Keywords: Synthèse organique, surface, fonctionnalisation, monocouches auto-assemblées, chimie bioorthogonale Organic synthesis, surface, functionalisation, Self-assembled monolayers (SAMs), bioorthogonal chemistry

The ability to control surface properties and produce molecularly engineered SAMs is the key aspect for a precise biomolecules immobilisation in a preferred manner, which is of major importance for the development of many bioapplications. In this work, we provide first the novel synthesis of different types of coupling agents used to provide two types of azideterminated SAMs: bearing an alkyl spacer or a urea spacer of different lengths. Then, we present in details the deposition of SAMs by using different methods and strategies in different solvents. Each change has been deeply studied to investigate its effect on the organic matter within the SAMs and the azide orientation, as well as the reactivity of the azido group towards the click reactions (CuAAC). The chemical modifications of the surfaces have been investigated by the PM-IRRAS, XPS, AFM and the contact angle measurements. Thus, this work ends up with a relatively big list of well-defined azide-terminated SAMs, as good candidates for various bioapplications. Some of azide-terminated SAMs have been successfully used to immobilise GNLs, resulting in differences of immobilised GNLs quantity on the surface depending on the nature of SAMs and the quantity of azide groups. This platform shows promising results when tested for stem cells culture.

Titre : Ingénierie moléculaire de SAMs constituées d'organosilanes terminées azoture pour des applications en biologie

Résumé : La capacité à contrôler les propriétés de surface et à réaliser l'ingénierie moléculaire de SAMs est déterminant pour permettre une immobilisation contrôlée des biomolécules, ce qui est d'une importance majeure pour le développement de nombreuses applications dans le domaine de la biologie. Dans ce travail, nous présentons d'abord la synthèse de différents types d'agents de couplage utilisés pour fournir deux types de SAM à terminaison azoture : portant un espaceur alkyle ou un espaceur urée de différentes longueurs. Ensuite, nous présentons en détail le dépôt des SAMs en utilisant différentes méthodes et stratégies dans différents solvants. Chaque changement a été étudié en profondeur pour étudier son effet sur la matière organique dans les SAMs et l'orientation des azotures, ainsi que la réactivité du groupe azido vis-à-vis de la réaction de clic CuAAC. Les modifications chimiques des surfaces ont été étudiées par les mesures PM-IRRAS, XPS, AFM et d'angle de contact. Ainsi, ce travail se termine par une liste relativement longue de SAMs bien définies à terminaison azoture, ce qui en fait de bons candidats pour diverses applications dans le domaine de la biologie. Certains des SAMs à terminaison azoture ont été utilisées avec succès pour immobiliser les GNL. La quantité de GNL varie à la surface en fonction de la nature des SAMs et de la quantité de groupes azotures. Cette plateforme montre des résultats prometteurs lorsqu'elle est testée pour la culture de cellules souches.

which I desire to express my gratefulness.

To Dr. Emilie Genin and Dr. Luc Vellutini, my great supervisors, I am grateful for the trust deposited in my work and for the motivation demonstrated along this work. I am thankful to the patient guidance, encouragement, invaluable advice, and support. It was a real privilege and honour for me to share to them exceptional scientific knowledge but also of their extraordinary human quality.

I would like to express my special thanks to all collaborators, especially, Dr. Thierry Buffeteau for his collaborative effort during data collection and his contributions in results analysis. I wish to thank the members of my dissertation committee: Dr. Sophie Lecomte, Dr. Thibaud Coradin, Dr. Michel Wong CHi Man, and Dr. Vincent Humblot for generously offering their time and guidance throughout the review of this document. I acknowledge the financial support of my research study from the French government through the French Consulate in Jerusalem. And special thanks to Mr. Farid Saadoun the great person in Campus France who was always helpful and available when needed.

I would like to extend my sincere thanks to my colleagues and all members in the research group C2M/ISM for a cherished time that we spent together in the lab, and in social settings.

The moments of the leisure we shared together helped to overcome some difficult moments. I am also truly thankful for their prompt help, wherever I needed it. I would like to express my gratitude to my friends, to all my new friends who I recently met in France, it is their kind help and support that have made my study a wonderful time. And I wish to extend a huge thank to all my friends in Palestine who are always caring, affectionate, supportive, good listeners, attentive, and funny despite distance. I am very lucky to have a big list of nice friends who I don't want to name to avoid forgetting unintentionally some of them, they know themselves and know how much I love them.

At the end, for all people who inspired me along my journey, I would like to say thank you for a bright smile, positive attitude, and for pushing limits personally and professionally.

