tecnologico.

With the increasing importance that nanotechnologies have in everyday life, it is not difficult to realize that also a single molecule, if properly designed, can be a device able to perform useful functions: such a chemical species is called chemosensor, that is a molecule of abiotic origin that signals the presence of matter or energy. Signal transduction is the mechanism by which an interaction of a sensor with an analyte yields a measurable form of energy. When dealing with the design of a chemosensor, we need to take into account a “communication requirement” between its three component: the receptor unit, responsible for the selective analyte binding, the spacer, which controls the geometry of the system and modulates the electronic interaction between the receptor and the signalling unit, whose physico-chemical properties change upon complexation. A luminescent chemosensor communicates a variation of the physico-chemical properties of the receptor unit with a luminescence output signal. This thesis work consists in the characterization of new molecular and nanoparticle-based system which can be used as sensitive materials for the construction of new optical transduction devices able to provide information about the concentration of analytes in solution. In particular two direction were taken. The first is to continue in the development of new chemosensors, that is the first step for the construction of reliable and efficient devices, and in particular the work will be focused on chemosensors for metal ions for biomedical and environmental applications. The second is to study more efficient and complex organized systems, such as derivatized silica nanoparticles. These system can potentially have higher sensitivity than molecular systems, and present many advantages, like the possibility to be ratiometric, higher Stokes shifts and lower signal-to-noise ratio.

With the increasing importance that nanotechnologies have in everyday life, it is not difficult to realize that also a single molecule, if properly designed, can be a device able to perform useful functions: such a chemical species is called chemosensor, that is a molecule of abiotic origin that signals the presence of matter or energy. Signal transduction is the mechanism by which an interaction of a sensor with an analyte yields a measurable form of energy. When dealing with the design of a chemosensor, we need to take into account a “communication requirement” between its three component: the receptor unit, responsible for the selective analyte binding, the spacer, which controls the geometry of the system and modulates the electronic interaction between the receptor and the signalling unit, whose physico-chemical properties change upon complexation. A luminescent chemosensor communicates a variation of the physico-chemical properties of the receptor unit with a luminescence output signal. This thesis work consists in the characterization of new molecular and nanoparticle-based system which can be used as sensitive materials for the construction of new optical transduction devices able to provide information about the concentration of analytes in solution. In particular two direction were taken. The first is to continue in the development of new chemosensors, that is the first step for the construction of reliable and efficient devices, and in particular the work will be focused on chemosensors for metal ions for biomedical and environmental applications. The second is to study more efficient and complex organized systems, such as derivatized silica nanoparticles. These system can potentially have higher sensitivity than molecular systems, and present many advantages, like the possibility to be ratiometric, higher Stokes shifts and lower signal-to-noise ratio.

[ES] La presente tesis doctoral titulada "Detección óptica de especies químicas de importancia ambiental y biológica utilizando sensores moleculares y materiales híbridos" se centra en el diseño, preparación, caracterización y evaluación de sensores químicos moleculares. El trabajo realizado se puede dividir en dos partes: (i) síntesis de sensores de cationes metálicos en disolución y (ii) síntesis y caracterización de nanopartículas híbridas orgánico-inorgánicas para el reconocimiento de especies químicas y biológicas. En el primer capítulo se introduce el marco en el que se engloban los fundamentos teóricos de la química supramolecular en que se basan los estudios prácticos realizados durante la presente tesis doctoral. A continuación, en el capítulo dos, se presentan los objetivos generales de la tesis. En el tercer capítulo se presenta un quimiodosímetro cromo-fluorogénico, capaz de detectar selectivamente cationes trivalentes entre cationes y aniones monovalentes y divalentes mediante una reacción de deshidratación en agua. En el cuarto capítulo se presenta una unidad (BODIPY) conectada electrónicamente con un macrociclo dithia-dioxa-aza. Las soluciones de acetonitrilo y agua-acetonitrilo 95: 5 v / v de la sonda mostraron una banda ICT en la zona visible y son casi no emisivas. Cuando se utilizó acetonitrilo como disolvente, la adición de Hg (II) y cationes metálicos trivalentes indujo un cambio hipsocrómico de la banda de absorción y mejoras moderadas de la emisión. Se obtuvo una respuesta altamente selectiva al utilizar medios competitivos como agua-acetonitrilo 95:5 v/v. En este caso, sólo el Hg (II) indujo un cambio hipsocrómico de la banda de absorción y una mejora marcada de la emisión. El quinto capítulo explora el desarrollo de sensores para berberina y amantadina. Dos moléculas de interés biológico por su uso como fármacos. Se han preparado tres sistemas de sensores basados en la aproximación de puertas moleculares. En concreto, sobre nanopartículas MCM-41 cargadas con rodamina B como unidad de señalización, se ha llevado a cabo la funcionalización con diversas aminas y el bloqueo de poros con cucurbituril CB7. Las aminas utilizadas son ii ciclohexilamina, bencilamina y amantadina. Los materiales obtenidos se han caracterizado por técnicas de difracción de rayos X y microscopía electrónica de transmisión confirmando la estructura mesoporosa de las nanopartículas. Los materiales preparados muestran una respuesta a la berberina y la adamantina, quitando el tapón y liberando el tinte fluorescente al medio. La respuesta de los materiales a las dos sustancias de interés (berberina y amantadina) depende de la estructura química de cada uno de los materiales en función de las constantes de afinidad entre el analito y CB7. Los resultados obtenidos abren el camino al uso de puertas moleculares como sensores de berberina y amantadina.
[CA] La present tesi doctoral titulada "Detecció òptica d'espècies químiques d'importància ambiental i biològica utilitzant sensors moleculars i materials híbrids" se centra en el disseny, preparació, caracterització i avaluació de sensors químics moleculars. El treball realitzat es pot dividir en dues parts: (i) síntesi de sensors de cations metàl·lics en dissolució i (ii) síntesi i caracterització de nanopartícules híbrides orgànic-inorgànics per al reconeixement d'espècies químiques i biològiques. En el primer capítol s'introdueix el marc en el qual s'engloben els fonaments teòrics de la química supramolecular en què es basen els estudis pràctics realitzats durant la present tesi doctoral. A continuació, en el capítol dos, es presenten els objectius generals de la tesi. En el tercer capítol es presenta un quimiodosímetro crom-fluorogénic, capaç de detectar selectivament cations trivalents entre cations i anions monovalents i divalents mitjançant una reacció de deshidratació en aigua. En el quart capítol es presenta una unitat (BODIPY) connectada electrònicament amb un macrocicle dithia-dioxa-aza. Les solucions de acetonitril i aigua-acetonitril 95:5 v/v de la sonda van mostrar una banda ICT a la zona visible i són gairebé no emisivas. Quan es va utilitzar acetonitril com a dissolvent, l'addició de Hg (II) i cations metàl·lics trivalents va induir un canvi hipsocròmic de la banda d'absorció i millores moderades de l'emissió. Es va obtenir una resposta altament selectiva a l'utilitzar mitjans competitius com aigua-acetonitril 95:5 v/v. En aquest cas, només el Hg (II) va induir un canvi hipsocròmic de la banda d'absorció i una millora marcada de l'emissió. El cinquè capítol explora el desenvolupament de sensors per berberina i amantadina. Dues molècules d'interès biològic pel seu ús com a fàrmacs. S'han preparat tres sistemes de sensors basats en l'aproximació de portes moleculars. En concret, sobre nanopartícules MCM-41 carregades amb rodamina B com a unitat de senyalització, s'ha dut a terme la funcionalització amb diverses amines i el bloqueig de porus amb cucurbituril CB7. Les amines utilitzades són ciclohexilamina, bencilamina i amantadina. Els materials obtinguts s'han caracteritzat per tècniques iv de difracció de raigs X i microscòpia electrònica de transmissió confirmant l'estructura mesoporosa de les nanopartícules. Els materials preparats mostren una resposta a la berberina i la adamantina, llevant el tap i alliberant el tint fluorescent a l'mig. La resposta dels materials a les dues substàncies d'interès (berberina i amantadina) depèn de l'estructura química de cada un dels materials en funció de les constants d'afinitat entre l'anàlit i CB7. Els resultats obtinguts obren el camí a l'ús de portes moleculars com a sensors de berberina i amantadina.
[EN] The present doctoral thesis entitled "Optical detection of chemical species of environmental and biological importance using molecular sensors and hybrid materials" focuses on the design, preparation, characterization and evaluation of molecular chemical sensors. The work carried out can be divided into two parts: (i) synthesis of metal cation sensors in solution and (ii) synthesis and characterization of hybrid organic-inorganic nanoparticles for the recognition of chemical and biological species. The first chapter introduces the framework that encompasses the theoretical foundations of supramolecular chemistry on which the practical studies carried out during this doctoral thesis are based. Next, in chapter two, the general objectives of the thesis are presented. In the third chapter, a chromium-fluorogenic chemodosimeter is presented, capable of selectively detecting trivalent cations by means of a dehydration reaction in water. The fourth chapter presents a new compound containing a BODIPY unit electronically connected with a dithia-dioxa-aza macrocycle. Acetonitrile and water-acetonitrile 95:5 v/v solutions of the probe showed an ICT band in the visible zone and were nearly non-emissive. When acetonitrile was used as a solvent, addition of Hg(II) and trivalent metal cations induced an hypsochromic shift of the absorption band and moderate emission enhancements. A highly selective response was obtained when using competitive media such as water- acetonitrile 95:5 v/v. In this case only Hg(II) induced a hypsochromic shift of the absorption band and a marked emission enhancement. The fifth chapter explores the development of sensors for berberine and amantadine; two molecules of biological interest due to their use as drugs. Three sensing systems based on a "molecular gate" approximation have been prepared. Specifically, MCM-41 nanoparticles were loaded with Rhodamine B as a signalling unit, functionalized with various amines and capped with cucurbituril CB7. The amines used are cyclohexylamine, benzylamine and amantadine., The materials obtained were characterized by X-ray diffraction techniques and transmission vi electron microscopy, confirming the mesoporous structure of the nanoparticles. The prepared materials showed a response to berberine and adamantine, which induced release of the fluorescent dye to the medium. The response of the materials to the two substances of interest (berberine and amantadine) depends on the chemical structure of the capping ensemble and it is a function of the affinity constants between the analyte and CB7. The results obtained open the way to the use of gated materials as berberine and amantadine probes.
We thank the Spanish Government (MAT2015-64139-C4-1-R) and Generalitat Valenciana (PROMETEOII/2014/047). M. L. P. thanks Generalitat Valenciana for her Grisolia fellowship. Thanks are also due to Fundação para a Ciência e Tecnologia (Portugal) for financial support to the Portuguese NMR network (PTNMR, Bruker Avance III 400-Univ. Minho), FCT and FEDER (European Fund for Regional Development)-COMPETEQREN- EU for financial support to the research centre CQ/UM [PEst-C/ QUI/UI0686/2013 (FCOMP-01-0124-FEDER-037302)], and a post- doctoral grant to R. M. F. Batista (SFRH/BPD/79333/2011).
Lo Presti, M. (2021). Optical detection of chemical species of environmental and biological relevance using molecular sensors and hybrid materials [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/172664
TESIS

