En terme de performance, le biocapteur idéal doit avoir très grande sensibilité, basse limite de détection et temps d’analyse qui est extrêmement court. Les biocapteurs sans marquage à base de résonance de plasmons de surface (biocapteurs SPR) possèdent naturellement le temps d’analyse le plus court parmi différent types de biocapteurs. Leur limite de détection n’est cependant pas la plus impressionnante. Il y a donc un besoin pour augmenter considérablement la sensibilité intrinsèque des biocapteurs SPR afin de permettre de plus basses limites de détection. Quelques approches pour exalter la sensibilité optique des biocapteurs SPR dans la configuration « traditionnel » de Krestchmann telles que film SPR bimétallique, plasmons à longues portées et détection dans l’infrarouge proche sont examinées dans ce travail. Des configurations « non traditionnelles » comme guides optiques planaires avec couplage par réseau et structures sub-longueur d’ondes ont été aussi théoriquement étudiées. Nouvelle stratégie de fonctionnalisation de surface à base de graphène qui augmente la sensibilité de reconnaissance biomoléculaire et peut être appliquée à quasiment toute structure SPR a été également démontrée. / In terms of performance, the ideal biosensor should have high sensitivity, low limits of detection, and extremely short analysis time. Label-free surface plasmon resonance (SPR) biosensors naturally offer the shortest analysis time compared to other types of biosensors. On the other hand, the limits of detection of SPR biosensors are not the most impressive. The inherent sensitivity of SPR biosensors thus needs to be significantly improved to allow lower limits of detection. Several approaches for the enhancement of optical sensitivity of SPR biosensors in the “traditional” attenuated total reflection (ATR) Kretschmann configuration such as the use of bimetallic SPR film, long-range surface plasmons, and near-infrared operating wavelength have been investigated in this work. In addition, some “non traditional” configurations for SPR biosensors including grating-coupled planar optical waveguides and arrays of sub-wavelength structures have been theoretically studied. Novel graphene-based surface functionalization strategy with enhanced biorecognition sensitivity that can be applied to virtually any SPR structure has also been demonstrated.

Plasmonic materials constitute one of the most explored platforms in the past decade for biological sensing, as they offer a wide range of advantages in respect to the currently available tests employed in either screening or medical diagnosis.The detection of leukaemia cancer markers was chosen as the medical applications in the evaluation of the sensing capabilities of these platforms. Particularly, nanohole arrays on gold films have already been demonstrated to be efficientsensing elements for the study of protein protein interactions. In this work,nanohole arrays platforms were optimized by studying the combinations of shape, diameter, aspect ratio, polarization and periodicity that lead to the highest sensitivity. In addition, different nanohole arrays substrates fabricated by UV-nanolithography and interference lithography were characterized and compared to the structures made by conventional focus ion beam (FIB) milling. Analytes derived from blood sample of leukemia cancer patients were detected on these structures with great sensitivity and specificity, demonstrating a large potential for medical applications. Furthermore, the development and characterization of a cost-effective system capable of detecting leukaemia cancer markers with comparable limit of detection and sensitivity as commercial platforms was started. With future development, this platform could provide advantages in terms of miniaturization, analysis time and the integration as an easy-to-use lab-on-chip device for diagnostics. / Graduate / 0494 / cvalsecc@gmail.com

Cancer is a leading cause of death and some types of cancer are hard to diagnose at early stages. An accurate method for subtype classification of cancer types is also critical for patients to receive effective treatments. Many cancer biomarkers (e.g., EGFR for lung cancers and CD19/CD20 for leukemia) have been found with potential of being used for cancer diagnosis and subtype cancer classification. A biosensing technique being able to detect biomarkers with a miniaturized system, based on extraordinary light transmission (EOT) through nanohole arrays on metal films, is promising for cancer diagnosis and subtype classifications. In this research, the detection of different biomarkers (EGFR, CD19 and CD20) was demonstrated using a surface plasmon resonance (SPR) setup with EOT. The concentration of EGFR from cell lysate solution was determined using the SPR setup and compared with a current analytical method (ELISA). The SPR setup gave a detection limit concentration of 0.77 g/mL for the EGFR. The EGFR concentration from the cell lysate was determined to be greater than 10 g/mL from SPR experiments; while a lower concentration of 0.604 g/mL was found from ELISA indicating some problems with the calibration curves obtained in the SPR experiments. A whole lung cancer cell capture experiment was also conducted using microscopy imaging and the SPR setup. A number of 11 2 cells/mm2 was captured from a pre-modified metal surface, which was confirmed by SPR. / Graduate / 0752 / 0541