VIII IX

Résumé

X XI

Résumé

Dans le domaine de la chimie des surfaces, les monocouches auto-assemblées (SAMs) constituent une méthode intéressante pour la préparation de surfaces peu rugueuses et ordonnées avec une fonctionnalité soigneusement contrôlée [1]. En particulier, les SAMs à base de siloxane forment des monocouches chimiquement et physiquement stables, ce qui les rend très intéressantes pour être utiliser sur des plateformes de détections ou dans l'étude des événements biochimiques. La possibilité d'adapter les caractéristiques de la surface, comme la présentation des biomolécules, a conduit à l'utilisation des SAMs dans une grande variété d'applications biologiques [2], y compris les biocapteurs, les substrats de culture cellulaire et comme interfaces pour analyser les interactions biomoléculaires [3] [4]. Les performances finales des systèmes utilisés dans les bioapplications dépendent fortement de l'état de surface du substrat et de la capacité du support à immobiliser efficacement les biomolécules d'intérêt, ainsi que de sa biocompatibilité [5].

Dans de nombreux cas, l'immobilisation des biomolécules sur des surfaces est nécessaire : (1) pour contrôler l'orientation et la densité, (2) pour établir une liaison forte avec le substrat, (3) pour favoriser une activité élevée et (4) une excellente stabilité à long terme. En outre, la propriété de sélectivité [1] est essentielle dans ce domaine. Une autre considération importante est que l'immobilisation dépend de l'accessibilité du groupe fonctionnel à la surface et de la densité de surface du groupe fonctionnel terminal, qui est un paramètre important car il détermine la quantité, la conformation et les propriétés physico-chimiques des systèmes immobilisés ainsi que la stabilité après la fixation. Pour faire face à ces défis, le contrôle du processus d'immobilisation peut être réalisé en contribuant à l'homogénéité de la couverture de la surface, de sorte que les dispositifs soient générés avec des caractéristiques uniformes et bien définies, fournissant un résultat reproductible et précis. Ce travail vise à développer des SAMs fonctionnelles avec des propriétés contrôlables et bien définies permettant l'attachement des biomolécules de manière bien contrôlée [5]. Le contrôle des propriétés des SAMs pourrait être réalisé par une étude approfondie du comportement des molécules utilisées pour la formation des SAMs, comme leur orientation, leur stabilité et leur réactivité [2].

Au cours des dernières décennies, les stratégies d'immobilisation des biomolécules ont permis d'utiliser de nouvelles réactions bioorthogonales qui se déroulent dans des conditions physiologiques et sélectives entre des groupes chimiques différents des fonctionnalités endogènes des biomolécules et qui ne réagissent pas avec elles [6] [7]. Parmi les différentes stratégies chimiques utilisées pour effectuer des réactions interfaciales sur les SAMs, la chimie XII dite "clic" a été l'une des plus employées. Le terme "click chemistry" a été introduit pour décrire des réactions chimiques spécifiques, qui sont rapides, fiables et peuvent être appliquées de manière sélective à la synthèse de matériaux fonctionnels et de conjugués de biomolécules [8] [9]. Sans aucun doute, la variante la plus souvent utilisée de la chimie clic est la réaction de cycloaddition 1,3-dipolaire catalysée par le Cu(I) (CuAAC) entre un azoture et un alcyne qui répond aux critères de bioorthogonalité et de chimiosélectivité. [10] La réaction peut être effectuée avec des azotures ou des alcynes sur les surfaces, l'utilisation d'azoture sur des surfaces offre l'avantage de pouvoir utiliser deux techniques spectroscopiques (PM-IRRAS et XPS) permettant de révéler sa présence [11].

Dans les domaines des SAMs et de l'immobilisation des biomolécules, les SAMs mixtes offrent des architectures intéressantes, afin d'offrir la possibilité de contrôler la densité en fonction terminales utilisables pour l'immobilisation des biomolécules. Les SAMs mixtes sont généralement constituées de deux composés, un composé avec un groupe principal fonctionnel et un autre composé non fonctionnel pour la dilution pouvant empêcher une adhésion de surface non spécifique. Le rôle d'un diluant est donc de réduire la concentration de surface du principal composant fonctionnel pour réduire l'encombrement stérique lors de l'immobilisation d'espèces biologiques [12] [13].

De manière générale, dans ce travail, nous avons choisi l'approche des SAMs utilisant des organosilanes avec un groupe terminal azoture pour la fonctionnalisation de la surface. Cette plateforme moléculaire pourra permettre l'immobilisation des biomolécules par la chimie bioorthogonale pour des bioapplications. L'approche générale utilisée dans ce travail est illustrée schématiquement dans la figure1. Chapitre 5 : Ce cinquième chapitre décrit l'utilisation des SAMs élaborées pour immobiliser les molécules GNL bioinspirées (un nouveau gélifiant de faible poids moléculaire) et fournir des SAM-GNL, qui seront utilisées comme plateformes de culture de cellules souches. À ce stade, la croissance des cellules souches dépend des propriétés physico-chimiques du système produit.

Le contrôle de l'orientation et de la distribution des biomolécules sur la surface artificielle est un grand défi dans des bioapplications telles que l'ingénierie tissulaire, les essais cellulaires ou les biocapteurs, etc.

