La fabrication de nano-cristaux métalliques suscite un effort de recherche en constante augmentation depuis plusieurs années. Cet immense intérêt est motivé par les propriétés uniques et fascinantes qui apparaissent à l’échelle des tailles nanométriques. En effet, le confinement des électrons au sein d’un nanocristal est un moyen puissant de moduler les propriétés électroniques, optiques et magnétiques d’un matériau. Les synthèses par voies chimiques sont des stratégies très rependues pour fabriquer des nanoparticules métalliques avec des morphologies originales en exploitant la versatilité des milieux réactionnels liquides pour contrôler les mécanismes de formation. Cependant, si la chimie employée lors de ces synthèses n’est pas très compliquée, la compréhension des processus de nucléation/croissance en milieu liquide complexe et l’influence de chaque espèce chimique est un tout autre challenge. Pour y répondre, nous avons utilisé la microscopie électronique en transmission en milieu liquide pour visualiser des phénomènes de croissance à l’échelle nanométrique. Cette récente technique de microscopie in situ nous a permis d’étudier en temps réel la dynamique de croissance de nanoparticules d’or et d’or-cuivre dans des milieux réactionnels de composition contrôlée. Le premier objectif de cette thèse était de distinguer les effets cinétiques (liés aux flux de matière) et les effets thermodynamiques (liés à l’équilibre des nanostructures en fonction de leur environnement) qui dictent tous les deux la forme finale des nanoparticules. De plus, des études systématiques nous ont permis de séparer les inévitables effets du faisceau d’électron, des effets de paramètres spécifiques de la synthèse, comme la forme des germes ou la fonctionnalisation organique, qui sont de toute première importance en chimie des colloïdes. Enfin, des phénomènes induits par le faisceau ont aussi été exploité pour comprendre l’influence de l’irradiation sur la chimie du milieu réactionnel, qui peut induire des réactions d’oxydo-réductions réversibles et contrôlables dans les nano-systèmes bimétalliques. / Recent years have seen a remarkable increase in research activities related to the synthesis of metallic nanocrystals. This intense interest is fueled by the unique and fascinating properties delivered at such size domains. Indeed, electrons confinement by nanocrystals is a powerful means to modulate electronic, optical and magnetic properties of a material. Most current strategies employ chemical synthesis to formulate unique nanoparticle morphologies by exploiting the versatility of liquid reaction media to control the formation mechanisms. Although the chemistry of metal nanocrystal synthesis is not complicated, understanding the nucleation and growth processes in complex liquid media and the influence of each chemical species is altogether a different challenge. It is in this regard, that we have utilized liquid cell transmission electron microscopy to visualize relevant growth phenomenon at the nanoscale. This recent in situ technique allowed us to study in real time the dynamics of growth of Au and Au-Cu nanoparticles in reaction media of controlled composition. The primary goal of this thesis was to distinguish the kinetics effects (related to the flow of matter) and the thermodynamics effects (related to the environment-dependent equilibrium of nanostructures) on final nanoparticle shapes. In addition to this, systematic studies were performed to separate the inevitable beam effects from the influence of specific synthesis parameters such as the seed crystal morphology and the organic functionalization that are of primary importance for colloidal chemists. Beam induced phenomena were also utilized to understand the solution chemistry of the exposed solvent which is in turn responsible for driving reversible redox reactions in bimetallic nano-systems.