El presente proyecto de investigación está enfocado al desarrollo de sensores químicos fluoro-cromogénicos, para la detección y determinación de especies químicas de interés biológico, industrial y medioambiental de forma selectiva y con alta sensibilidad. En forma general, se busca el diseñar nuevos sistemas sensores basados en compuestos (receptores) formados por dos unidades: una unidad coordinante que interacciona con el anión a determinar y una unidad generadora de señal que alerta del reconocimiento molecular efectuado. Durante este estudio se están preparando diversas moléculas receptoras funcionalizandas con grupos modificadores de estructura para evaluar su influencia sobre las capacidades de detección y selectividad como receptores de especies específicas en diferentes condiciones y medios. Las diferentes aproximaciones en prueba implican a su vez el diseño y síntesis molecular, así como el análisis de las diferentes señales ópticas producidas en el reconocimiento, con el fin de diseñar sistemas de alta eficacia y eficiencia, y con posibilidades reales de aplicación.
Santos Figueroa, LE. (2014). New approaches for the development of chromo-fluorogenic sensors for chemical species of biological, industrial and environmental interest [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/43216
TESIS
Premiado

The identification and quantification of atoms, molecules, or ions concentrations in gaseous samples are in great demand for medical, environmental, industrial, law enforcement and national security applications. These applications require in situ, high-resolution, non-destructive, sensitive, miniature, inexpensive, rapid detection, remotely accessed, real time and continuously operating chemical sensing devices. The aim of this work is to design a miniature optical sensing device that is capable of detecting and measuring chemical species, compatible with being integrated into a large variety of monitoring systems, and durable enough to be used under extreme conditions. The miniature optical sensor has been realized by employing technologies from the optical communication industry and spectroscopic methods and techniques. Fused silica capillary tubing along with standard communication optical fibers have been utilized to make miniature gas sensor based on near-infrared spectroscopy for acetylene gas detection. In this work, the basic principles of infrared spectroscopy are reviewed. Also, the principle of operation, fabrication, testing, and analysis of the proposed sensor are discussed in details.
Master of Science

The advances in nanoscience and nanotechnology in recent decades have renewed the interests in the optical properties of metals. Today, the field known as Plasmonics studies the control and manipulation of the electromagnetic near-fields of metallic nanostructures in order to realize novel subwavelength optical applications. In particular, this thesis explores the phenomenon of plasmon resonance for molecular sensing. Surface plasmon resonance (SPR) on flat metal surfaces is used for index-of-refraction sensing and localized surface plasmon resonance (LSPR) on metal nanospheres can produce surface-enhanced Raman scattering (SERS). The operation principles and the experimental evaluation of two SPR sensing devices are presented. An integrated sensor and a 2D wavelength-angle modulated version were estimated to have an angular sensitivity of 126°/RIU and 91°/RIU, respectively. Furthermore, through an implementation of a full vector multiple-multipole light scattering method, useful for the calculations of the field focusing efficiency between assemblies of metal nanospheres, we showed that optical frequency electric fields can be enhanced in gold nanoparticle assemblies by an order of 450 in nanometer volumes. Keywords: surface plasmons, plasmonics, optical biosensors, surface-enhanced Raman scattering, optics of metals, nanophotonics, nanomaterials, classical electrodynamics
L'avancée des nanosciences et de la nanotechnologie au cours des dernières décennies a suscité un renouvellement de l'intérêt pour les propriétés optiques des métaux. Aujourd'hui, la Plasmonique cherche à contrôler les champs proches électromagnétiques des nanostructures métalliques afin de bénéficier des nouvelles applications reposant sur l'optique de sous-longueur d'onde. En particulier, ce mémoire explore l'utilisation de la résonance à plasmons pour la capture des molécules. L'étude se divise en la résonance à plasmons de surface (SPR) pour des surfaces métalliques planes et pour des nanosphères métalliques. Ces deux méthodes permettent de créer des capteurs sensibles aux variations d'indice de réfraction et d'autres qui reposent sur des effets non-linéaires tels que la diffusion Raman exaltée de surface (SERS). Suite à l'introduction des bases, l'opération ainsi que des résultats expérimentaux de deux capteurs à plasmons de surface sont présentés. Un capteur intégré et une version 2D avec modulation de longueur d'onde et de l'angle possèdent une sensibilité angulaire d'environ 126°/RIU et 91°/RIU, respectivement. Par la suite, la réalisation de la méthode du multiple-multipole, utile pour évaluer l'efficacité de la concentration des champs entre des nanosphères métalliques, est discutée. Une amélioration de la concentration de champ optique de l'ordre de 450 par des nanospheres d'or est présentée. Mots-clés: plasmon de surface, plasmonique, biocapteurs optiques, diffusion Raman exaltée de surface, optique des métaux, nanophotonique, nanomatériel, électro-dynamique classique