Dans cette thèse, nous avons conçu des monocouches auto-assemblées (SAMs) avec des groupes terminaux azotures sur des surfaces de silice et évalué leurs caractéristiques XV structurelles, ainsi que leur performance pour l'immobilisation de biomolécules en utilisant la cycloaddition 1,3-dipolaire entre un azoture et un alcyne catalysée par Cu(I) (CuAAC). Deux types de SAM à terminaison azoture ont été préparés : Les SAMs portant un espaceur alkyle et les SAM portant un espaceur urée.

Les SAMs à terminaison azoture portant un espaceur alkyle ont été déposées avec succès en utilisant trois méthodes de dépôt différentes (post-fonctionnalisation et greffage direct par immersion ainsi que par spin coating) dans trois solvants différents (acétonitrile, toluène et chloroforme). Les surfaces chimiques modifiées ont été étudiées à l'aide de la spectroscopie d'adsorption par réflexion infrarouge à modulation de polarisation (PM-IRRAS) et de la spectroscopie photoélectronique à rayons X (XPS). La microscopie à force atomique (AFM) a également été utilisée pour obtenir des images de la topographie de la surface. Nous avons constaté que la méthode de dépôt et la nature du solvant de greffage pouvait affecter la quantité de matière organique dans la monocouche et impacter l'orientation de l'azoture. De plus, nous avons conclu que la réactivité du groupe azido en surface avec l'alcyne en solution n'est pas négligeable et semble être étroitement liée à l'orientation de l'azoture. En effet, plus l'azoture est orienté verticalement, plus il est accessible et réactif (figure 2). L'orientation des dipôles azoture à la surface dépend fortement de la méthode utilisée pour préparer la monocouche. La méthode de post-fonctionnalisation permet d'avoir des groupes d'azotures en surface avec une meilleure orientation verticale que celle obtenue par greffage direct par des méthodes de dépôt par immersion ou par spin coating. Quel que soit le type de SAM à terminaison azoture, la réactivité des groupes azido verticaux accessibles est totale. Cette étude démontre clairement qu'il est possible de contrôler la quantité d'azotures réactifs et, par conséquent, la quantité de molécules immobilisées sur la surface après la réaction de clic en choisissant la méthode de dépôt appropriée. En effet, pour chaque composé, la qualité de l'auto-assemblage dépend de la méthode de dépôt.

Par immersion, la force de la liaison hydrogène augmente avec le nombre d'atomes de carbone, alors que pour le procédé spin coating, c'est l'inverse. En général, pour les deux méthodes, les composés ayant le plus petit nombre de carbone (C6) semblent être plus favorables à un dipôle azoture vertical. A partir de ces SAMs uréido à terminaison azoture l'immobilisation de biomolécules par cycloaddition catalysée par Cu(I) a été réalisée avec succès. Les deux méthodes (immersion et spin coating) conduisent à la même réactivité du groupe azido terminal, soit environ 80 % de taux de réactivité, pour toutes les SAM uréido à terminaison azoture.

Toutefois, il y a une exception avec le composé avec le plus petit nombre de carbone (C6) préparé par la méthode d'immersion, qui présente un pourcentage de seulement 50 %. Cela montre que le minimum de 11 atomes de carbone est nécessaire pour avoir une bonne accessibilité du groupe azoture terminal dans la monocouche. En revanche, six atomes de carbone sont préjudiciables à l'accessibilité et donc à la réactivité. Cependant, l'auto-assemblage par liaison hydrogène reste trop faible pour résister au protocole de lavage, ce qui entraîne la perte totale du composé non silylé. En outre, l'analyse AFM semble montrer qu'il n'y a pas de ségrégation de phase entre les deux types de molécules, ce qui assure une distribution homogène des deux molécules dans la monocouche. Les deux types de SAMs pures à terminaison azoture avec un espaceur alkyle ou urée ont été utilisés avec succès pour immobiliser les GNL (figure 5). La quantité de GNL varie à la surface en fonction de la nature des SAMs et de la quantité de groupes azotures, ce qui pourrait affecter l'utilisation ultérieure en culture de cellules souches. En fin de compte, un système de GNL immobilisés sur des SAMs à terminaison azoture portant un espaceur alkyle a été évalué comme plateforme de culture de cellules souches pour deux types de cellules. Les résultats préliminaires ont montré que le processus de stérilisation des surfaces est efficace et que les surfaces fonctionnalisées ne sont pas toxiques pour les cellules. Nous avons également observé une amélioration de l'adhérence ainsi qu'une prolifération et un maintien du phénotype. Tous ces résultats sont prometteurs pour la suite des recherches. Pour conclure, ces travaux de thèse présente une étude approfondie des paramètres permettant l'ingénierie des SAMs terminées par un groupe azoture, comme un outil utile pour contrôler l'immobilisation des biomolécules et donc les applications souhaitées en biologie. Les perspectives de ce travail sont les suivantes :

1) La méthode spin coating pour réaliser une monocouche est une avancée qui devrait à terme être plus attractive que la méthode d'immersion classique, pour des raisons de mise en oeuvre bien plus facile.