Depuis quelques années, le développement et l’utilisation des nanomatériaux suscitent une attention croissante pour la communauté scientifique. L’intérêt pour ces matériaux s’explique par l’apparition de nouvelles propriétés qui, à l’échelle nanométrique, deviennent modulables en fonction de leur taille et leur forme.De par leur taille, les microplasmas sont particulièrement adaptés à la synthèse de nanomatériaux. En effet, les microplasmas représentent une classe de décharges électriques dont au moins l’une des dimensions est réduite sous l’échelle millimétrique. Leurs propriétés uniques en font également un excellent choix dans le cadre de la synthèse en phase liquide.Dans le cadre de ce travail, les mécanismes de formation et de croissance de nanoparticules d’or et d’argent synthétisées par microplasma à pression atmosphérique ont été étudiés. La première partie de ce travail s’est concentrée sur la mise en évidence des paramètres clés pour le contrôle de la nucléation et de la croissance des nanoparticules.Les études menées lors de la synthèse de ces nanoparticules, par spectroscopie d’absorption UV-visible, microscopie électronique en transmission, diffusion dynamique de la lumière et spectroscopie des photoélectrons X, ont permis de mettre en évidence le rôle de l’agent stabilisant ainsi que l’effet du temps de traitement et du courant de la décharge sur le diamètre et la distribution de taille des particules. Il a également été observé que la concentration en électrons, contrôlée par le courant injecté, et la concentration en précurseur métallique influençaient de manière opposée le diamètre moyen des particules. En effet, aux concentrations élevées en précurseur, les phénomènes de croissance et la réduction directe à la surface des noyaux métalliques sont favorisés alors qu’aux courants élevés, la nucléation est majoritaire.Dans la deuxième partie du travail, la réduction de l’acide tétrachloroaurique et du nitrate d’argent a été étudiée dans le but d’élucider le rôle des différentes espèces impliquées dans les mécanismes de formation des nanoparticules d’or et d’argent.Dans un premier temps, des études par spectroscopie d'absorption UV-visible, par conductivité ionique et par potentiométrie ont mis en évidence que la réduction des deux métaux est directement dépendante de la quantité de charge injectée par le plasma dans le système et donc du nombre d’électrons.Cependant, plusieurs différences ont été observées entre la synthèse des nanoparticules d’or et d’argent. D’une part, l’efficacité faradique de la réduction du nitrate d’argent est supérieure à celle de l’acide tétrachloroaurique. D’autre part, il a pu être montré que le complexe d’or continuait à être réduit après que le plasma soit éteint. Ces différences nous ont menés à l’hypothèse que d’autres espèces que les électrons, de temps de vie plus longs, pouvaient être impliquées dans le mécanisme de réduction.C’est pourquoi dans un second temps, les phases aqueuse et gazeuse ont été caractérisées par spectroscopie d’absorption UV-visible, chromatographie ionique, spectrométrie d’émission optique et spectrométrie de masse et ce, afin d’étudier l’influence des différentes espèces formées dans les deux phases sur la synthèse des nanoparticules et particulièrement, le peroxyde d’hydrogène.Il a alors pu être montré que les électrons étaient impliqués dans d'autres processus de transfert de charge que la réduction des sels métalliques tels que l’oxydation de l’eau mais aussi la formation du peroxyde d’hydrogène, des nitrites et des nitrates.Finalement, le rôle du peroxyde d’hydrogène dans le mécanisme de synthèse des nanoparticules d’or a été démontré, en opposition aux résultats observés pour le sel d'argent qui suggèrent que les électrons solvatés sont les principales espèces réductrices et qu'une voie de réduction plus directe a lieu dans ce cas. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Anion recognition is a topical area of research warranted by the potential applications of anion receptors in environmental and biological monitoring. Anions are indeed widespread in nature, are involved in many biochemical processes and are also major aqueous pollutants. Molecular receptors able to recognize anions with high affinity and selectivity in organic solvents are well documented in the literature but only few systems are efficient in aqueous media. Water is undeniably a particularly challenging solvent to work with due to the competition of water in the recognition process. Moreover, most of the receptors that are known to bind anions with high affinity and selectivity in organic solvents are not soluble in water. One strategy used to make hydrophobic molecular receptors “water-compatible” is micellar incorporation. This strategy is straightforward as no synthetic modifications of the receptor are required and has furthermore been seen to enhance the apparent properties, in particular binding properties, of the receptors. Following previous work undertaken in the laboratory, this thesis was devoted to the study of the micellar incorporation of different anion receptors. The first part of this thesis focused on the potential of Nuclear Magnetic Resonance (NMR) and more precisely Paramagnetic Relaxation Enhancement (PRE) experiments to provide robust information on the localisation of receptors within micelles. We studied the effect of various parameters and were able to rationalize the effect of the nature and concentration of the counterion and of the surfactant concentration on the PRE values obtained with cationic cetyltrimetylammonium (CTAX) micelles. By applying a normalization procedure we were then able to compare different receptor/micelle systems. This work has been reported in the Journal of Physical Chemistry (“Paramagnetic Relaxation Enhancement Experiments: a Valuable Tool for the Characterization of Micellar Nanodevices”, F. Keymeulen, P. De Bernardin, A. Dalla Cort, and K. Bartik, J. Phys. Chem. B, 2013, 117, 11654–11659).The second part of our work consisted in the study of the role played by the surfactant on the efficiency of the supramolecular system formed. Using UV-vis and/or NMR titrations, we studied the impact of the nature of the surfactant (cationic, zwitterionic or neutral) as well as its concentration on the apparent binding affinity for fluoride of two uranyl-salophen receptors. We showed that the supramolecular systems formed with cationic micelles are the most efficient, due to the favourable electrostatic interaction between the positively charged micelle and the fluoride despite the competition of the surfactant counter-ion. The concentration of the cationic surfactant does however have an impact as the apparent affinity decreases with surfactant concentration as a consequence of this non-specific interaction of the guest with the micelles. PRE and DLS (Dynamic Light Scattering) experiments allowed us to better understand the differences between the different types of micelles. This work has been reported in Organic & Biomolecular Chemistry (“Fluoride binding in water with the use of micellar nanodevices based on salophen complexes”, F. Keymeulen, P. De Bernardin, I. Giannicchi, L. Galantini, K. Bartik, and A. Dalla Cort, Org. Biomol. Chem. 2015, 13, 2437–2443).The final part of our study was devoted to the investigation of the applicability of micellar incorporation to other receptors. Two other uranyl-based receptors were studied in cationic CTAX and neutral Triton X-100 micelles. One suffered from chemical stability issues and the other receptor did not perform any better than the ones previously studied. We also studied trimesitylborane which can bind fluoride via Lewis acid-base interactions. This system, which is highly efficient in organic solvents, was shown to be ineffective once incorporated into micelles, probably because the change in the hybridization of the boron atom upon fluoride binding is not favourable in the confined micellar environment. Indolocarbazole-based anion receptors, which recognize acetate and benzoate via hydrogen bonding, were successfully incorporated into DPC micelles, albeit at low concentrations, and were observed to be efficient as the apparent binding affinity measured in water is of the same order of magnitude or higher as the one observed in organic solvents. / Doctorat en Sciences de l'ingénieur et technologie / info:eu-repo/semantics/nonPublished

Today, development of accurate early diagnosis of cancers thus became the number one challenge of medicine during the 21st century as the current techniques relies on imaging methods that suffer from low sensitivity and misdiagnosis.For these reasons, in this work, we aimed at developing a no-wash colorimetric biosensor for the detection of the oncoprotein Mdm2. Indeed, abnormal levels of Mdm2 could be related to the early formation of tumors.This thesis is devoted to the conception of a no-wash colorimetric biosensor for the detection of the oncoprotein Mdm2. This work can be divided in four parts:(i) The detection strategy and the design of the recognition elements (Chapter I).(ii) The conjugation of gold nanoparticles with the recognition elements (Chapter II, III, IV, V and VI).(iii) The modification of the metallic core of the nanoparticles (Chapter VII).