Diese Dissertation befasste sich mit der Entwicklung von optischen Sensormaterialien für anionische Zielmoleküle durch die Kopplung der herausragenden Erkennungsfähigkeiten von molekular geprägten Polymere (molecularly imprinted polymers, MIPs) mit der Empfindlichkeit fluorometrischer Nachweisverfahren. In dieser Arbeit wurde dabei der direkte Einschritt-Nachweis für das Design der Sensormaterialien adaptiert. Hierbei wird eine Fluoreszenzsonde für die Signalübertragung kovalent in die Hohlräume der MIP-Matrix eingebaut. MIP-Sensormaterialien wurde in monolithischen, Dünnfilm- und Kern/Schale-Partikel-Formaten hergestellt. Die hergestellten Materialien wurden unter Verwendung unterschiedlicher Techniken charakterisiert. Die Performanz der Sensormaterialien wurde auch in Bezug auf die Sensitivität, Selektivität sowie Ansprechzeit bewertet. In dieser Arbeit wurden dabei Systeme untersucht, bei denen die Signalerzeugung sowohl auf dem „Einschalten“ als auch auf dem „Ausschalten“ der Fluoreszenz beruhte. Mit den hergestellten Materialien wurden dabei viele Ziele des Projekts erreicht. Sowohl die synthetisierten dünnen Filme als auch die Kern/Schale-Partikel zeigten eine hohe Selektivität für die geprägten Aminosäuren, auch in Bezug auf die Unterscheidung von Enantiomeren. Diese Sensormaterialien waren ebenfalls durch eine niedrige Nachweisgrenze bis 60 µM und eine schnelle Ansprechzeit von 20 Sekunden gekennzeichnet. Insbesondere die Kern/Schale-Partikel können mit verschiedenen Detektionstechniken gekoppelt werden und sind potentiell für die Entwicklung von miniaturisierten Messinstrumenten für die on-line-Detektion sowie Point-of-Care-Diagnostik (patientennahe Labordiagnostik) einsetzbar.
This dissertation derives from the DFG project aimed on preparing optical sensor material for anionic target through combing the outstanding recognition of Molecularly imprinted polymer (MIPs) and sensitive fluorescence technique. A single step direct sensing strategy is adopted to prepare the sensor material in this thesis. Here, a fluorescence probe is covalently embedded into the MIP cavity for signal transduction. MIP sensor material are prepared in forms of bulk, thin film and particles. The material is characterized using various techniques. The performance of the sensor materials is also assessed in terms of sensibility, selectivity as well response time. Both the switching on and off signaling methods are tested in this thesis. The prepared material achieves the goal of the project. Both the prepared thin film as well as core-shell particle show prominent selectivity even a strong enantioselective discrimination. These sensing materials also have low LOD to 60 µM and fast sensing response of 20 seconds. Especially the core-shell sensing particle can be coupled with various detection techniques and is potentially applicable for developing miniaturized sensing instrument for on-line detection as well as point of care diagnose.

L’obésité et les maladies cardiométaboliques sont des problèmes de santé publique dans les populations nordiques du Canada ainsi qu’à travers le monde. Il est actuellement proposé que l’augmentation de ces désordres est en partie causée par divers facteurs environnementaux qui génèrent des changements importants du microbiote intestinal. Cette communauté microbienne qui peuple notre tractus gastrointestinal joue un rôle clé dans le métabolisme de nutriments, mais peut aussi avoir des effets néfastes lorsque son équilibre avec l’hôte est perturbé. Cette compréhension a mis en évidence le manque d’outils prédictifs permettant un diagnostic rapide et efficace dans le domaine biomédical. L’analyse actuelle du microbiote est réalisée à posteriori au niveau des selles, ce qui requiert du personnel hautement qualifié de même que des procédures longues et dispendieuses. L’objectif de ce projet est de concevoir un capteur optique qui, une fois implanté dans l’intestin, permettra de détecter en temps réel des biomarqueurs clés produit par le microbiome intestinal. Dans le cadre d’une preuve de concept, une architecture fibrée simple permettant de mesurer quantitativement des variations de pH est démontrée. Contrairement aux capteurs fibrés traditionnels, la sonde optique de ce projet exploite l’onde évanescente générée sur la périphérie de l’interface pour exciter des nanomatériaux greffés dont les propriétés de fluorescence varient selon leur environnement chimique. Les mesures sont possibles grâce à un système optique mobile contrôlé par un logiciel convivial qui permet à un utilisateur nonexpert d’utiliser l’appareil. Les résultats confirment qu’avec un étalonnage préalable il est possible avec cette sonde modèle de prendre des mesures quantitatives du pH en temps réel in vitro. Les expériences préliminaires suggèrent que la sonde permet aussi de mesurer le pH en temps réel dans l’intestin in vivo.
L’obésité et les maladies cardiométaboliques sont des problèmes de santé publique dans les populations nordiques du Canada ainsi qu’à travers le monde. Il est actuellement proposé que l’augmentation de ces désordres est en partie causée par divers facteurs environnementaux qui génèrent des changements importants du microbiote intestinal. Cette communauté microbienne qui peuple notre tractus gastrointestinal joue un rôle clé dans le métabolisme de nutriments, mais peut aussi avoir des effets néfastes lorsque son équilibre avec l’hôte est perturbé. Cette compréhension a mis en évidence le manque d’outils prédictifs permettant un diagnostic rapide et efficace dans le domaine biomédical. L’analyse actuelle du microbiote est réalisée à posteriori au niveau des selles, ce qui requiert du personnel hautement qualifié de même que des procédures longues et dispendieuses. L’objectif de ce projet est de concevoir un capteur optique qui, une fois implanté dans l’intestin, permettra de détecter en temps réel des biomarqueurs clés produit par le microbiome intestinal. Dans le cadre d’une preuve de concept, une architecture fibrée simple permettant de mesurer quantitativement des variations de pH est démontrée. Contrairement aux capteurs fibrés traditionnels, la sonde optique de ce projet exploite l’onde évanescente générée sur la périphérie de l’interface pour exciter des nanomatériaux greffés dont les propriétés de fluorescence varient selon leur environnement chimique. Les mesures sont possibles grâce à un système optique mobile contrôlé par un logiciel convivial qui permet à un utilisateur nonexpert d’utiliser l’appareil. Les résultats confirment qu’avec un étalonnage préalable il est possible avec cette sonde modèle de prendre des mesures quantitatives du pH en temps réel in vitro. Les expériences préliminaires suggèrent que la sonde permet aussi de mesurer le pH en temps réel dans l’intestin in vivo.
Obesity and cardiometabolic diseases (CMD) are major public health issues among Canada’s northern population and throughout the world. It is believed that the exponential rise in CMD incidence is due to numerous environmental factors, which are driving important changes in the gut microbiome. This microbial community which populates our intestinal tract plays a key role in nutrient and energy metabolism, but can also drive pathogenic mechanisms when its interaction with the host is disrupted. This understanding has highlighted the lack of predictive tools and biomarkers for rapid and efficient diagnostic of various diseases within the medical field. Current analysis of the gut microbiota is mostly based on sequencing technologies to determine microbial composition and gene expression, while functional analyses are limited to surrogate markers of microbial activities through stool metabolites. The goal of this study is to develop a “Sensor-in-Fibre” probe with the capacity to detect key microbiome-derived molecules relevant to CMD pathogenesis in real time in vivo. The optical probe takes advantage of evanescent fields generated on its peripheral interface to excite species-selective surface-grafted sensing nanomaterials that have varying fluorescent properties based on the target molecules present in the surrounding environment. As a model system, FITC functionalized with (3-aminopropyl)triethoxysilane was grafted on the periphery of an optical fiber, leading to qualitative pH measurements revealed through fluorescence emission qualities. These measurements are possible due to the use of a mobile signal collection apparatus in conjunction with custom software made to enable a non-expert technician to use it. The experimental results demonstrate that, with the appropriate preparation, it is possible to quantitatively measure pH with this probe structure in vitro and preliminary studies suggest that the probe is also capable of measuring pH in vivo in real time.
Obesity and cardiometabolic diseases (CMD) are major public health issues among Canada’s northern population and throughout the world. It is believed that the exponential rise in CMD incidence is due to numerous environmental factors, which are driving important changes in the gut microbiome. This microbial community which populates our intestinal tract plays a key role in nutrient and energy metabolism, but can also drive pathogenic mechanisms when its interaction with the host is disrupted. This understanding has highlighted the lack of predictive tools and biomarkers for rapid and efficient diagnostic of various diseases within the medical field. Current analysis of the gut microbiota is mostly based on sequencing technologies to determine microbial composition and gene expression, while functional analyses are limited to surrogate markers of microbial activities through stool metabolites. The goal of this study is to develop a “Sensor-in-Fibre” probe with the capacity to detect key microbiome-derived molecules relevant to CMD pathogenesis in real time in vivo. The optical probe takes advantage of evanescent fields generated on its peripheral interface to excite species-selective surface-grafted sensing nanomaterials that have varying fluorescent properties based on the target molecules present in the surrounding environment. As a model system, FITC functionalized with (3-aminopropyl)triethoxysilane was grafted on the periphery of an optical fiber, leading to qualitative pH measurements revealed through fluorescence emission qualities. These measurements are possible due to the use of a mobile signal collection apparatus in conjunction with custom software made to enable a non-expert technician to use it. The experimental results demonstrate that, with the appropriate preparation, it is possible to quantitatively measure pH with this probe structure in vitro and preliminary studies suggest that the probe is also capable of measuring pH in vivo in real time.
Résumé en espagnol
Résumé en espagnol

This thesis aimed at elaborating an optical sensor to detect molecules in a biological fluid. Two steroids and a xenobiotic were identified as biomarkers released in some body fluids: cyproterone acetate, cortisol and 2,4-dichlorophenoxyacetic acid respectively. On one hand, detection was performed by Molecularly Imprinted Polymers (MIPs). These tailor-made synthetic receptors display numerous qualities that foster their integration in sensors. MIPs were therefore developed against the targeted analytes. Formulation optimization was led thanks to experimental designs. On the other hand, optical transduction was made possible thanks to the structuring of a polymer into a photonic crystal. Opals were manufactured with a new process suitable for large scales and were used to mold MIPs in inverse opals. Thus, submicron structures of the polymer are responsible for the color of the sensor. A change of color is triggered by the recognition of the analyte by the polymer (upon swelling). Polymers studied displayed sufficient swelling observed by spectrophotometry. Finally, the work of this thesis consisted in elaborating polymer formulations and their integration in a sensor so as to detect an analyte with direct, rapid and unobtrusive means.