2) Le nouveau concept de SAMs mixtes pourrait être modifié en essayant des molécules non silylées ayant une plus grande affinité pour la surface ou en introduisant une unité permettant une polymérisation latérale telle que les diacétylènes.

3) Les résultats préliminaires de l'utilisation des GNL pour la culture des cellules souches encouragent à réaliser davantage d'études sur de telles surfaces pour les cellules souches, en créant des systèmes avec différentes densités de GNL sur la surface pour voir l'effet de ce changement sur la croissance des cellules.

4) Enfin, cette thèse s'est terminée par une liste relativement longue de SAMs à terminaison azoture et ces SAMs sont bien étudiées et analysées, ce qui en fait de bons candidats pour diverses applications dans le domaine de la biologie ou dans d'autres domaines. Notre principale préoccupation étant les domaines biologiques, ces SAMs pourraient être testées pour immobiliser une variété de biomolécules (telles que l'ADN, les enzymes, les protéines et les peptides) ainsi que des polymères et des nanoparticules fonctionnalisées ou d'autres espèces terminées par un groupe fonctionnel approprié pour réagir avec l'azoture.

General introduction

General introduction

In the domain of surface chemistry, self-assembled monolayers (SAMs) provide a powerful method for the preparation of smooth, ordered surfaces with carefully controlled functionality [1]. In particular, siloxane-based SAMs form chemically and physically stable covalently attached monolayers on technologically relevant surfaces, which makes them an important tool in the study of biochemical events. The ability to tailor surface features, such as biomolecule presentation, has led to SAMs use in a wide variety of bioapplications [2], including biosensors, cell culture substrates and as interfaces to analyse biomolecular interactions [3] [4]. The final performance of bioapplications strongly depends on the surface condition of the substrate and the ability of the support to effectively immobilise the biomolecules of interest, as well as its biocompatibility [5].

In many cases, biomolecules immobilisation onto surfaces is needed with: (1) controllable orientation and density, (2) high bonding strength, (3) high activity and ( 4) with excellent longterm stability. Moreover, central to this field is the property of selectivity [1]. Another important consideration is that the immobilisation depends on the accessibility of the terminal functional group on the surface, and surface density of this terminal functional group, which is an important parameter as it determines the quantity, conformation, and physico-chemical properties of immobilised systems and the stability after the attachment. To face those challenges, control over the immobilisation process can be achieved by contributing to the homogeneity of the surface coverage, such that devices are generated with uniform and welldefined characteristics, providing reproducible and accurate output. This work aims to develop functional SAMs with well-controlled and well-defined properties enabling biomolecules attachment in a controllable way [5]. The control of SAMs properties could be achieved by a deep investigation (at the molecular level) of the behaviour of molecules during and after SAMs formation, such as their orientation, stability, and reactivity [2].

In the past few decades , the developments in the strategies for biomolecules immobilisation comprise the discovery of new bioorthogonal reactions that proceed under physiological conditions between chemical groups that are absent in, and do not cross-react with, endogenous functionalities in biomolecules [6][7]. Among the several chemical strategies reported to perform interfacial reactions on SAMs, the so-called "click chemistry" has been one of the most employed. The term "click chemistry" has been introduced to describe specific chemical reactions, which are fast, reliable and can be selectively applied to the synthesis of functional materials and biomolecule conjugates [8] [9]. Without question, the most often employed variant of click chemistryand the reaction most closely associated with the termis the Cu(I)-catalysed 1,3-dipolar azide-alkyne cycloaddition (CuAAC) between azides and alkynes that meets the criteria of bioorthogonality and chemoselectivity. [10] The reaction can be run with either azides or alkynes on surfaces, the use of azides on surfaces offers the advantage of using two spectroscopic techniques (PM-IRRAS and XPS) [11].

In the fields of SAMs and biomolecules immobilisation, mixed SAMs provide attractive architectures, e.g., with an enhanced ability to immobilise biomolecules. Mixed SAMs are typically constitute of two compounds, one compound with a functional headgroup and another compound devoid of functional end-group for dilution that is supposed to prevent nonspecific surface adsorptionadhesion. The role of a diluent is also to reduce the surface concentration of the main functional component and thus prevent steric hindrance due to the binding of biomolecules at excessive density [12] [13].

Broadly speaking, in this work, we chose the approach of siloxane-based SAMs with an azide terminal group for surface functionalisation, as a helpful tool, in cooperation with bioorthogonal This strategy involves three main steps. First, the synthesis of azide-terminated coupling agents bearing either alkyl or urea spacers will be implemented. In this part the incorporation of a urea unit is to facilitate the synthesis and the self-assembly capacities of such molecules [14].

Second, we will investigate the elaboration of siloxane monolayers with tunable azide densities using the classical immersion method and the newly investigated spin coating method.

