Les capteurs biologiques basés sur l’électronique nanométrique ont la propriété intéressante d’être à l’échelle des molécules étudiées. Plus spécifiquement, grâce à leurs propriétés électroniques exceptionnelles, les transistors à effet de champ à base de graphène (TECG) permettent des mesures électriques locales à grandes vitesses d’acquisition et sur de longues durées, offrant un cadre idéal pour la biodétection et l’étude de la cinétique moléculaire. Le présent mémoire traite de l’analyse, la mesure et la fonctionnalisation des TECG dans l’optique d’en faire des biocapteurs performants. En introduction, nous décrirons les propriétés électroniques du graphène ainsi que les principaux concepts reliés aux transistors de graphène et à la détection biologique. Puis, nous établirons les trois objectifs qui seront élaborés en autant de chapitres. Dans le premier chapitre, nous présenterons une revue de littérature critique qui cible l’analyse statistique et l’assemblage de l’interface de détection comme facteurs déterminants de la performance à l’aide d’analyses originales et d’une description approfondie de l’état du domaine. Dans le deuxième chapitre, nous présenterons des ajustements concrets aux sysèmes expérimentaux basés sur les recommandations émises dans la revue. D’abord, nous améliorons la productivité de la fabrication des transistors, puis développons une instrumentation permettant de mesurer plusieurs capteurs en parallèle. Dans le troisième chapitre, nous prendrons avantage de ces modifications pour présenter dans le deuxième article une méthode permettant une fonctionnalisation du graphène à la fois contrôlée et solide. En utilisant le voltage de grille, nous initions et suspendons la fonctionnalisation covalente du graphène aux sels de diazonium afin d’obtenir le taux de greffage désiré, tout en observant la réaction en temps-réel. Ainsi, par nos avancées méthodologiques et d’instrumentation, nous résolvons un enjeu critique du développement de la chimie de surface, centrale à la performance de biodétection. / Nanoscale electronics are a promising tool for biosensing as they fit their target’s size and allow for local, fast-paced measurements over long time scales. Because of their exceptional electronic properties, graphene field-effect transistors (GFETs) are excellent candidates for biosensing and studying molecular kinetics. This work discusses the analysis, measurement, and functionalization of GFETs as optimized biosensors. In the introduction, we describe the electronic properties of graphene and the main concepts related to GFETs and biodetection. We also establish the three aims of the project, elaborated in three chapters. The first chapter contains a critical literature review that uses original analyses and a thorough state-of-the-field to target statistical analysis and the biorecognition interface assembly as determining factors in sensing performance. In the second chapter, we present the practical adjustments to the experimental systems based on the review’s recommendations. First, we increase the productivity of device fabrication, then we develop a multiplexed electrical measurement setup. In the third chapter, we take advantage of these modifications to present in the second article a method for stable and controlled functionalization. Using the gate voltage, we start and stop the covalent functionalization of graphene with aryldiazonium salts to get the desired grafting level, while observing the reaction in real-time. Thus, with our advances in methodology and instrumentation, we solve a critical aspect of surface chemistry, central for biodetection performance

Recently, there has been a shift in the global healthcare paradigm, which is prioritizing a more patient-centric approach causing an increase in the demand for rapid and point-of-care (PoC) biomolecular detection. Electrochemical (EC) signal transduction has been used to great effect to meet some of this demand by constructing biosensors with high sensitivity and low limit-of-detection (LOD). However, signal generation in EC biosensors requires input bias potentials to activate electrochemical redox reactions. This means EC systems are inherently built-in with high background noise that limits the performance of biosensors. Biosensors with photoelectrochemical (PEC) signal transduction have recently shown great promise in being able to deliver biomolecular detection on par with, if not better than, EC biosensors. PEC biosensing directly improves upon EC signal transduction by combining EC signal readout with optical excitation as the bias input, and generally being able to achieve similar performance with simpler bioassay designs. In this scheme, the input and output of the signal transduction are decoupled from each other, significantly reducing background signal in biosensors to enhance their sensitivity. Despite being highly effective, PEC biosensors have yet to find commercial breakthrough as they have so far only shown quantitative analysis on a limited set of biomarkers and have not shown to be