This thesis demonstrates comprehensive research on novel sensors of multifunctional applications. It presents novel materials (dye-coated paper strips, functionalized gold nanoparticles/gold nanostructured substrates, antibody-coated conductive polymers, SERS tags) for the determination of bioactive molecules. A novel thiol chemistry approach, bioconjugation of recognition molecules, and surface functionalizations were developed for the ultrasensitive detection of target analytes. The newly developed materials, multimodal sensors (optical, fluorescence, SERS, electrochemical) of high sensitivity and selectivity were utilised and validated for the rapid screening/quantification of bioactive molecules (diseases biomarkers, amino acids, and toxic substances) in biological and environmental matrices.

Cette thèse est organisée en trois chapitres basés sur trois publications et deux manuscrits en cours de soumission. Les polymères à empreintes moléculaires (MIP) sont des récepteurs produits à façon qui sont obtenues par polymérisation en présence de molécules modèles. Le premier papier décrit l'utilisation de la spectroscopie Raman pour la détection et la quantification de molécules modèles au sein d'une particule unique de polymères a empreintes moléculaires de taille micrométrique. Les particules de polymères ont eté imprimées avec comme molécules modèles S-propranolol. Des particules de taille nanométrique et micrométrique ont été respectivement utilisées pour une analyse en masse et sur particule unique. La quantification de molécules cibles est possible sur particule unique et une limite de détection de 1µM a été obtenue sur l’analyse en masse. Le second papier décrit un capteur chimique de taille nanométrique composé d’une particule en core-shell composite. Cette particule comprend un cœur polymérique, une enveloppe inorganique composée de nanoparticules d’or pour l’exaltation du signal, recouvert d’une couche de polymères à empreintes moléculaires pour la reconnaissance. La méthode d’analyse utilisée est une exaltation su signal Raman appelé « surface enchanced Rama scattering ». Ces particules ont aussi été utilisées comme capteur avec la résonance plasmonique localisé comme méthode de transduction (papier n°3). Afin de résoudre certains problèmes associés aux polymères à empreintes moléculaires, tels que l’inhomogénéité en terme de morphologie et de distribution en site de reconnaissance, il a été suggéré d’utiliser des méthodes de polymérisations radicalaire contrôlées. Ces méthodes facilitent aussi leurs synthèses sous forme de nanomatériaux, nanocomposites et films minces. Le quatrième papier considère les récents avantages et inconvénients de ces nouvelles méthodes de polymérisation rapportés aux polymères à empreintes moléculaires. Le dernier papier décrit la première utilisation d’un récente méthode basée sur la polymérisation par transfert à l’iode (ITP), « reversible chain transfer catalyzed polymerization » (RTCP) adapté aux polymères à empreintes moléculaires. Nous décrivons un exemple de synthèse de MIP spécifiques pour la molécule propanolol par RTCP, produisant des polymères sous forme de « bulk » ou particules ayant la même capacité de reconnaissance que ceux synthétisés par polymérisation radicalaire (FRP). La RMN à l’état solide a révélée que la conversion en double liaison intramoléculaire était plus élevée dans le cas de polymères synthétisés par RTCP que par FRP, en particulier à haute concentration en amorceur
This thesis is organized in three chapters and is based on three published papers, and two manuscripts about to be submitted. Molecularly imprinted polymers (MIPs) are tailor-made synthetic receptors that are obtained by polymerization in the presence of a molecular template. The first paper describes the use of Raman spectroscopy to detect and quantify the presence of the imprinting template in single molecularly imprinted polymer microspheres. The polymers were imprinted with the Beta-blocking drugs propranolol and atenolol, and precipitation polymerization was used to obtain spherical particles. The nanoparticles were used for bulk detection whereas with micrometer-sized particles, quantitative measurements on single particles were possible. Relatively low detection limits down to 1µM have been reached for the detection of S-propranolol through bulk measurements on MIP nanoparticles. The second paper describes chemical nanosensors with a submicron core-shell composite design, based on a polymer core, a molecularly imprinted polymer (MIP) shell for selective analyte recognition, and an interlayer of gold nanoparticles for signal amplification. SERS measurements on single nanosensors yielded a detection limit of 10-7 M for the Beta-blocker propranolol, several orders of magnitude lower than on plain MIP spheres. These particles were also used as sensor materials with localized surface plasmon resonance measurements as the transduction method (Paper III), for the determination of the Beta-blocking drug propranolol. The sensors were used in suspension and were measured using a standard UV-Vis spectrophotometer. In order to solve general problems associated with MIPs, in particular their heterogeneity in terms of inner morphology and distribution of binding site affinities, it has been suggested to use modern methods of controlled/living radical polymerization for their synthesis. This also facilitates their generation in the form of nanomaterials, nanocomposites, and thin films, a strong recent trend in the field. The fourth paper reviews recent advances in the molecular imprinting area, with special emphasis on the use of controlled polymerization methods, their benefits, and current limitations. In the last paper, we have for the first time used a recently developed CRP method based on iodide mediated polymerization, reversible chain transfer catalyzed polymerization (RTCP), for the synthesis of MIPs. We show on the example of MIPs specific for the Beta-blocking drug propranolol that RTCP is compatible with MIP synthesis, both for the synthesis of bulk polymers and nanospheres, and that it yields polymers with the same binding capacity as the standard FRP method used for comparison. Solid-state NMR measurements revealed that the conversion of pendant vinyl groups was higher with RTCP than with polymers synthesized by FRP, in particular at higher initiator concentrations

The scientific and social increasing demand for quantitative/qualitative determination of natural and artificial chemical species in different sector of human life (health, living environment, food, etc.) highlights the need for new strategies and new measurement methods. In particular, the agro-food industries need quick and sensitive tools to identify molecules in its final products or to monitor them during production processes. This need is due to the increase of food frauds. Indeed, in last decades, adulteration and falsification of food become a very serious problems also as regard human health. Biosensors are cutting-edge and the cheapest tools that can satisfy the monitoring of food quality because they represent an intelligent combination of biological components, such as enzymes or bacteria, and technological components that detect physical and chemical changes and transmit them in the form of data. Among agro-food chain products, wine plays an important role in world trade. Wine is one of the most appreciated beverages and it is a complex mixture of sugars, acid and odours that concourse to give it characteristic flavour. Concerning flavours, specific volatile organic compounds (VOCs) in wine are able to give it fruity o floral aroma. These VOCs belong to terpenes class (monoterpenes and sesquiterpenes) and they all together contribute to wine aroma. Analysis of wine aroma is at the base of quality assessment and physiochemical parameters are important to be monitored in order to limit frauds or adulterations. In an automated food production system, devices as biosensors are important to monitor and maintain product quality and uniformity based on aroma characteristics. The nature is the principal source to discover new molecules that could be used to realize biosensors. In this context, insects represent the main source from which to be inspired because they constitute the largest animal phyla. Insects have an important part in all ecosystems considering their role in pollination, biological control and bioconversion. They are a resource of genes, molecules and processes that can inspire several technological innovations. Considerably sophisticated in insect is the “chemoreception” that is a process by which organisms respond to chemical stimuli in their environments that depends primarily on the senses of taste and smell. Chemoreception plays an essential role throughout the life cycle of insects that respond to different arrays of chemical, biological and environmental signals to locate and select food, mates, oviposition sites and avoid predators. To interpret these signals, insects use a range of molecular components that are located in specialized structures called chemosensilla. The last ones are characterized by a variety of forms and by one or more pores on their surface, that allow the access of molecules to the internal aqueous phase, the sensillar lymph, which surrounds the dendrites of sensory neurons. In the sensillar lymph, a large amount of small soluble proteins, Odorant Binding Proteins (OBPs) and Chemosensory Proteins (CSPs) are present. Some aphids OBPs are able to bind the terpene (E)-β-Farnesene that it is used as alarm pheromone. In Acyrthosiphon pisum, OBP3 and OBP7 are able to bind (E)-β-Farnesene and they are responsible of alarm response. Recently, Acyrthosiphon pisum OBP9 was found to be involved in perception of this compound. Even Megoura viciae use (E)-β-Farnesene as alarm pheromone and its perception is mediate by OBP3. Acyrthosiphon pisum OBP3 are able to bind also farnesol, structurally related to (E)-β-Farnesene and Megoura viciae OBP3 is able to bind limonene. Farnesol and limonene are listed as wine terpenes. Starting from this consideration, aphids OBPs were produced through recombinant procedures and used to develop a biosensor able to bind terpenes in wine. Beside farnesol and limonene, already tested with aphids recombinant proteins, others wine terpenes were tested with Acyrthosiphon pisum OBP3 and Megoura viciae OBP3. A preliminary in silico analysis of aphid OBPs, including also Acyrthosiphon pisum OBP9 and OBP7, was conducted to select other terpenes useful for a rational design of the biosensor. Terpenes selected were geraniol, nerol and citronellol. Concerning biosensor assembly, the choice was to produce optical silicon biosensors. Optical sensors are more versatile than others because they can be made from different materials (silicon, glass, polymers, etc.) and among materials, silicon has gained attention and popularity in recent years because of its ease fabrication, special optical properties and its versatile surface chemistry. Moreover, silicon is readily available, low cost and biocompatible. Acyrthosiphon pisum OBP3 was the first protein used to optimize the optical biosensor assembly protocol because of its previous functional characterization. For biosensor development, Porous Silicon (PSi) surfaces were used and the flow sensing was used as a strategy to verify the behavior of sensing in real time. OBP was adsorbed onto oxidized surface simply flowing the protein on surface and farnesol was tested in different concentration (range: 10 μM – 400 μM). Even if results are not conclusive, they are encouraging considering that terpenes are very small molecules that give a small change in the refractive index.