Moreover, a new concept of mixed SAMs will be investigated in this work for the family of urea compounds, by suggesting a model of mixed SAMs made of two types of molecules: silylated molecules with no functional end-group and non-silylated molecules with an azide functional group. This concept aims to enhance tune the distribution of molecules and space the functional groups in the SAMs by relying on the hydrogen bonds as the driving force of SAMs formation. All resulted SAMs will be characterised by various techniques, mainly Atomic Force Microscopy (AFM), X-ray Photoelectron Spectroscopy (XPS) and PM-IRRAS (Reflectionabsorption spectroscopy by polarisation modulation). Then, azide-terminated SAMs will react with alkyne-terminated species of interest by Cu-catalysed azide-alkyne cycloaddition (CuAAC). This step is to evaluate the azide reactivity and to be further used in particular for the immobilisation of Glycoside Nucleoside Lipids (GNLs) to create a new platform for stem cells culture.

List of abbreviations

Introduction

One known effective method for nanofabrication and material design is surface modification.

It can be simply described as changing the physical, chemical or biological characteristics (e.

g. conductivity, corrosion resistance, adhesion properties, biocompatibility, wettability, etc.) [1] [2] , aiming at improving specific responses of a material when interacting with its environment.

Notably, many fields have been significantly benefitted from surface modification to design innovative devices for a broad range of applications, but the enormous effect is on the biological applications (e. g. biosensing, cell culture, tissue engineering, drug delivery). But, in this fields, an ideal reaction used for surface biofunctionalisation should meet some requirements such as simplicity, efficiency, universality, and biocompatibility, etc [3] [4].

Among all surface modification techniques, chemical surface functionalisation is the simpler and most efficient way to tailor surfaces properties, both morphological properties and chemical composition, by applying functional coatings (organic, inorganic, or hybrid) to produce a welldefined set of functions on the surface [5]. Some methods have been explored using polymer and protein coatings [6] [7], acid/base treatment [8], and heat treatment as methods to modify surface properties but they suffer from some limitations [9]. Polymer and protein coatings are performed from large molecules with many reactive sites [6] [7]. However, it is difficult to introduce specific and appropriated orientation of chemical groups on a surface with welldefined concentration and spatial control [10]. Treating a surface with an acid or a base is also an effective means of introducing a specific functional group on the surface but it is limited to only a few chemical groups such as acidic, basic, or hydroxyl groups. While heat treatment such as flame annealing affects the physical properties but not the chemical properties of the surface [11] [12]. On the other hand, Self-Assembled Monolayers (SAMs) constitute a good way to chemically modified the surfaces of materials for several reasons: first their easy and rapid formation on different variety of surfaces, including glass, silicon, gold [13], amorphous carbon, diamond [14] and metals [15]. The second reason is the variety of terminal functional groups that can be used to tailor the surface for further modification. Those features make SAMs as an important tool in the study of biochemical events and to be used in a wide variety of applications, including biosensors, cell culture substrates, and as interfaces to analyse biomolecular interactions [3].

This chapter presents an introduction to both biomolecules immobilisation on surfaces and SAMs. It starts with an overview of the chemistry and strategies of biomolecules immobilisation to surfaces for bioapplications. Followed by a description of SAMs chemistry (definition, formation, properties, characterisation methods). We will pay special attention to presenting a state of the art of azide-terminated SAMs preparation and their chemistry as well as mixed SAMs elaboration.

Immobilisation of biomolecules to SAMs

Attaching biomolecules to surfaces has been developed in the last decades using several methods. All these methods can be classified into two categories based on the attachment mechanism : either an attachment through non-covalent interactions between biomolecules and the terminal groups of the SAM (such as electrostatic, hydrophilic, hydrophobic or polar ) or an attachment through covalent bonds between biomolecules and the end groups of the SAMs.

The method is chosen depending on the biomolecule of interest, the purpose of the immobilisation and the importance of the biomolecules orientation or deformation of the native structure after attaching to the surface [17].

Non-covalent immobilisation

Non Covalent or nonspecific adsorption processes often occur between the biomolecules and the solid surface, resulting in the loss of interface's functionalities. In this category, the attachment of biomolecules to the surface depends on charges or dipolar interactions. This category contains a lot of successful examples like the immobilisation of proteins, peptides, DNA, amino acids, sugar moieties and others on different types of SAMs with a variety of different functional groups [18] [19] [20]. The attachment can be achieved through two major methods:

Physical adsorption

This method relies on electrostatic interactions, van der Waals interactions, hydrophobic or polar interactions [20]. For example, the attachment of a robust protein BSA on a carboxylterminated SAM has been performed using electrostatic interactions between -CO2 -of the SAM end groups and pendent -NH3 + on BSA protein (figure 1.1) [17].