PoC-capable. In this thesis, we developed new strategies to improve PEC signal transduction so that it could be applied to build robust ultrasensitive PoC biosensors with high dynamic range, simple operation, and low LOD for detecting a wide variety of different disease biomarkers. The most popular photoactive materials used in the fabrication of PEC biosensors are TiO2 nanomaterials on account of their availability, chemical stability, high catalytic efficiency, tunable morphology, and ideal band energy levels for driving useful EC reactions. However, unmodified TiO2 suffers from several drawbacks that limit its photocurrent generation efficiency, such as poor visible range absorbance due its wide bandgap and fast charge carrier recombination. Alongside the additional difficulty of biofunctionalization, PEC biosensors fabricated from TiO2 nanomaterials are limited in their bioanalytic performance. In order to make improvements on PEC biosensors, we modified the surface of TiO2 nanomaterials by chelating them with catecholate molecules. The surface modification with catecholates formed charge transfer complexes on TiO2, which resulted in enhanced photoexcitation due to enhanced electron injection attributable to intermolecular orbital excitations in the catecholate molecules. The catecholate ligands also added improved colloidal stability and additional functional groups that aided with biofunctionalization. This resulted in multifunctional TiO2 nanoparticles with improved photocurrent signal generation and enhanced visible range photoabsorption. We took this one step further by taking advantage of the high binding affinity of catecholates on TiO2 surfaces to create novel synthesis methods that created high surface area nanostructures. Photoelectrodes fabricated from these new TiO2 nanostructures had nanoporous morphology and were able to capture biomolecules more efficiently. Using our novel TiO2 nanomaterials, we fabricated signal-off biosensors that were able to detect DNA biomarkers and IL-6 protein (cancer and inflammatory biomarker) in urine with an LOD of 1.38 pM and 3.6 pg mL-1, respectively. We further explored hybrid semiconductor structures by combining TiO2 nanomaterials with other materials such semiconductors with different bandgaps or plasmonic metal nanoparticles (NP). Using the aforementioned catechol-assisted synthesis techniques, we were able to produce different morphologies of TiO2 nanomaterials with distinct phases: anatase TiO2 nanorod assemblies and rutile TiO2 NP. The two different TiO2 nanomaterials have different bandgaps and can be used to form semiconductor heterostructures. By combining rutile TiO2 NPs with DNAzymes, a type of synthetic functional nucleic acid, we created a photoactive molecular switch that worked by making and breaking heterostructures between the two TiO2 nanomaterials. We used DNAzymes specific to E. coli bacteria to develop a highly sensitive signal-on bacterial detection platform that was able to detect E. coli in lake water samples with an LOD of 18 CFU mL-1. Using catecholate-assisted photoreduction synthesis, we developed an efficient and novel method for decorating TiO2 NP with silver (Ag) NP. The resultant nanomaterial featured TiO2 NP surfaces modified with Hematoxylin (HTX) dyes and covered with sub-nanometer sized silver NP. The band structure of TiO2/HTX/Ag NP hybrid material involved high energy electron generation through decay of surface plasmons in the Ag NP and then enhancing the photoelectron injection process between HTX and TiO2. This significantly enhances the photoexcitation and photoabsorbtion, resulting in the material with the highest photocurrent generation as presented in this thesis. By taking advantage of thiol-metal bonds, we used the TiO2/HTX/Ag NP material system in the fabrication of a highly sensitive signal-off microRNA (prostate cancer biomarker) sensor with an LOD of 172 fM in urine. Special attention was paid to the design of PEC bioassays in this work so that they are miniaturized and easy to use, and thus suitable for PoC applications. Because PEC signal transduction generates ultrahigh signals compared to other transduction methods, it allows bioassay designs to remain simple without sacrificing performance. This allowed us to create bioassays with very few operational steps, that excel in reliability and ease-of-use. To further improve PoC capability, we explored multiplexing with the biosensor made from TiO2/HTX/Ag NP. Here we were able to demonstrate multiplexing with PEC signal transduction for the first time. Another major barrier to PEC biosensors becoming widespread is the requirement of large benchtop instrumentation such as potentiostats and light sources. To address this challenge, we designed a portable smartphone-interfacing potentiostat with a built-in LED light source to support PEC biosensing. This device, named the PECsense was as versatile as any commercial potentiostats, having features such as adjustable recording periods, variable illumination periods, automatic data processing and being