This thesis describes the development of highly selective fiber optic sensors using molecularly imprinted polymers (MIPs) as recognition elements associated with fluorescence for detection. Additionally, we extended the study to the development of other MIP-based optical sensors and sensing methods. MIPs are synthetic biomimetic receptors possessing specific cavities designed for a target molecule. Produced by a templating process at the molecular level, MIPs are capable of recognizingand binding target molecules with selectivities and affinities comparable to those of natural receptors. Compared to biological recognition elements, MIPs are more stable, cheaper and easier to integrate into standard industrial fabrication processes. Hence, MIPs have become interesting alternatives to biomolecules as recognition elements for biosensing. In the first part of this thesis (Chapter 2), MIPs were synthesized by in-situ laser-induced photopolymerization in only a few seconds, as a micrometer-sized tip at the extremity of a telecommunication optical fiber. Photonic and physico-chemical parameters were optimized to tailor the properties of the polymer micro-objects. Gold nanoparticles were incorporated into the MIP microtip for signal enhancement. To prove the efficiency of the sensor, initial studies were performed with a MIP templated with N-carbobenzyloxy-L-phenylalanine (Z-L-Phe) and the fluorescent amino acid derivative dansyl-L-phenylalanine as analyte. The fluorescence was collected either externally at the tip level by an optical fiber connected to a spectrofluorimeter or by collection of the fluorescent signal re-emitted into the fiber through the second arm of a Y-shaped bifurcated fiber. The fluorescent analyte could be detected in the low nM concentrations. In order to monitor nonfluorescent analytes, a naphthalimide-based fluorescent monomer was incorporated into the MIP during its synthesis; fluorescence enhancement was observed when analyte binding occurs. Using this system, the sensor containing a MIP specific for the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), could detect and quantify this analyte at concentrations as low as 2.5 nM. The signaling MIP-based sensor was also applied to analytes of interest for food safety and biomedical applications, such as the mycotoxin citrinin and the sphingolipid, D-erythro-sphingosine-1-phosphate. In the second part of the thesis (Chapter 3), a different type of fiber optic sensor: cheap, fast and made for "single-use", was developed by using 4-cm long disposable polystyrene evanescent wave optical fiber waveguides. The coating of the MIP was either performed ex-situ, by dip-coating the fiber in a suspension of MIP particles synthesized beforehand, or in-situ by evanescent-wave photopolymerization directly on the fiber. The resulting fiber optic sensor could detect 2,4-D in the low nM range and demonstrated specific and selective recognition of the herbicide over its structural analogues and other non-related carboxyl-containing analytes. Additionally, we demonstrated the versatility of the system by applying the evanescent wave fiber optic sensor to detect citrinin, a mycotoxin, by simply coating the waveguide with a MIP specific for citrinin. This type of technology could possibly be extended to detect other carboxyl-containing analytes, as long as a specific MIP for the concerned analyte is available. In parallel, the technique of evanescent-wave photopolymerization was used for the synthesis of signaling MIP microdots on continuous and nanostructured gold films. This study lays the foundations for future development of plasmonic MIP nanosensors and microchips. In the last part of the thesis (Chapter 4), an innovative sensing method, based on the use of MIPs and analysis by fluorescence polarization, was developed in order to allow the fast and directquantification of analytes in food and environmental samples.

The work embodied in this dissertation is specifically focused on the evanescent interaction of light with thin-films which has lead to two related instrument based projects: i) the Electroactive Fiber-Optic Chip (EA-FOC) and ii) Attenuated Total Reflectance (ATR) spectroscopy of novel materials. The EA-FOC combines the sensitivity of an electroactive total internal reflection element (20 to 50 times more sensitive than a transmission experiment) with the ease of use of fiber-optic based CCD spectrometers. A side-polished optical fiber, in a V-groove glass mount, forms the planar platform, which allows access to the evanescent field escaping from the fiber core. The exposed evanescent field, which was used to probe molecules or molecular assemblies supported by the platform, has an interaction area ca. 0.05 cm squared. Thin-film and bulk absorbing samples, and waveguide modeling calculations were initially used to evaluate the sensitivity of the FOC platform, which was found to be analogous to ATR instrumentation. The wavelength range of the FOC platform was increased to include the near-UV and applied to monitor adsorption of a protein film. Fluorescence applications of the FOC were demonstrated using a fluorescence bioassay and a drop cast nanoparticle film. Finally, a transparent conducting oxide film, ITO, was added to the surface of the platform to complete the EA-FOC for spectroelectrochemical applications. A methylene blue redox couple and an electrodeposited ultra-thin PEDOT film were used to probe the capabilities of the EA-FOC. The EA-FOC was shown to be a multifunctional platform for advanced sensor technologies requiring absorbance, fluorescence, and electrochemical detection or a combination thereof.ATR spectroscopy of novel materials included the evaluation of two architectures: i) a pH sensitive polyelectrolyte film and ii) surface capture of a nanoparticle film. Absorbance spectra of a polyaniline/polyacetic acid self-assembled bilayer were evaluated with respect to pH and potential using ATR spectroscopy; the ultimate application of the polymer signal transduction layer was to monitor proton transport across a lipid-bilayer. Additionally, ATR spectroscopy was used to monitor adsorption of pyridine capped nanoparticles on a silyl-propyl-thiol modified surface.

Für den Nachweis dieser Schadstoffe in niedrigen Konzentrationsbereichen sind schnelle und empfindliche Analysemethoden erforderlich. Molekular geprägte Polymere (MIPs) wurden als synthetische Materialien entwickelt, um die molekulare Erkennung von natürlichen Rezeptoren nachzuahmen, aufgrund ihrer Fähigkeit, selektiv eine Vielzahl von Analyten zu erkennen, ihre Stabilität und ihrer einfachen Herstellung. Sie sind zunehmend in der chemischen Sensorik als Rezeptormaterial für den Nachweis bestimmter Analyten bei niedrigen Konzentrationen zu finden, insbesondere in Kombination mit Fluoreszenz aufgrund dessen hoher Empfindlichkeit. Ziel dieser Arbeit war die Entwicklung von optischen Sensormaterialien unter Verwendung von MIPs als Erkennungselemente im Zusammenhang mit Fluoreszenz zum sensitiven Nachweis von Herbiziden und Antibiotika in Wasser- und Lebensmittelproben and deren Kombination mit verschiedenen Vorrichtungsformaten für die zukünftige Detektion einer breiten Palette von wichtigen Analyten.
For the detection of these contaminants in low concentration ranges fast and sensitive analytical tools are required. Molecularly imprinted polymers (MIPs) have been used as synthetic materials mimicking molecular recognition by natural receptors due to their ability to recognize selectively a wide range of analytes, their stability and ease of synthesis. They have gained more and more attention in chemical sensing as receptor material for the detection of suitable groups of analytes at low concentrations especially in combination with fluorescence due to the latter’s high sensitivity. This work aimed the development of optical sensor materials using MIPs as recognition elements connected with fluorescence for the sensitive detection of herbicides and antibiotics in water and food samples and their combination with various device formats for the future detection of a wide range of analytes.