Affinity recognition

Sometimes the physical attachment process may involve pairs of molecules with very strong affinity interactions, verging on covalent force levels, such as biotin with streptavidin. The affinity of streptavidin for biotin is the strongest noncovalent biological interaction. Each streptavidin monomer can bind four biotin molecules. In addition to the strength of the interaction, this binding is highly specific, rapid on-rate, resistant to changes in temperature or pH and can withstand the presence of organic solvents and denaturing agents. Streptavidin and biotin are used extensively in a wide range of laboratory applications, and there are many ways in which their strong biological interaction can be exploited such as bio-molecules tagging or recognition, and biochemical sensing applications, especially in the identification of possible new drug targets [24]. One of the most crucial disadvantages of the non-specific immobilisation is the insufficient exposure of functional domains, largely due to a variety of unpredictable orientations that the immobilised biomolecules can adopt upon binding to the surface. This often results in binding of an unnecessary fraction of biomolecules with improper orientation, thus impeding their binding with ligands and subsequent downstream biological assays. Another possible drawback is that the noncovalent binding by hydrophobic interactions may cause biomolecules (such as protein or DNA) denaturation and the loss of their functional activity. The molecules immobilised on the surface may also be vulnerable to further manipulation, which may result in the graduate depletion of biomolecules adsorbed noncovalently [26].

Covalent immobilisation

To ensure that all biomolecules are functionally active, it is imperative that they are aligned uniformly and optimally upon immobilisation onto the surface. A variety of covalent immobilisation techniques have therefore been developed in the past few years.

In this category, the covalent bond is formed via a chemical reaction between the exposed functional groups on side-chains of the biomolecule and the functional groups on the surface.

Those bonds are stronger and more stable than the non-covalent bonds. For example, they resist to buffer solutions. The advantage of a covalent link is that the molecule is tethered at one site onto the surface rather than in contact over a significant part of its surface as in the case of physisorption. In comparison to non-covalent interactions, the covalent bonding of molecules on a surface can greatly shorten the time and provides a distinct, more robust, and stable way of tethering biomolecules to the surface.

Many chemical reactions have been reported for a covalent immobilisation, such as NHS (Nhydroxysuccinimide) ester amino ligation, aldehyde-assisted ligation, epoxy-amino ligation, thiol-maleimide, thiol-vinyl sulfone, amino-vinyl (Michael additions), and azide-alkyne (click)

conjugation reactions [20][27]. This type of conjugation can be used for various types of chemical and biological entities such as proteins oligonucleotides, polymers, nanoparticles (NPs), and cells [28]. In general, this category (the covalent immobilisation) can be classified into two types depending on whether the reactive function of biomolecules is naturally present (using native functions) or not site-specific chemoligation (using non-native functions).

Using native functions

In general, some biomolecules like proteins offer many functional groups naturally present, mainly in the amino acid side chains, that are suitable for immobilisation purposes. Such functional groups can be used to covalently couple proteins to surfaces by a range of different reactions. Suitable complementary groups should be installed on the solid support in advance to the biomolecule attachment. Figure 1.4 shows some typical examples of compatible groups attached to surfaces and the corresponding biomolecules reactive functional groups they react with. By far the most common method to covalently attach proteins to surfaces uses the amine group of the lysine side chain which can react with: surface-bound N-hydroxysuccinimide (NHS)-activated carboxylic acids forming stable peptide bonds [29], with aldehyde-derivatised glass slides forming imines [30], with isothiocyanate-functionalised surfaces forming thiourea [31] and with epoxy-modified surfaces forming aminoalcohol [32]. Amine-reactive surfaces have also been developed to attach proteins by their carboxylic acid sites via an amide bond [33]. It is important to note that protein attachment can occur simultaneously through many residues, thereby restricting degrees of conformational freedom (and thus possibly activity) and also increasing heterogeneity in the population of immobilised proteins [28].

Figure 1.4. Examples of method used for nonspecific covalent protein immobilisation [28] In this approach, immobilisation efficiency depends on several parameters: pH value, concentration, ionic strength, and reaction time. In some cases, conditions need to be optimised for several classes of proteins. To conclude, even though covalent immobilisation creates a strong bond between the biomolecule and the SAM, the attachment is random and nonspecific in nature. It is important to early mention that to get better control in immobilisation, ''bioorthogonal'' reaction can be used.

Using non-native functions

This approach depends on introducing groups functionalities are not present naturally (such as aldehydes, azides, or alkynes) into the biomolecule of interest, then the chemical immobilisation reaction can occur with another functional group on a surface or nanoparticles or another probe. The development of these chemical reactions has greatly been driven by the desire to selectively label a target molecule in complex mixtures of biomolecules, such as cells or living organisms. What is noteworthy is that these approaches, which have been described

as bioorthogonal chemistry, depend on the fact that the functional groups which are employed in the different reactions are absent and do not cross-react with the biological activities.

Typically, bioorthogonal reactions are exothermic by virtue of their highly energetic reactants or strongly stabilised products, and most involve the formation of carbon-heteroatom (mostly N, O, and S) bonds. The reactions are typically fusion processes that leave no byproducts.

Moreover, most bioorthogonal reactions reported in the literature are chemoselective [34].