able to record both anodic and cathodic photocurrents. The PECsense was demonstrated to be used successfully as a signal reader in a PEC DNA detection assay. Ultimately, we designed several ultrasensitive PEC biosensors used for the detection of four different diagnostic biomarkers. Combined with the exploration of miniaturized design, multiplexing and portable signal-reading, our designed PEC biosensors were made PoC-capable. The work in this thesis presented innovations in areas of nanotechnology, material synthesis, solid-state physics, biotechnology and embedded systems for the advancement of biomolecular detection and PoC diagnostics. / Thesis / Doctor of Philosophy (PhD) / Biosensors show great promise for use in point-of-care diagnostics and health monitoring systems. Such deceives combine biorecongition with signal transduction for analyzing biologically relevant targets. Photoelectrochemical (PEC) mode of signal reading, particularly those based on TiO2 nanomaterials, have shown great promise in delivering point-of-care biosensors that have excellent diagnostic performance. In this thesis, our goal was to develope new techniques for creating low-cost, easy-to-use and ultrasensitive photoelectrochemical biosensors. To achieve this goal, our work here can broadly be split into three objectives. Firstly, we focused on developing new material synthesis methods to improve traditional TiO2 nanomaterials so they can be more useful in PEC biosensors. These methods involved combining TiO2 with organic molecules known as catecholates and metal nanoparticles. This work created material systems that are able to generate high signals and more easily interface with biomolecules for improving PEC biosensor sensitivity. For the second objective, we used our newly developed enhanced TiO2 nanomaterials as the foundation for designing various bioassays for the detection of a wide range of different biological targets such as DNA, RNA, proteins and bacteria. This served to demonstrate the robustness of PEC signal reading as a tool for various markers of diseases. Despite PEC biosensors being a powerful tool in healthcare, they have seen very little commercial breakthrough, which can primarily be attributed to needing bulky benchtop instruments and light sources for signal reading. For the last objective, we worked on designing a handheld smartphone-operated signal-reader for PEC biosensing with its own built-in light source.

Despite the increasing number of electrochemical and photoelectrochemical biosensors reported in the research literature, few have achieved success outside of a laboratory setting. This can partly be attributed to accessibility issues with commercially available readout instruments. Consequently, low-cost and portable readout instruments have been developed by researchers, but these devices fail to address other key compatibility and accessibility challenges. Much like the commercial systems, these devices are not natively compatible with multiplexed signal assays consisting of two or more working electrodes, cannot control optical excitation sources for photoelectrochemical biosensing, nor can they interface with auxiliary instruments such as heaters and electromagnets. To this end, we have developed a low-cost smartphone-operated electrochemical and photoelectrochemical readout system for point-of-care biosensing. Our readout system can perform standard voltammetric techniques and is capable of synchronously controlling an optical excitation source to support photoelectrochemical biosensing. This device is compatible with standard three-electrode assays as well as dual signal assays with two working electrodes. We have also created a portable sample heater that can be controlled by this readout system to facilitate on-site sample heating and have also integrated a portable electromagnet to perform away-from-lab magnetic manipulation. / Thesis / Master of Applied Science (MASc) / Early and prompt detection of disease biomarkers is crucial in order to develop effective disease management strategies. Unfortunately, many gold-standard diagnostic techniques for infectious diseases, cancers, heart diseases, among other conditions prove to be time-consuming, costly, and reliant on trained professionals in a laboratory setting. Electrochemical and photoelectrochemical detection are two sensing modalities that show promising potential for point-of-care applications, as they are easily miniaturized, inexpensive, and can be used to detect both the presence of and the amount of analyte present. However, up until now, these sensing modalities have mostly been confined to research settings. To expedite the commercialization of such sensors and to facilitate their translation to point-of-care diagnostics, we have developed a low-cost smartphone-operated electrochemical and photoelectrochemical readout system. Through the integration of peripheral instruments including a sample heater, electromagnet, and optical excitation source, this system is compatible with a number of different biosensors.