Plasmonic materials constitute one of the most explored platforms in the past decade for biological sensing, as they offer a wide range of advantages in respect to the currently available tests employed in either screening or medical diagnosis.The detection of leukaemia cancer markers was chosen as the medical applications in the evaluation of the sensing capabilities of these platforms. Particularly, nanohole arrays on gold films have already been demonstrated to be efficientsensing elements for the study of protein – protein interactions. In this work,nanohole arrays platforms were optimized by studying the combinations of shape, diameter, aspect ratio, polarization and periodicity that lead to the highest sensitivity. In addition, different nanohole arrays substrates fabricated by UV-nanolithography and interference lithography were characterized and compared to the structures made by conventional focus ion beam (FIB) milling. Analytes derived from blood sample of leukemia cancer patients were detected on these structures with great sensitivity and specificity, demonstrating a large potential for medical applications. Furthermore, the development and characterization of a cost-effective system capable of detecting leukaemia cancer markers with comparable limit of detection and sensitivity as commercial platforms was started. With future development, this platform could provide advantages in terms of miniaturization, analysis time and the integration as an easy-to-use lab-on-chip device for diagnostics. / Graduate / 0494 / cvalsecc@gmail.com

Cancer is a leading cause of death and some types of cancer are hard to diagnose at early stages. An accurate method for subtype classification of cancer types is also critical for patients to receive effective treatments. Many cancer biomarkers (e.g., EGFR for lung cancers and CD19/CD20 for leukemia) have been found with potential of being used for cancer diagnosis and subtype cancer classification. A biosensing technique being able to detect biomarkers with a miniaturized system, based on extraordinary light transmission (EOT) through nanohole arrays on metal films, is promising for cancer diagnosis and subtype classifications. In this research, the detection of different biomarkers (EGFR, CD19 and CD20) was demonstrated using a surface plasmon resonance (SPR) setup with EOT. The concentration of EGFR from cell lysate solution was determined using the SPR setup and compared with a current analytical method (ELISA). The SPR setup gave a detection limit concentration of 0.77 µg/mL for the EGFR. The EGFR concentration from the cell lysate was determined to be greater than 10 µg/mL from SPR experiments; while a lower concentration of 0.604 µg/mL was found from ELISA indicating some problems with the calibration curves obtained in the SPR experiments. A whole lung cancer cell capture experiment was also conducted using microscopy imaging and the SPR setup. A number of 11 ± 2 cells/mm2 was captured from a pre-modified metal surface, which was confirmed by SPR. / Graduate / 0752 / 0541

Du fait de leur haute stabilité et bas coût de production, les aptamères suscitent un intérêt croissant, depuis près de 20 ans, dans le design de biocapteurs en tant qu'élément de reconnaissance idéal. Le but de ce travail de thèse est de démontrer l'intérêt et la pertinence d'un outil tel qu'une biopuce à aptamères, associant les avantages des sondes aptamères à ceux d'une détection par SPRi (Surface Plasmon Resonance imaging), permettant une détection sans marquage et en temps réel d'interactions moléculaires. Dans ce but, deux aptamères anti-thrombine (APT1 = 5′- GGT-TGG-TGT-GGT-TGG -3′ et APT2 = 5′-AGT-CCG-TGG-TAG-GGG-AGG-TTG-GGG-TGA-CT-3′) ont été choisis comme objets d'étude modèles. Ce choix a permis d'orienter différents axes de recherche : utilisés indépendamment comme sondes lors de l'élaboration de notre biopuce, ils ont tout d'abord permis de réaliser une détection cinétique optimisée de la thrombine, avec des performances remarquables pour une détection de ce type, ainsi que le calcul de constantes de dissociation en solution et à la surface des biopuces. Mais au-delà d'un simple biocapteur, la biopuce a également pu être utilisée comme véritable plateforme d'étude de la thrombine et de ses interactions, au sein de structures plus complexes telles que la structure « sandwich » entre les deux aptamères, ou d'autres interactions impliquant la thrombine en tant qu'acteur de la cascade de coagulation (inhibition de la thrombine par l'antithrombine III et le cofacteur II de l'héparine, transformation de la prothrombine au sein du complexe prothrombinase). / For 20 years, aptamers have been raising an increasing interest for biosensor applications as replacements for antibodies, due to their high stability and low cost. The main objective of this Ph.D. thesis is to show the great capacities of an aptamer biochip that combines the advantages of aptamer probes associated with a SPRi (Surface Plasomn Resonance imaging) detection to monitor, in real-time and in a label-free manner, molecular interactions occurring on the surface of the biochip. Two aptamers selected against the thrombin protein (APT1 = 5′- GGT-TGG-TGT-GGT-TGG -3′ and APT2 = 5′-AGT-CCG-TGG-TAG-GGG-AGG-TTG-GGG-TGA-CT-3′) were chosen as models for our study. This choice led to the exploration of different lines of research. First, both aptamers were used independently to develop a kinetic biosensor with remarkable performances for the quantification of thrombin. This tool served to determine independently, and compare, both the solution- and surface-phase affinities of the trombin-APT2 interaction. But more than a simple and effective biosensor, this kind of biochip represents a true platform to study the protein and its interactions within complex structures, such as the sandwich-like architecture with APT1 and APT2, or its interactions with other factors of the coagulation cascade (inhibition of thrombin by antithrombin III and heparin cofactor II, conversion of prothrombin into thrombin by the prothrombinase complex).