Philosophiae Doctor - PhD
Electroanalytical and optical properties of nanoscale materials are very important for biosensing applications as well as for understanding the unique one-dimensional carrier transport mechanism. One-dimensional semiconductor nanomaterials such as semiconductor quantum dots are extremely attractive for designing high-density protein arrays. Because of their high surfaceto-volume ratio, electro-catalytic activity as well as good biocompatibility and novel electron transport properties make them highly attractive materials for ultra-sensitive detection of biological macromolecules via bio-electronic or bio-optic devices. A genosensor or gene based biosensor is an analytical device that employs immobilized deoxyribonucleic acid (DNA) probes as the recognition element and measures specific binding processes such as the formation of deoxyribonucleic acid-deoxyribonucleic acid (DNA-DNA), deoxyribonucleic acid- ribonucleic acid (DNA-RNA) hybrids, or the interactions between proteins or ligand molecules with DNA at the sensor surface.In this thesis, I present four binary and two ternary-electrochemically and optically modulated selenide and telluride quantum dots, all synthesised at room temperature in aqueous media. Cationic gallium (Ga3+) synthesized in form of hydrated gallium perchlorate salt[Ga(ClO4)3.6H2O] from the reaction of hot perchloric acid and gallium metal was used to tailor the optical and electrochemical properties of the selenide and telluride quantum dots. The synthesized cationic gallium also allowed successful synthesis of novel water soluble and biocompatible capped gallium selenide nanocrystals and gallium telluride quantum dots. Cyclic voltammetric studies inferred that presence of gallium in a ZnSe-3MPA quantum dot lattice improved its conductivity and significantly increased the electron transfer rate in ZnTe-3MPA.Utraviolet-visible (UV-vis) studies showed that incorporation of gallium into a ZnSe-3MPA lattice resulted in a blue shift in the absorption edge of ZnSe-3MPA from 350 nm to 325 nm accompanied by decrease in particle size. An amphiphilic bifunctional molecule, 3-Mercaptopropionic acid (3-MPA) was used as a capping agent for all quantum dots. It was found that 3-MPA fully solubilised the quantum dots, made them stable, biocompatible, non agglomerated and improved their electron transfer kinetics when immobilized on gold electrodes.Retention of the capping agent on the quantum dot surface was confirmed by Fourier transform infrared spectroscopy (FTIR) which gave scissor type bending vibrations of C-H groups in the region 1365 cm-1 to 1475 cm-1, stretching vibrations of C=O at 1640 cm-1, symmetric and asymmetric vibrations of the C-H in the region 2850 cm-1 to 3000 cm-1 as well as stretching vibrations of –O-H group at 3435 cm-1. The particle size and level of non-agglomeration of the quantum dots was studied by high resolution transmission electron microscopy (HRTEM). The optical properties of the quantum dots were studied using UV-vis and fluorescence spectroscopic techniques.Quantum dot/nanocrystal modified gold electrodes were prepared by immersing thoroughly cleaned electrodes in the quantum dot/nanocrystal solution, in dark conditions for specific periods of time. The electrochemical properties of the modified electrodes were characterized by cyclic voltammetry (CV), square wave voltammetry (SWV), electrochemical impedance and spectroscopy (EIS). Six sensing platforms were then prepared using quantum dot/nanocrystal, one of which was used for detection of dopamine while the rest were used for detection of a DNA sequence related to 5-enolpyruvylshikimate-3-phosphate synthase, a common vector gene in glyphosate resistant transgenic plants.The first sensing platform, consisting of ZnSe-3MPA modified gold electrode (Au|ZnSe-3MPA) gave rise to a novel method of detecting dopamine in presence of excess uric acid and ascorbic acid. Using a potential window of 0 to 400 mV, the ZnSe-3MPA masked the potential for oxidation of uric and ascorbic acids, allowing detection of dopamine with a detection limit of 2.43 x 10-10 M (for SWV) and 5.65 x 10-10 M (for steady state amperometry), all in presence of excess uric acid (>6500 higher) and ascorbic acid (>16,000 times higher). The detection limit obtained in this sensor was much lower than the concentration of dopamine in human blood(1.31 x 10-9 M), a property that makes this sensor a potential device for detection of levels of dopamine in human blood.The other sensing platforms were prepared by bioconjugation of amine-terminated 20 base oligonucleotide probe DNA (NH2-5′-CCC ACC GGT CCT TCA TGT TC-3′) onto quantum dot modified electrodes with the aid of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The prepared DNA electrodes were electrostatically hybridized with different sequences which included 5′-GAA CAT GAA GGA CCG GTG GG-3′ (complementary target), 5′-CATAGTTGCAGCTGCCACTG-3′ (non complementary target) and 5′-GATCATGAAGCACCGGAGGG-3′ (3-base mismatched target).The hybridization events were monitored using differential pulse voltammetry (DPV) and SWV by monitoring the guanine oxidation signal or using EIS by monitoring changes in the charge transfer resistance. The quantum dot genosensors were characterized by low detection limits (in the nanomolar range), long linear range (40 - 150 nM) and were able to discriminate among complementary, non-complementary and 3-base mismatched target sequences.

Optical tools to control neuronal activity include synthetic photoswitchable ligands of receptors and ion channels. Photoswitches can act either as soluble molecules (photochromic ligands, PCLs) or tethered to the protein (photoswitchable tethered ligands, PTLs), and they have been used to photocontrol many ion channels and receptors such as voltage-gated potassium channels, acetylcholine or glutamate receptors. Recognizing both the need for new optical tools in neuroscience and the opportunities offered by photoswitches, this work is focused on the use of light gated glutamate receptors to control neuronal activity and neurotransmission. In the first chapter of results of the thesis, we demonstrate that the Ca2+-permeable LiGluR can be used as a tool to reversibly control neurosecretion by directly affecting the intracellular [Ca2+]. To achieve this goal, LiGluR was expressed in cultured bovine chromaffin cells and cultured hippocampal neurons. We measured secretion in chromaffin cells using two techniques, amperometry and membrane capacitance, and current-clamp recordings to assess neurotransmission in cultured neurons. The results indicated that the magnitude of LiGluR-mediated Ca2+ influx is sufficiently large to trigger regulated exocytosis in chromaffin cells and neurons. In addition, LiGluR induced secretion can be modulated with the wavelength of illumination. This new application of LiGluR opens the possibility to reversibly control the activity of individual synapses, which might help to understand the computational properties of neurons and to unravel how brain circuits work. To use LiGluR as an effective method to interrogate the neuronal function it should support high-spatial 3D resolution and tissue penetration. Multiphoton excitation with near-infrared light enables stimulation in intact tissue with cellular and subcellular resolution, and it has been extensively applied to optical actuators such as caged compounds and more recently to optogenetics. However, two-photon stimulation of synthetic photoswitches had not been explored before. In the second section of the results, the two-photon stimulation of LiGluR is investigated. Two new photoswitches were designed (MAG2p and MAGA2p) based on the structure of the original photoswitch (MAG) and intended to enhance the two-photon absorption ability of the azobenzene switch. The three PTLs, including MAG, successfully activate LiGluR under two-photon stimulation, suggesting that multiphoton excitation can be applied to other azobenzene-based molecules. Interestingly, the rationally designed photoswitches were more efficient in opening LiGluR as lower power and shorter simulation time were required. Finally we validated MAG2p and MAG as new tools to control the activation of neurons and astrocytes with cellular and subcellular resolution. In the last chapter, a new method based on the affinity labeling approach is presented in order to confer light sensitivity to endogenous receptors. Glutamate-azobenzene-reactive PTLs with different lengths and reactive groups were tested on kainate receptors. These reactive PTLs successfully and functionally conjugate to the ionotropic glutamate receptor subunit GluK1, thus enabling to photoswitch its activity, as evidenced from photocurrent recordings of mammalian cells overexpressing the non-mutated receptor. These results are also supported by the photocontrol of GluK1 currents in dorsal root ganglion neurons, where GluK1 is the main glutamate subunit that is endogenously expressed. The new strategy proposed is versatile and we suggest that it can be extended to label other endogenous receptors, giving rise to novel optpharmacological therapies. The new methods here developed improve the LiGluR performance, and address photoswitches to use endogenous neuronal receptors to optically control neuronal activity, being able to stimulate them in small volumes corresponding to the neuronal functional unit (i.e. synaptic terminals). In this way, light would emerge as a unique tool to dissect neuronal physiology and to understand the function of neuronal circuits.
L’estudi de la neurotransmissió requereix noves eines moleculars, i els fotocommutadors ofereixen grans possibilitats. Aquesta tesi està centrada en l’ús de receptors de glutamat activables per llum (LiGluRs) pel control de l’activitat neuronal i dels processos de neurosecreció. Al primer bloc de resultats, la permeabilitat a Ca2+ dels receptors de glutamat s’aprofita per manipular de manera directa -independentment del voltatge de membrana- i reversible la concentració intracel•lular de Ca2+ amb llum. Així, és possible desencadenar els processos de secreció en cèl•lules cromafins i de neurotransmissió en neurones hipocampals. A la segona part dels resultats de la tesi, s’investiga l’estimulacio multifotó del receptor de glutamat modificat químicament amb fotocommutadors basats en l’azobenzè. Els resultats mostren l’estimulació per dos fotons del LiGluR, incloent-hi dos fotocommutadors nous, que milloren l’absorció multifotó del commutador azobenzè. Finalment, s’aplica aquesta tècnica per estimular neurones i astròcits amb una resolució a l’escala d’una cèl•lula o de compartiment subcel•lular. Al tercer capítol dels resultats, es descriu un nou mètode per aconseguir el fotocontrol de receptors neuronals endògens, utilitzant lligands covalents. Amb l’aplicació d’aquest mètode es pot fotocommutar l’activitat del receptor de kainat subtipus 1 (GluK1) no mutant, quan se sobreexpressa el receptor en cèl•lules de mamífer. Aquesta estratègia també permet obtenir fotocorrents en cultius de neurones dels ganglis de l’arrel dorsal, on GluK1 és la subunitat de glutamat endògenament més expressada. Els nous mètodes desenvolupats en aquesta tesi milloren la utilització dels LiGluRs, encaminant l’ús dels fotocommutadors cap al control òptic dels receptors endògens, amb la possibilitat d’estimular cèl•lules individuals o estructures subneuronals, fet que situaria els fotocomutados com a eines indispensables per l’estudi del cervell, des de la fisiologia fins als circuits neuronals.