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

The rise of internet connected devices has enabled the home with a vast amount of enhancements to make life more convenient. These internet connected devices can be used to form a community of devices known as the internet of things (IoT). There is great value in IoT devices to promote healthy independent living for older adults. Fall-related injuries has been one of the leading causes of death in older adults. For example, every year more than a third of people over 65 in the U.S. experience a fall, of which up to 30 percent result in moderate to severe injury. Therefore, this thesis proposes an IoT-based fall detection system for smart home environments that not only to send out alerts, but also launches interaction models, such as voice assistance and camera monitoring. Such connectivity could allow older adults to interact with the system without concern of a learning curve. The proposed IoT-based fall detection system will enable family and caregivers to be immediately notified of the event and remotely monitor the individual. Integrated within a smart home environment, the proposed IoT-based fall detection system can improve the quality of life among older adults. Along with the physical concerns of health, psychological stress is also a great concern among older adults. Stress has been linked to emotional and physical conditions such as depression, anxiety, heart attacks, stroke, etc. Increased susceptibility to stress may accelerate cognitive decline resulting in conversion of cognitively normal older adults to MCI (Mild Cognitive Impairment), and MCI to dementia. Thus, if stress can be measured, there can be countermeasures put in place to reduce stress and its negative effects on the psychological and physical health of older adults. This thesis presents a framework that can be used to collect and pre-process physiological data for the purpose of validating galvanic skin response (GSR), heart rate (HR), and emotional valence (EV) measurements against the cortisol and self-reporting benchmarks for stress detection. The results of this framework can be used for feature extraction to feed into a regression model for validating each combination of physiological measurement. Also, the potential of this framework to automate stress protocols like the Trier Social Stress Test (TSST) could pave the way for an IoT-based platform for automated stress detection and management.

In dieser Arbeit wurde eine neuartige Möglichkeit zur Funktionalisierung von beliebigen Oberflächen entwickelt und getestet. p-(N-maleimido)phenyl- (MP) und p-Aminophenyl-Reste (AP) wurden auf CVD gewachsenem Graphen via Elektroreduktion der jeweiligen Diazoniumkationen abgeschieden. Die so funktionalisierten Graphenschichten wurden auf verschiedenste Materialien transferiert. Die Präsenz der funktionellen Gruppen wie auch ihre kovalente Anbindung wurde durch Infrarotellipsometrie und Ramanspektroskopie bestätigt. Für das MP-funktionalisierte Graphen wurde mittels optischer Simulation der IRSE-Daten die Dicke der MP-Schicht zu 4.4 nm vor und 4.8 nm nach dem Transfer bestimmt. Die Dicke nach Transfer wurde zusätzlich durch Infrarot-gekoppelte Rasterkraftmikroskopie zu 4.4 nm