Ferrocene-peptide conjugates represent a hybrid area between organometallic chemistry and biochemistry. In these bioconjugates, the ferrocene (Fc) moiety can serve as molecular scaffold, chromophore, sensitive probe, biological marker, redox active site, etc. Disubstituted Fc systems, in which both cyclopentadienyl rings are substituted, provide influence over the supramolecular structure of the assemblies, and serve as starting materials for the design of electronic biomaterials. Recently, 1'-amino-ferrocene-1-carboxylic acid (Fca) and 1,1'-diaminoferrocene Fc[NH2]2 were recognized as useful tools in bioorganometallic chemistry. This work sketches some novel preparative and structural aspects of Fc-peptide conjugates and explores their applications as biosensors and as polymeric materials. <p>First, I demonstrated that Fca invariably induces a turn-like structure, which is stable in solution and the solid state. The obtained results showed different behaviour for Fca peptides depending on the chirality and position of the attached amino acid. The axial chirality of the Fca is completely dependent on the chirality of the first amino acid attached to the amino terminal of the Fca group.<p>Second, I was able to develop a surface based sensor for the electrochemical detection of papain based on Fc-peptide conjugates. The idea was to place a surface-bound redox probe in close proximity to the electrode surface. In addition, the redox-active Fca label will be part of the recognition site but will not interfere with the recognition process. My sensor provides an attractive alternative for the electrochemical detection of non-labelled non-redox active proteins, which under current detection schemes remains a significant challenge.<p>This work represents a truly important proof of concept for establishing this novel bioorganometallic approach for the electrochemical detection of important biological targets.<p>Last, I was successful in developing a very convenient method to synthesize 1,1'-bis(tert-butoxycarbonylamino)ferrocene as a stable derivative of Fc[NH2]2. This new synthetic approach has circumvented the problems encountered with the explosive diazide usually used as a precursor in the conventional synthons of Fc[NH2]2. Building on this achievement, a series of novel peptide-like oligomeric and polymeric ferrocenyl-amides were synthesized and fully characterized. The electrochemical investigations on these polymers suggested unresolved or no electronic interaction between the ferrocene groups in all systems. These results may reveal the influence of the amide groups on suppressing the electronic interaction between the iron centers in my polymers. <p>Thus, my systematic work on Fc-peptide conjugates lays solid foundations in the fields of structural control, biosensors, and material science.

Ferrocene-peptide conjugates represent a hybrid area between organometallic chemistry and biochemistry. In these bioconjugates, the ferrocene (Fc) moiety can serve as molecular scaffold, chromophore, sensitive probe, biological marker, redox active site, etc. Disubstituted Fc systems, in which both cyclopentadienyl rings are substituted, provide influence over the supramolecular structure of the assemblies, and serve as starting materials for the design of electronic biomaterials. Recently, 1'-amino-ferrocene-1-carboxylic acid (Fca) and 1,1'-diaminoferrocene Fc[NH2]2 were recognized as useful tools in bioorganometallic chemistry. This work sketches some novel preparative and structural aspects of Fc-peptide conjugates and explores their applications as biosensors and as polymeric materials. <p>First, I demonstrated that Fca invariably induces a turn-like structure, which is stable in solution and the solid state. The obtained results showed different behaviour for Fca peptides depending on the chirality and position of the attached amino acid. The axial chirality of the Fca is completely dependent on the chirality of the first amino acid attached to the amino terminal of the Fca group.<p>Second, I was able to develop a surface based sensor for the electrochemical detection of papain based on Fc-peptide conjugates. The idea was to place a surface-bound redox probe in close proximity to the electrode surface. In addition, the redox-active Fca label will be part of the recognition site but will not interfere with the recognition process. My sensor provides an attractive alternative for the electrochemical detection of non-labelled non-redox active proteins, which under current detection schemes remains a significant challenge.<p>This work represents a truly important proof of concept for establishing this novel bioorganometallic approach for the electrochemical detection of important biological targets.<p>Last, I was successful in developing a very convenient method to synthesize 1,1'-bis(tert-butoxycarbonylamino)ferrocene as a stable derivative of Fc[NH2]2. This new synthetic approach has circumvented the problems encountered with the explosive diazide usually used as a precursor in the conventional synthons of Fc[NH2]2. Building on this achievement, a series of novel peptide-like oligomeric and polymeric ferrocenyl-amides were synthesized and fully characterized. The electrochemical investigations on these polymers suggested unresolved or no electronic interaction between the ferrocene groups in all systems. These results may reveal the influence of the amide groups on suppressing the electronic interaction between the iron centers in my polymers. <p>Thus, my systematic work on Fc-peptide conjugates lays solid foundations in the fields of structural control, biosensors, and material science.