The new term of bioorthogonal reaction was first coined by Carolyn Bertozzi in 2003, it is a rapid and reliable bioconjugation that refers to any chemical reaction that can occur inside living systems without interfering with native biochemical processes [35] [34]. Prescher and Bertozzi [36] defined bioorthogonal reactions as -non-native, non-perturbing chemical handles that can be modified in living systems through highly selective reactions with exogenously delivered probe. Most importantly, the covalent reaction between these two compounds needs to proceed rapidly and selectively in a physiological environment with biocompatible pH (6)(7)(8), and temperature (37 °C), have less byproducts and be nontoxic. The chemical reporter should be inert in vivo, have no reaction with the biological environment and small enough to modify the target substrate without any functional and spatial interference. Therefore, so far, only a handful of bioorthogonal reagents have been developed for biological use [37]. Commonly, this concept (bioorthogonal) is incorporated with the term click chemistry and provides a powerful highly specific tools that can be used for investigating for the efficient conjugation of biomolecules, nanomaterials, and living cells [38].

Click chemistry can be broadly defined as a ligation reaction in which two reactants are joined under ambient conditions to provide the desired product in high chemical yield and short time [39]. In biology, highly complex biological systems rely on a modest library of monomers linked together by click reactions. The objective of click chemistry is to establish an ideal set of easily performed, straightforward, and highly selective reactions with high yield and no side products. The term "click chemistry" was coined by K. Barry Sharpless in 1998, and was first fully described by Sharpless, Hartmuth Kolb, and M.G. Finn in 2001 [40] [41]. Afterwards, the popularity of click chemistry significantly increased and the number of publications in this field has increased dramatically [42] [43]. The commonly studied click reactions include, Cu(I) catalysed alkyne-azide (CuAAC) and Diels-Alder reactions as well as thiol-yne click, and thiol-Michael addition (Figure 1.5) [28]. The combination of high yield and outstanding selectivity afforded by these reactions has allowed the simple fabrication and functionalisation of biomaterials, importantly under conditions sufficiently mild. For example, Diels-Alder reaction is used to immobilise diene-modified carbohydrates (galactose, glucose, mannose, lactose, and maltose) and proteins onto a solid surface. Houseman reported a process using the Diels-Alder reaction to connect a saccharide-cyclopentadiene to a benzoquinone group on the SAM. A carbohydrate array was successfully made and was capable of identifying specific lectins [44] [45]. Over the last two decades, tremendous development and progress has been achieved in these conjugation reactions to encompass wide substrate scopes in the click reaction. Among all mentioned processes, most attention has been focused on the alkynes and azides click reactions to yield 1,2,3-triazoles, due to the general ease of execution, facile reaction conditions, and impressive orthogonality of this reaction [41]. water [47]. It is a significant tool for the detection and quantitation of azide-labelled modifications. However, the phosphines are reducing agents, easily oxidised by air or metabolic enzymes to generate phosphine oxide byproducts. When comparing to strain-promoted cycloaddition, it exhibits better sensitivity. For studies involving live organisms, the Staudinger ligation and copper-free azide-alkyne cycloaddition seem to be the better choices [37] [48].

The most primitive azide-alkyne cycloaddition was thoroughly explored by Huisgen and coworkers in the 1950-70s. It is a cycloaddition reaction of an azide and an alkyne to form a 1,2,3-triazole (figure 1.6). Huisgen 1,3-dipolar cycloaddition is exothermic and experienced very low reaction rates and yields. The high pressures and temperatures required in this reaction were the biggest constraint for its application in living systems. Furthermore, this cycloaddition has low regioselectivity since it gives two different regioisomers, namely the 1,4-and 1,5triazole, which are extremely difficult to separate. It had a great impact on further research.

These issues were later overcome by Tornøe and Meldal [START_REF] Meldal | Cu-catalyzed azide-alkyne cycloaddition[END_REF], who introduced Cu(I) catalysis in alkyne-azide coupling reactions, rendering the reaction faster and more selective. The addition of copper as a catalyst favours the formation of only the 1,4-regioisomer. The CuAAC reaction, i.e., the copper-catalysed cycloaddition reaction between alkynes and azides yielding triazoles, occurs effectively under an extensive range of environments and with many Cu(I) sources. Usually, copper (II) salts are used, such as copper sulphate pentahydrate or copper acetate, in combination with metallic copper or sodium ascorbate which act as reducing agents of copper(II) to copper(I) [47].

The term copper-catalysed azide-alkyne cycloaddition (CuAAC), a widely well-known example of click chemistry, was coined by Sharpless in 2002, it was described as a better version of Huisgen's [2 + 3] cycloaddition with little solvent dependence and better adherence of click chemistry principles. The presence of copper greatly changed the reaction mechanism and improved the yields, dramatically accelerated the cycloaddition reaction and most significantly, it can be performed below room temperature. On the other hand, due to the toxic features of copper, which can catalyse atmospheric oxygen reactions to form reactive oxygen species (ROS), strain-promoted alkyne-azide cycloaddition (SPAAC) has been developed by using cyclic alkynes, typically cyclooctyne derivatives. Nonetheless, compared to CuAAC, the copper-free click chemistry is still limited by its low rates and researchers are exploring new ways to increase the rate of strain-promoted cycloadditions. Moreover, for some in vitro studies, especially some proteomic applications, CuAAC seems to be the most efficient and convenient strategy [37] [48] [47]. The CuAAC reaction proceeds at considerably faster rates than the Staudinger ligation (more than 25-fold) and is usually 10-100 times faster than copper-free strain-promoted azide-alkyne with second-order rate constants of 10-200 M -1 s -1 in the presence of 20-500 μM Cu [50] [51].