bestimmt. MP-Graphen wurde via MICHAEL-Addition sowohl mit p-Nitrobenzylmercaptan (NBM), als auch mit Cystein-terminierter Peptidnukleinsäure (PNA) modifiziert. Die jeweiligen Moleküle wurden nach dem Transfer per IRSE nachgewiesen. Die Modifikation mit NBM wurde darüber hinaus in einem Mikrofluidikaufbau in-situ verfolgt. Die Zeitabhängigkeit der Anreicherung an der MP-Oberfläche wurde mit der auf einer Goldoberfläche verglichen. AP-Graphen wurde mittels Zero-Length-Crosslinking mit p-Nitrobenzoesäure (NBA), sowie Carboxyl-funktionalisierten Nanokristallen (Quantum Dots) modifiziert. Die kovalente Anbindung wurde durch Vorhandensein der Amid-I-Bande nachgewiesen und der erfolgreiche Transfer mittels Photolumineszenz-Spektroskopie. Um das herausragende Potential des Transfers von funktionalisiertem Graphen zu zeigen, wurde PNA-modifiziertes MP-Graphen auf ein vorkontaktiertes Sensor-Array transferiert. Durch einfache Widerstandsbestimmung des Graphens konnte die Hybridisierung mit komplementärer DNA von 1b-mismatch DNA unterschieden werden. / A new pathway for the functionalization of arbitrary surfaces was developed and tested. p-(N-maleimido)phenyl (MP) and p-Aminophenyl (AP) residues were deposited on CVD grown graphene on copper, via electroreduction from the respective diazonium cation. The functional graphene layers were subsequently transferred to a variety of substrates, comprising metals (gold), insulators (SiO2, glass) and flexible ones (PTFE tape). The presence and covalent attachment of the desired groups was confirmed via infrared ellipsometry and Raman backscattering spectroscopy. The thickness of the Maleimidophenyl layer was determined via optical simulation of the IR ellipsometry data. The resulting 4.4 nm prior to and 4.8 nm after transfer to a gold film on silicon are in good agreement. The thickness after transfer was additionally measured using AFM, amounting as well to 4.4 nm. All results are in good agreement and confirm the possibility to transfer covalently functionalized graphene without noticeable loss. MP functionalized graphene was modified wet-chemically. MP-functionalized graphene was modified using the MICHAEL addition, attaching p Nitrobenzylmercaptane (NBM) or cysteine terminated peptide nucleic acid (PNA) to the surface. The success of modification and subsequent transfer was confirmed via infrared ellipsometry. MP graphene was furthermore integrated in a microfluidic setup. The time dependence of the accumulation of NBM on the functionalized graphene was compared to a similar on a plain gold surface. The different behaviour strongly hints at a covalent bonding between thiol and maleimide. AP functionalized graphene was modified via zero-length crosslinking with p Nitrobenzoic acid (NBA) and COOH containing Nanocrystals (Quantum Dots). The covalent attachment was shown by the presence of the amide bonds. Additionally, the successful transfer of the Quantum Dots was demonstrated via photoluminescence spectroscopy. To prove the outstanding potential of the transfer of functionalized graphene, PNA-modified MP-graphene was transferred on top of a pre-contacted sensor array. By simple current measurement, complementary DNA strands could be distinguished from the respective 1 b mismatch DNA.