(iv) The use of the optimized biosensor for the detection of Mdm2 (Chapter VIII).In the first part, we investigated the sensing strategy. An aggregation-based assay with plasmonic nanoparticles was selected, as the detection signal is a change of color of the suspension that can be observed to the naked eye or by UV-Vis spectroscopy. We designed the recognition elements, two peptide aptamers coming from endogenous proteins p53 and p14, and we grafted them separately on two batches of gold nanoparticles (AuNPs) via thiol end-groups. We used these latter for the detection of various concentrations of Mdm2 in buffer using our dual-trapping strategy with these two batches of functionalized AuNPs. We demonstrated that both peptides are able to interact with Mdm2 even after grafting onto the particles and that this detection strategy is highly specific. However, this first sensor presented some drawbacks, such as a poor colloidal stability of the AuNPs and a limited dynamic range.With the aim to encompass these issues we investigated, in the second part of this thesis, alternative strategies to conjugate the peptides to the particles. We investigated the functionalization of the particles with stabilizing ligands such as thiolated poly(ethyleneglycol) (HS-PEG). We first studied their simultaneous grafting with the peptides on the AuNPs. We observed that grafting HS-PEGs and peptides side-by-side allowed to control the density of peptides conjugated to the AuNPS and increased drastically the stability of the particles. However, the detection of Mdm2 was strongly hindered by the presence of PEG on the particles carrying the p14 peptide. In a second step, we investigated the conjugation of peptides on the top of a PEG layer carrying functional groups (HS-PEG-X where X is a carboxylate or an alkyne). AuNPs were first functionalized with mixtures of HS-PEG and HS-PEG-X, and the peptides were conjugated to the functional groups via amide bond formation or CuAAC coupling in a second step. However, we noticed that it was not possible to control the composition of the mixed layer of PEGs and thus the peptide grafting density.Due to the lack of recognized protocols in the literature for (i) the determination of the chemical and colloidal stabilities of AuNPs and (ii) the determination of the proportion of different ligands in the organic coating of the particles, we developed two interesting tools. The first one was a convenient method allowing to evaluate by UV-Vis spectroscopy the efficiency of the citrate exchange process using thiol-, alkyne- or diazonium-ligands from gold nanoparticles synthesized via a Turkevich method. The second protocol was a method allowing to quantify the proportion of two HS-PEGs ligands grafted in mixtures onto gold nanoparticles via 1H NMR spectroscopy.As we couldn’t find conditions in which the proportion of multiple thiolated ligands can be controlled on AuNPs, we decided to investigate another functionalization strategy based on the use of calix4arene-diazonium salts.We first studied the grafting on AuNPs of calixarenes bearing four PEG chains at the level of their small rim, one ended by a carboxylic acid and three by a methoxy group. The calixarene layer allowed to obtain AuNPs covered by a very dense PEG shell (with more PEG chains/nm2 that what was obtained previously with thiol anchoring). In addition to that, this PEG shell was strongly anchored to the AuNPs, conferring them a very high colloidal and chemical robustness. We then combined the grafting of this calixarene with the grafting of another non-functional calixarene, bearing four PEG chains ended by a methoxy group, and we quantified the conjugation capacity of such particles by amide bond formation. We demonstrated that this strategy allows to (i) increase drastically the stability of the AuNPs and (ii) control the proportion of peptide conjugated at their surface. Finally, we showed that calixarene-coated AuNPs to which to the p53 and p14 peptides have been conjugated could be used to detect Mdm2.With the evidence that the peptide conjugation density could be controlled using calixarene-coated AuNPs, we investigated the simultaneous grafting of two functional calixarenes on particles: one bearing four carboxylic acids groups and one bearing four PEG chains ended by alkyne groups. We optimized the grafting of these calixarenes in mixed layers on the AuNPs as well as their conjugation. We demonstrated that the grafting of two functional calixarenes led to the production of bi-functional AuNPs, capable of conjugation with two molecules via two distinct chemistries.In the third part, we optimized the composition of the metallic core of the biosensor. As it is well known that silver nanoparticles express better optical properties than gold nanoparticles of the same size, we aimed to incorporate silver nanoparticles (AgNPs) in the biosensor. This was a true challenge due to the intrinsic low chemical stability of silver nanoparticles that greatly limits their use in IVD. For this purpose, we developed an innovative in situ synthesis of silver nanoparticles in the presence of the calixarene-diazonium salts. After optimization of the synthesis, we observed that calixarenes bearing four carboxylic acids groups at the level of their small rim allowed the production of ultra-stable silver nanoparticles to which biomolecules can easily be conjugated. This in situ synthesis procedure even allowed us to produce alloy nanoparticles, with metallic cores whose composition could easily be tuned from pure silver to silver/gold alloys or pure gold. With this synthesis, the composition of the organic layer could also be easily tuned by using mixtures of calixarenes-diazonium salts.Finally, in the last part, we investigated the detection of Mdm2 with the optimized version of the biosensor, i.e. silver nanoparticles coated by a calixarene layer to which the p53 and p14 peptides were conjugated. With this novel class of nanoparticles, we could encompass the two initial drawbacks of the initial sensor. First, we were able to detect Mdm2 with a wider detection range and a lower limit. Secondly, the particles were sufficiently stable and robust to be dispersed in physiological fluids and we could detect Mdm2 in human serum without interference. / Doctorat en Sciences de l'ingénieur et technologie / info:eu-repo/semantics/nonPublished

The development of synthetic molecular receptors that can selectively bind anions, translocate them through a lipidic bilayer membrane and release them on the other side is a very topical and emerging field of supramolecular chemistry, warranted by the biological importance of transmembrane anion transport.The first part of this thesis is devoted to the study of the transmembrane transport of chloride and of the organic ion pair propylammonium chloride with calix[6]arene receptors functionalized with three (thio)urea arms on their small rim. The transport of chloride across the lipid bilayer of liposomes was monitored by fluorescence spectroscopy using the lucigenin assay. We report the first example of calix[6]arenes able to act as mobile carrier for the transport of chloride via a Cl-/NO3- antiport. We furthermore show that our calixarene systems are able to perform the cotransport of propylammonium chloride, with the chloride bound at the level of the (thio)urea groups and the ammonium included in the calixarene cavity. To provide direct proof of cotransport, we developed a 1H NMR methodology involving a thulium- complex shift reagent with which we were able to distinguish the signals of the ammonium transported inside the liposomes from those of the external ammonium. We also highlight the role of the complexing calixarene cavity for the cotransport by comparing the calixarenes to known transporters deprived of a cavity. The transmembrane transport organic ion pairs could find applications in the transport of biologically relevant ammonium compounds such as catecholamines and amino acids. Our results are reported in the publication “Repositioning Chloride Transmembrane Transporters: Transport of Organic Ion Pairs” Grauwels, G. Valkenier, H. Davis, A. P. Jabin, I. Bartik, K. Angew. Chemie - Int. Ed. 2019, 58, 6921–6925.The second part of this thesis is devoted to the study of binding of chloride to receptors embedded in a lipid membrane, the first step of the transmembrane transport process. Both 1H and 31P NMR spectroscopy proved to be inadequate to study the binding using liposomes or micelles as model membranes. With liposomes, the NMR signals are too broad to be exploited and in the case of micelles, the competition between the lipid headgroups and chloride made it impossible to obtain a NMR signature which unambiguously characterizes chloride binding. The 35Cl NMR signal is on the other hand strongly affected by the presence of anion receptors, both in organic solvents and when incorporated lipid bilayers. We developed a methodology to evaluate the binding of chloride, based on the monitoring of the chloride linewidth during titration experiments. A linear relationship between the linewidth and the concentration of receptors is observed and the slopes can be exploited to compare the binding strengths of different structurally related receptors. We show that 35/37Cl NMR is a versatile tool which can help in the understanding and development of new transporters by providing new insights of the physicochemical understanding of the transport process. / Doctorat en Sciences de l'ingénieur et technologie / info:eu-repo/semantics/nonPublished