"With the increased exposure to antibiotics and the matching selective pressure that it imposes, bacteria naturally have evolved to develop resistance to several antibiotics and, in the worst cases, multi drug resistance. This seriously affects the treatment of bacterial infections and the problem worsens when considering that the development of new antibiotic molecules is increasingly difficult and costly. Consequently, alternative antibacterial substances are being increasingly sought out, where antimicrobial lipids are being pointed as a promising solution. Fatty acids and monoglycerides are natural substances that possess antimicrobial properties, capable of destabilizing cell membranes, while causing several direct and indirect inhibitory effects, such as impairment of nutrient uptake, generation of peroxidation and auto-oxidation degradation products or direct lysis of bacterial cells and inhibition of enzyme activity. More importantly, evolution of resistant phenotypes is increasingly difficult. "

Measurement of molecular interactions is essential for fundamental biological studies as well as for the development of health diagnostics devices. Several label-free and labeled techniques to characterize and quantify molecular interactions have been developed. A major disadvantage of the labeled techniques is the requirement of an additional process step of labeling that increases the system complexity for completely automated systems. There are many label free molecular detections techniques such as electrical (nano wires and nano tubes based), mechanical (cantilever and quartz crystal micro balance based), electrochemical and optical (interferometry, waveguides, optical cavities and surface plasmon resonance (SPR) based). The requirements of an ideal bio-molecular sensing technique are a) cost effective, portable and simple to operate, b) high sensitivity and specificity, c) high reproducibility, d) real time measurement capability and e) ability to do parallel measurements in a Lab-on-Chip format. The goal of the work done in this thesis is to design an optical real time bio molecular sensing technique that should be cost effective and highly sensitive. In this work we describe the use of self-referenced measurement techniques to eliminate common mode signal drifts due to thermal and concentration gradients in the fluid flow cell and demonstrate a nearly 40-fold decrease in the system noise. It started with a reflectance modulation point by point scanning system. Polyelectrolytes (poly allylamine hydrochloride (PAH) and poly acrylic acid (PAA), negatively and positively charged polymers) microarray was created on a SiO2 grown silicon substrate. Reflectance modulation was analyzed from the SiO2 surface and polyelectrolyte microarray on the SiO2. But the limit of detection (LoD) was poor due to laser intensity fluctuations. Therefore, a self-referencing diffractive reflectance modulation system (DRMS) was proposed. There, diffractive microstructures were fabricated in the SiO2 layer on top of silicon substrate and created a flow cell on the diffractive microstructures. When these diffractive microstructures are illuminated by a laser beam, they produce a diffraction pattern in reflected and transmitted light. In the diffraction pattern the zeroth contains average background information whereas higher orders contain signal information of the liquid sample present on the microstructures. By using Si/SiO2 devices we demonstrated bulk (refractive index sensing for water ethanol mixtures) and surface adsorption (real time binding of polymer layers on micro structured Si/SiO2 devices) molecular detection measurements. Instead of doing bio molecular detection measurements on Si/SiO2 devices we started doing measurements on glass devices with the advantages, flexibility to do measurement in transmission and lower substrate cost. In the next part of the thesis, bulk and surface adsorption measurements were demonstrated for glass substrates. For this, a 2D periodic array of wells of 10-micron diameter was created in the glass cover slide with various well depths. A flow cell was constructed on the diffractive micro structured cover slide to inject liquid samples on the microstructures, and this micro patterned glass cover slide was used as our sensor device for bulk and surface detection measurements. With the glass sensor devices bulk and surface adsorption bio molecular detection measurements were demonstrated both in transmission and reflection. To demonstrate the bulk detection, we measured the refractive index of different concentrations of NaCl in water and for bulk measurements a limit of detection (LoD) of 3x10-6 RIU was achieved. For surface adsorption measurements, layer by layer (LBL) deposition of positively (PAH) and negatively (PAA) charged polymers was demonstrated at the glass micro array sensor surface. The experimental data were validated with the model developed using transfer matrix method in chapter 4. Bio-molecular detection measurements were not demonstrated with this technique because this technique is in very preliminary stage of development. This technique needs more work to be done for standardization and optimization of appropriate conditions of detection protocols for biological samples. Large number of trials are necessary to standardize this technique under various control conditions because detection of proteins and DNA samples will add a whole new dimension of complexities depending upon size, coverage, and orientation of biological samples. The same diffractive self-referencing concept can be applied for SPR also. For this, grating structures available on commercial DVDs were used for the excitation of surface plasmons. On these DVD samples, metal dielectric multi layers were coated and demonstrated to couple surface plasmons and give multiple SPR peaks in transmission as a function of incidence angle. By using diffractive self-referencing concept an intensity referenced SPR method was demonstrated for bio molecular detection measurements. With the intensity referenced SPR as a proof of concept for bulk measurements refractive index detection of different concentration of NaCl in water was demonstrated. Similarly, for surface adsorption measurements, polyelectrolytes PAH and PAA were used for the functionalization of surface and detection of Bovine Serum Albumin (BSA) was demonstrated. Only very preliminary experiments were done to demonstrate that the ratio metric self-referencing concept can be exploited in grating coupled SPR also for bulk and surface bio-molecular detection measurements. This technique also needs many trials and repetitions for the standardization and optimization for use with biological samples. A self-referencing method was introduced and demonstrated, and this method was applied to two different optical label-free sensing techniques, namely, interferometric reflectance modulation and SPR. As a proof of concept for bulk detection, real time refractive index measurements were done for different concentrations of ethanol and NaCl in water and for surface adsorption detection, real time layer-by-layer (LBL) assembly of polyelectrolytes PAH and PAA was demonstrated on Si/SiO2 and glass micro array sensor surfaces. These real time self-referencing techniques namely DRMS and intensity referenced SPR need lot more work to be done to realize their true potential for bio-molecular detection measurements. The performance of these techniques was studied from the point of view of simplicity, cost effectiveness, sensitivity, and robustness.

Thin film photovoltaic materials have garnered much interest recently due to their processability in addition to good properties for conversion of solar photons to usable energy. Amine-thiol chemistry has shown the ability to produce solution processed materials such as Cu₂ZnSn(S,Se)₄ (CZTSSe), a thin film absorber composed of earth abundant metals. Using similar solution processing methods as those used to produce CZTS, we wish to synthesize a phase pure solution processed material from molecular precursors of metals and metal chalcogenides into an Ag₂ZnSnSe₄ absorber which lacks the electronic defects that plague CZTSSe. Additionally, we will utilize the reactive dissolution of metal in amine-thiol solution chemistry for a more detailed understanding of how metal-sulfur complexes form and then decompose into films, to gain insight about the conditions that produce stable solutions and high quality films for a better ability to optimize processing conditions.