Les cucurbiturils (CBn) sont des molécules-cages synthétiques constituées d’un nombre n d’unités glycoluril et dont les applications en chimie, en biologie et en physique ont commencé à être exploitées au début des années 2000. Ces composés trouvent leur importance dans un grand nombre de domaines incluant la chimie en phase solide, le piégeage des contaminants en solution, la catalyse ou encore l’encapsulation de principes actifs pour des applications pharmaceutiques futures. Ces récentes molécules-cages rejoignent le large groupe des récepteurs synthétiques comprenant les cyclodextrines, les calixarènes, les cryptophanes, les carcérands et hémicarcérands. L’encapsulation des gaz dans ce type de structures est encore mal connue à l’heure actuelle et, dans ces travaux de thèse, notre intérêt s’est porté sur la complexation de gaz, notamment de xénon, dans les cucurbiturils. Ces travaux ont débuté par la synthèse de nouveaux cucurbiturils hydrosolubles dans le but d’étudier leur capacité d’encapsulation des gaz rares et de petits alcanes comme le méthane et l’éthane, notre objectif final étant de définir de nouvelles règles régissant l’encapsulation des gaz par les cucurbiturils. Les gaz rares allant de l’hélium au krypton ont été étudiés. Parmi ces gaz, le xénon hyperpolarisé a particulièrement retenu notre attention du fait de son intérêt dans la conception de biosondes pour le développement de nouvelles méthodes de diagnostic en IRM. Dans ce mémoire, la synthèse de nouveaux cyclohexylcucurbiturils mixtes hydrosolubles et leurs études en présence de xénon sont rapportées. Un nouveau cucurbituril mixte constitué de cinq unités glycoluril et d’une unité glycoluril à six chaînons a également été synthétisé. Les deux premiers chapitres constituent une introduction au phénomène d’encapsulation des gaz ainsi que des généralités sur la famille des cucurbiturils. Le troisième chapitre est consacré aux résultats obtenus au laboratoire sur la synthèse de nouveaux composés. Dans un quatrième chapitre, nous avons développé une méthode permettant la préparation de précurseurs d’analogues acycliques de cucurbiturils. Pour cela, la réaction de métathèse par ouverture de cycle associée à la métathèse croisée (ROM-CM) a été utilisée pour mener à bien cette synthèse. Enfin, un cinquième chapitre est consacré aux résultats concernant l’étude de l’encapsulation de gaz dans les nouveaux cucurbiturils synthétisés, étude effectuée en collaboration avec le Laboratoire de Structure et Dynamique par Résonance Magnétique du CEA de Saclay. / The cucurbituril family (CBn) constitutes a group of recent synthetic host-molecules. These compounds are formed by n glycoluril units and, since the beginning of the 2000’s, they have found their interest in multiple domains such as biology, chemistry and physics. They have joined the large group of synthetic receptors comprising cyclodextrins, calixarens, cryptophanes, carcerands and hemicarcerands. Gaz encapsulation in this family of compounds is misunderstood. In these PhD works, our interest concerned gaz complexation, especially xenon, into cucurbiturils. First, the synthesis of new hydrosoluble cucurbiturils to study their ability to encapsulate noble gaz and small alcans such as methan and ethan is described. Our final goal will be to define new rules concerning gaz encapsulation by cucurbiturils. Noble gaz from helium to krypton have been studied. Among these noble gaz, xenon is particularly interesting in the domain of biosensing, for the design of new diagnostic methods by MRI. In this manuscript, the synthesis of a family of hydrosoluble cyclohexylcucurbiturils and their ability to encapsulate xenon is described. Another new cucurbituril constituted by five glycoluril units and one glycoluril unit forming a six-membered ring, has also been prepared. The first two parts of the manuscript present the gaz encapsulation and the cucurbituril family. The third part describes our results concerning the new synthetized cucurbiturils. In the fourth part, we have developped a method based on metathesis reactions (ROM-CM) for preparing some precursors of acyclic congeneers of cucurbiturils. Lastly, the fifth part concerns results about gaz encapsulation. All of this study has been driven in collaboration with Laboratoire de Structure et Dynamique par Résonance Magnétique (CEA Saclay).