Ce document présente les notions de base permettant la conception d'un système de biodétection reposant sur la résonance des plasmons de surface. Il expose ensuite les grandes étapes de la conception du système, sa caractérisation, de même que son application à la mesure de l'activité cellulaire. Plus particulièrement, un système de biodétection basé sur la résonance des plasmons de surface a été conçu et réalisé.Ce système, intégrant deux types de détection, soit la modulation de l'angle de couplage aux plasmons de surface ainsi que la modulation de l'intensité d'un laser réfléchi sur le substrat, a été fabriqué sous la forme d'un goniomètre vertical. Un prisme sur lequel était déposé le substrat assurait le couplage de ce dernier avec un laser, permettant la mesure de la résonance des plasmons de surface.Ce système a été caractérisé quant à sa fidélité, sa sensibilité, sa plage dynamique et sa résolution. Finalement, le système a été appliqué à la mesure de l'activité de cellules vivantes induite par l'injection d'agents reconnus pour leur effet sur la morphologie des cellules. Cette application démontre qu'un biocapteur à large plage de détection peut être formé par la combinaison de la résonance des plasmons de surface et d'une monocouche de cellules vivantes.Ce biocapteur permet de détecter en temps réel des agents chimiques ou biologiques induisant des changements morphologiques dans la cellule.

L'accessibilité aux techniques d'analyse avancées est souvent un problème pour l'établissement des diagnostics, posés par le personnel médical. Les techniques classiques requièrent souvent des installations considérables (laboratoires d'analyses) ou utilisent des équipements volumineux et difficilement disponibles. Dans le but de résoudre ce problème, l'usage d'une plateforme technologique composée de l'intégration partielle d'un biodétecteur sur une structure auto-émettrice est un intéressant point de départ. Cette plateforme considère le problème d'accessibilité à la technologie directement en réduisant la taille et le coût relié à celle-ci. L'usage d'une structure compatible aux procédés de microfabrication couramment utilisés dans l'industrie de la microélectronique indique la possibilité d'augmenter l'échelle de production aisément et à faible coût. En contrepartie, les systèmes non intégrés sont généralement plus polyvalents sur les procédés de détection possibles mais aussi plus sensible, grâce à des systèmes optiques complexes. L'intégration d'un système interférométrique et son couplage à la plateforme technologique déjà existante permettraient l'implémentation d'une mesure basée sur la détection de phase additionnelle à la mesure classique de l'intensité, menant ainsi à l'augmentation de la sensibilité du biodétecteur. En se basant sur la théorie électromagnétique des modes couplés dans les structures laminaires pour créer le tissu théorique et sur des simulations FEM (modélisation par éléments finis) pour effectuer les démonstrations préliminaires, l'objectif de cette maîtrise consiste à étudier les caractéristiques d'un biodétecteur SPR (résonance de plasmons de surface) dont la mesure d'indice de réfraction de surface utilise une approche interférométrique. Pour ce faire, une microstructure est ajoutée à la surface du biodétecteur pour coupler une lumière incidente cohérente aux modes de surface. Ces modes de surface sont le produit de l'interaction et de l'interférence des plasmons diffractés par les différentes composantes de la microstructure. Dans un cas de microstructure simple, par exemple une paire de réseaux finis adjacents, l'analyse détaillée de l'interaction des plasmons diffractés est possible, comme il sera démontré dans ce document. Cette interaction sera ensuite liée à la forme de la résonance de la structure et comparée à d'autres cas simples, dont une structure classique de SPR. Cette transformation de la forme de la résonance augmente la précision globale de la mesure du biodétecteur sans en augmenter grandement la complexité. La méthode interférométrique promet des résultats très intéressants sous certain paramètres, aussi mis en évidence dans ce mémoire.