We find we are able to individually dissolve zinc metal, tin metal, and silver sulfide precursors to produce solutions of metal thiolate complexes. Based on results from electrospray ionization mass spectrometry (ESI-MS), proton nuclear magnetic resonance (¹H-NMR), and extended X-ray absorption fine structure (EXAFS)/ X-ray absorption near edge spectra (XANES) we propose that these structures contain thiolate molecules coordinated with Ag, Zn, and Sn in the +1, +2, and +2 oxidation states respectively. However, mixing these produces an AZTS solution which is only stable for 3 hours, due to a redox reaction between Ag⁺ and Sn²⁺ which forms Sn⁴⁺ and insoluble Ag metal. To solve this, we synthesize SnS₂ and show this produces a different Sn-thiolate complex with fully oxidized Sn⁴⁺. This is then used to produce the first stable AZTS solution, an essential step to fabricating reproducible films. We use this AZTS solution to fabricate films containing AZTS, and selenize these films in a tube furnace to produce films which contain AZTSe as well as secondary phases. We then use rapid thermal processing furnace to remove some of these secondary phases, and discuss ways to further improve our material quality.

In this thesis work, a systematic quantitative study has been undertaken, on the performance of etched fiber Bragg Grating (FBG) sensors in the investigation of surface molecular adsorption in real-time; it is shown that the limit of detection (LOD) of FBGs etched below 2 microns diameter, is better compared to prominent optical label-free molecular sensing techniques such as Surface Plasmon Resonance (SPR). Novel fiber optic sensors based on FBG and etched FBG with various nano materials (polyelectrolytes, carbon nanotubes, hydrogel, metals and chalcogenides) coated on the surface of the core or cladding, have been proposed for sensing multi parameters such as pH, protein, humidity, gas, strain, temperature, and light etc. Besides being reproducible and repeatable, the proposed methods are fast, compact, and highly sensitive. A novel optical instrument has also been developed to measure angular deviation, binocular deviation and refractive index of glass slabs, and liquids, based on a shadow casting technique. This method uses the deviation in the geometrical shadow cast by a periodic dot pattern trans-illuminated by a distorted light beam from the transparent test specimen relative to a reference pattern.

In this thesis work, a systematic quantitative study has been undertaken, on the performance of etched fiber Bragg Grating (FBG) sensors in the investigation of surface molecular adsorption in real-time; it is shown that the limit of detection (LOD) of FBGs etched below 2 microns diameter, is better compared to prominent optical label-free molecular sensing techniques such as Surface Plasmon Resonance (SPR). Novel fiber optic sensors based on FBG and etched FBG with various nano materials (polyelectrolytes, carbon nanotubes, hydrogel, metals and chalcogenides) coated on the surface of the core or cladding, have been proposed for sensing multi parameters such as pH, protein, humidity, gas, strain, temperature, and light etc. Besides being reproducible and repeatable, the proposed methods are fast, compact, and highly sensitive. A novel optical instrument has also been developed to measure angular deviation, binocular deviation and refractive index of glass slabs, and liquids, based on a shadow casting technique. This method uses the deviation in the geometrical shadow cast by a periodic dot pattern trans-illuminated by a distorted light beam from the transparent test specimen relative to a reference pattern.

Στην παρούσα εργασία ερευνάται η τρίτης τάξης μη γραμμική απόκριση διαφόρων υλικών τα οποία μελετήθηκαν σε μορφή διαλυμάτων ή λεπτών υμενίων. Αρχικά περιγράφονται βασικές έννοιες της μη γραμμικής οπτικής, μερικών σημαντικών φυσικών διαδικασιών που σχετίζονται με αυτή, καθώς και των διαφόρων μηχανισμών που μπορούν να συνεισφέρουν στο μη γραμμικό δείκτη διάθλασης. Στη συνέχεια παρουσιάζεται η μη γραμμική οπτική απόκριση νανοδομών Au, Pd και Ag. Με τη βοήθεια πολυμερών αποτρέπεται η συσσωμάτωση και καθίζηση του μετάλλου και επιτυγχάνεται η δημιουργία μεταλλικών νανοσωματιδίων συγκεκριμένω διαστάσεων. Επίσης μελετάται η μη γραμμικότητα TiO2, φουλλερενικών παραγώγων και μοριακών μηχανών. Η μεγάλη απόκριση των συστημάτων αυτών σε συνδυασμό με την έντονη εξάρτησή της από διάφορες μορφολογικές/δομικές παραμέτρους καθιστά τα συστήματα αυτά πολύ χρήσιμα για φωτονικές εφαρμογές.
In this work the third order nonlinear optical response of several photonic materials, has been investigated. These materials were in the form of solutions, colloids or thin films. Initially some basic concepts of nonlinear optics, the physical processes related with it, as well as the physical mechanisms related to the nonlinear refractive index are presented. Then, the nonlinear optical response of Au, Pd and Ag nanoparticles is presented. By using polymers, formation of nanoparticles exhibiting specific sizes can be achieved. Furthermore the polymer does not allow metal aggregation in the system. The nonlinearity of TiO2 films, fullerene derivatives and molecular engines is also investigate. The large response of these systems, combined with the strong dependence on several morphological/structural parameters makes them very promising candidates for several photonic applications.

博士
國立臺灣大學
電機工程學研究所
97
In the biochip applications, the mechanism of specific molecular recognition plays a fundamental role for molecular diagnosis. Traditionally, the dynamics of such an interaction are obtained by spatial and temporal summations of measured signals at macroscopic scale. However, it should be feasible to acquire such information in microscopic environment at single molecule level. The goal of this research is to develop platform technologies for a novel "digital nanoarray", with feature size down to 50 nm, to explore the microscopic measurement of stochastic behaviour at single molecule level and the feasibility studies of acquiring molecular dynamics by spatial-temporal summations of non-labelling biomolecules. These platform technologies include (1) nanofabrication of zero dimensional (0 D) nanodot array for developing a biomimicking platform and one-dimensional (1 D) nanowire array of conducting polymer for NO gas sensing; (2) a multi-functional dark-field microscopy (DFM) with nano-resolution down to 40 nm and spectroscopic function for characterizing observed nanostructure; and (3) a theoretical model for simulation and analysis of one and two dimensional of stochastic molecular interaction. First of all, we have used dip-pen nanotechnology (DPN) to fabricate nanoarray and nanowires for the studies of their nonlinear optical and electrical properties at large scale. With the fabricated nanodot array of 50 nm in diameter, we were able to immobilize bio-molecules on to prepared surface and acquire both 1 D and 2 D signals for further calculation of the spatial-temporal summations. The non-labeling detection of biomolecular recognition has been done by scanning images of AFM, and the resultant height of streptavidin is about 4 nm, which is close to the theoretical value. To improve the quality of surface preparation, we have used the atomic layer deposition (ALD) to deposit a 2 nm thickness of alumina oxide as a passivation layer to minimize non-specific binding to the native glass surface. We have compared the performance of both oxide surface of germanium and silicon substrate with the bare silicon surface. In the prepared 1 Dnanowire of both 68 nm and 300nm in line width, we are able to precisely align the nanowires across the gap of two microelectrodes. The nonlinear I-V curves of single nanowire were measured and analyzed at room temperature for practical applications. Several parallel and series circuitries of nanowire are measured for the verification of theoretical calculation. Finally, the nanowire array is applied for NO detection. In order to observe the stochastic behavior of single molecule on the digital nanoarray, it is essential to develop an optical microscopy beyond the diffraction limits. Herein, we reported a multifunctional optical microscopic system that combined the features of dark-field microscopy (DFM) with a spectrophotometric function to characterize the nanoarray for both qualitative and quantitative analysis. We have used various formats of optical storage discs, which have different feature sizes in the pitch of track patterns, to demonstrate the resolving power of this system. The calculated resolution is down to 40 nm. Moreover, a thin gold layer is deposited on the disc to study the optical enhancement of pit features and the change of absorption peaks due to surface Plasmon resonance (SPR) effect. A significant change of absorption peak is obtained by depositing a thin film of biolmolecules on the gold film. These results indicate the possible use of this nanoarray platform on medical diagnosis. The stochastic model of behavior of biomolecular interactions is similar to the bi-state (on-off) of channel proteins. Our simulation results show that 100 of molecular interactions are the same in both 1 D and 2 D signals. After this simulation process, we are able to determine the proper size of nanoarray by evaluating the reasonable errors of possibility. We have used deposited 1 D data from internet for our own purposes. This model can be applied to the multi-state systems in the future.