This dissertation explores the different ways to create thin film-based biosensors that are capable of rapid and label-free detection of cortisol, a non-specific biomarker closely linked to stress, within the physiological range of 10pM to 10 uM. Increased cortisol levels have been linked to stress-related diseases, such as chronic fatigue syndrome, irritable bowel syndrome, and post-traumatic stress disorder. It also plays a role in the suppression of the immune system as well. Therefore, accurate measurement of cortisol in saliva, serum, plasma, urine, sweat, and hair, is clinically significance to predict physical and mental diseases. In this dissertation, thin film-based electrochemical immunosensors were fabricated using a self-assembled monolayer (SAM) functionalized by cortisol specific antibodies to detect cortisol at 10 pM level sensitivities in the presence of a redox probe. The fabricated electrochemical cortisol immunosensors were able to detect cortisol in human saliva samples and the outcomes were validated using the standard Enzyme Linked Immuno Sorbent Assay (ELISA) technique. With the aim of improving signal amplification and label-free cortisol detection, copper nanoparticles were incorporated on screen-printed carbon electrodes (SPCE) for the fabrication of electrochemical cortisol immunosensor. This SPCE-based sensor showed a sensitivity of 4.21µA/M and the limit of detection 6.6nM. Both the SAM and SPCE-based immunosensors were not thermally stable due to the instability of antibodies at room temperature. To address this issue, an antibody-free immunosensor was fabricated. Molecular Imprinted Polymer (MIP) was used to template the target cortisol molecule. The MIP-based sensing platform was prepared using polypyrrole, a thermally stable conducting polymer. The conductivity of the polymer ensured good electrical performance. The polypyrrole-based MIP was synthesized by means of electrochemical polymerization and was used to detect cortisol within the physiological range at room temperature. MIP-based sensors exhibited the detection limit of 1 pM, and were cost-effective, easy to fabricate, temperature stable, and reusable. The sensing performance of the resulting sensors was comparable to those of commercially available technologies, such as ELISA. Aiming to perform cortisol sensing at point-of-care (POC), an Extended Gate Field Effect Transistor (EGFET) was integrated with a developed MIP cortisol sensor. The as developed MIP-EGFET sensor was used to detect the cortisol concentration in the range of 1 pM to 100 nM. A few of the major advantages of the developed sensor are its ability to provide a direct readout and simpler electronic systems, which are necessary for miniaturized Point of Care devices.

Proteins possess a broad range of structural and functional properties and, therefore, can be employed in a variety of biomedical applications. While a good number of protein-based biosensing systems and biosensors for target analytes have been developed, the search for versatile, highly sensitive and selective sensors with long term stability able to provide fast detection of target analytes continues to be a challenge. To that end, we now report the design and development of modified proteins with tailored characteristics and their further utilization in the development of biosensing systems. We take advantage of binding proteins that undergo a change in conformation upon binding to their respective target ligand analytes for the development of highly selective biosensing systems. The first class of binding proteins that was explored for this purpose was antibodies. A non-canonical site in the variable region of a monoclonal antibody was tagged with a fluorescent probe to sense the binding of analyte to its corresponding antigen-binding site. The strategy employed for designing antibodysensing molecules is universal as it can be employed for sensing any biomolecule of interest provided that there is an available antibody against the target ligand analyte. In a second strategy, we utilized designer glucose recognition proteins (GRPs) that were prepared by incorporation of unnatural amino acids in the glucose/galactose binding protein (GBP) of Escherichia coli and its truncated fragments. By taking advantage of the global incorporation method, we were able to fine-tune the binding affinity and thermal stability of the proteins, thus, allowing for the development of a reagentless fluorescence based fiber optic glucose biosensor capable of monitoring glucose in the hypoglycemic, normal, and hyperglycemic range, as well as in the hypothermic and hyperthermic temperature range.

Cette thèse décrit la synthèse de nanoparticules de polymères à empreintes moléculaires (MIP, de l’anglais molecularly imprinted polymer) pour la reconnaissance de protéines, par une approche de synthèse en phase solide. Les polymères à empreintes moléculaires sont des récepteurs biomimétiques synthétisés sur mesure par un processus de nanomoulage du polymère autour de la molécule unique. Ils possèdent ainsi des cavités de reconnaissance spécifiques pour leur molécule cible. La technique de l'impression moléculaire pour les petites molécules cibles est bien établie, alors que l'impression de protéines reste encore un défi en raison de la flexibilité et complexité de leur structure native et de leurs nombreux sites fonctionnels, mais aussi en raison de leur faible stabilité dans des conditions inhabituelles. Par conséquent, une approche de synthèse en phase solide a été développée ici où la protéine est immobilisée sur un support avant la synthèse de nanoparticules hydrosolubles de MIP par polymérisation radicalaire. Les MIPs obtenus ont des affinités comparables à celles des anticorps, et des réactivités croisées faibles. Ils possèdent des avantages tels qu'une stabilité meilleure, un coût plus faible et peuvent potentiellement être régénérés et réutilisés, devenant ainsi des alternatives prometteuses aux anticorps naturels. Nous avons fabriqué des MIPs contre des protéases à sérine, telles la trypsine et la kallikréine, mais aussi contre un épitope peptidique de la protéine gp41 du VIH. Des nanogels de MIP thermosensibles ont été synthétisés dans un réacteur sous la forme d’une colonne thermostatée ou une boîte de Pétri, par polymérisation radicalaire initiée par voie thermique ou photochimique. Un simple changement de la température permet de libérer les MIPs de la protéine immobilisée. Ces MIPs sont hydrosolubles en fonction de la température et ont un diamètre inférieur à 100 nm. Leur affinité pour leur cible est élevée, avec un Kd du nano ou picomolaire. Ces 'anticorps synthétiques' ont été appliqués dans des tests d'adsorption sur microbalance à cristal de quartz, mais également comme 'chaperons synthétiques'. Des études préliminaires de la protection des protéines d'une dénaturation thermique ou par un pH défavorable ont été effectuées. L'utilisation d'un iniferter pour initier la photopolymérisation vivante du MIP a permis de synthétiser des nanogels de type core-shell. En introduisant des marqueurs fluorescents dans les MIPs, les tests d’immunoessai dans des fluides biologiques ont été démontrés, ce qui indique le grand potentiel de ces MIPs dans le diagnostic clinique. En conclusion, nous avons développé une nouvelle approche de synthèse de nanoparticules de MIP hydrosoluble ayant une haute affinité pour une protéine, utilisables à la place des anticorps dans des applications dans le monde réel tel que la détection de protéines biomarqueurs dans des échantillons complexes, et potentiellement comme principe actif in vivo. / This thesis describes the synthesis, by a solid-phase synthesis approach, of nanoparticles of molecularly imprinted polymers (MIPs) for the recognition of proteins. Molecularly imprinted polymers are biomimetic receptors synthesized by a nanomolding process of the polymer around single molecules. They therefore possess specific recognition cavities for their target molecule. The technique of molecular imprinting for small target molecules is well established, while protein imprinting remains a challenge due to the flexibility and complexity of their native structure and functional sites, but also because of their low stability under unusual conditions. Therefore, a solid-phase synthesis approach has been developed where the protein is immobilized on a support before the synthesis of water-soluble MIP nanogel particles by radical polymerization. The MIPs obtained have affinities comparable to those of antibodies, and low cross-reactivities. They have advantages such as better stability, lower cost, and can potentially be regenerated and reused, thus becoming promising alternatives to real antibodies. We have synthesized MIPs against serine proteases such as trypsin, and kallikrein, but also against a peptide epitope of the HIV gp41 protein. Thermosensitive MIP nanogels were synthesized in a thermostated column-type reactor or a petri dish, by thermally or photo-initiated radical polymerization. Their thermosensitivity allows the MIPs to be released from the immobilized protein by a simple temperature change. They are water-soluble as a function of temperature and have a diameter of less than 100 nm. Their affinity for their target is strong, with a Kd in the nano or picomolar range. These 'synthetic antibodies' have been applied in binding assays with quartz crystal microbalance, but also as 'synthetic chaperones'. Preliminary studies of the protection of proteins from thermal denaturation or from denaturation by an unfavorable pH have been carried out. The use of an iniferter to initiate the living photopolymerization of MIP made it possible to synthesize nanogels of core-shell type. By introducing fluorescent markers into MIPs, immunoassay applications in biological fluids have been demonstrated, indicating the great potential of these MIPs in clinical diagnostics. In conclusion, we have developed a novel approach to the synthesis of soluble MIP nanoparticles having high affinity for a protein, usable in place of antibodies in real world applications such as the detection of biomarker proteins in complex samples, and potentially as an active principle in vivo.