The appealing bioorthogonal features of azide have enabled its applications in many areas of biology. The azide-based bioorthogonal click chemistry has demonstrated its advantages within the last 10 years [52] [53]. Undoubtedly, it will continue to play an irreplaceable role in biology research in the future. In click chemistry field the CuAAC reaction is known to be "cream of the crop" of the family. This cycloaddition is an ideal candidate for surface attachment because it provides a simple, rapid, versatile and attractive route to impart surfaces and nanomaterials with improved features in terms of selectivity and electrocatalytic properties. It allows the incorporation of nanomaterials and/or biological compounds in a selective and controlled manner as well as quantitative and proceeds rapidly in aqueous and organic solvents under ambient atmospheric conditions. The resulting 1,2,3-triazole is unique in that the link is an aromatic heterocycle. This link has superior stability to linkages resulting from other couplings, being resistant to both hydrolysis and redox reactions. Of course, depending on the application, this irreversible reaction can be viewed as an advantage or a disadvantage. In addition, the triazole provides good electronic coupling between organic molecules when it is required by the application. To summarise, the triazole product of the CuAAC reaction is thermally, oxidatively, reductively and hydrolytically stable [54] [55] .

The application of CuAAC to SAMs on silica surfaces was demonstrated soon after reports on gold. The disappearance of the azide-IR stretch was used to demonstrate quantitative conversion to the triazole. There have been numerous reports of creating tailored silica surfaces for various applications through attaching specific molecules via triazole formation [START_REF] Meng | Affinity mass spectrometry from a tailored porous silicon surface[END_REF] [57] [START_REF] Prakash | Click" Modification of Silica Surfaces and Glass Microfluidic Channels[END_REF]. Though there are numerous interesting applications of nanomaterials, one of the stumbling blocks is controlling their surface chemistry. A number of groups have demonstrated that single stranded DNA can be immobilised on glass [54] [59]. On the other hand, protein microarrays have been less worked on compared with DNA microarrays. This is partially due to the challenge of incorporating site-specific immobilising strategies rather than opportunistic ligation with free amine or thiol functional groups that are inherently present on the surface of the protein. This is an important consideration given that the activity of the protein being immobilised will be dependent on its orientation and where it has been chemically modified.

Poulter and Lin have independently explored the application of CuAAC to this problem [START_REF] Gauchet | Regioand Chemoselective Covalent Immobilization of Proteins through Unnatural Amino Acids[END_REF] [61] and both have developed methodology to site-specifically introduce an azide or an alkyne on the protein's C terminus. Acetylene-modified proteins could be readily attached to azidefunctionalised surfaces via CuAAC. It is notable that low protein concentrations (1 mM) in the deposition solution were enough to easily create detectable levels of protein coverage. Lin also demonstrated higher protein activity if immobilised site-specifically via CuAAC as compared to random protein amine coupling with activated N-hydroxysuccinimide slides [START_REF] Lin | Site-Specific Protein Modification through CuI-Catalyzed 1,2,3-Triazole Formation and Its Implementation in Protein Microarray Fabrication[END_REF].

Thus, as self-assembled monolayer (SAMs) are popular surfaces for the biomolecules immobilisation, the following section will present a description of SAMs chemistry.

Self-Assembled Monolayers 1.3.1. Self-Assembled Monolayers definition

The term Self-Assembled Monolayers (SAMs) is used to describe the spontaneous formation of a thin film of molecules onto a solid surface. In this process disordered components in solution organise themselves near the surface as a result of specific interactions and without external directions. When the constitutive components are molecules, the process is termed molecular self-assembly (figure 1.7) [13]. Despite the early article in 1946 for Bigelow et al, [START_REF] Bigelow | Oleophobic monolayers: I. _lms adsorbed from solution in non-polar liquids[END_REF] the researchers payed attention to self-assembled monolayers only in the 80ths. In this year Sagiv [START_REF] Sagiv | Organized monolayers by adsorption. 1. Formation and structure of oleophobic mixed monolayers on solid surfaces[END_REF] showed the possibility of producing chemically attached monolayers on silicon dioxide. Then later in 1983, Nuzzo and Allara [START_REF] Nuzzo | Adsorption of bifunctional organic disulfides on gold surfaces[END_REF] exploited the chemical interaction between sulphur atoms and noble metal to fabricate alkanethiolated SAMs on Au and this work promoted the investigations of SAMs on gold substrates until today. SAMs have been used on a wide variety of substrates including gold, silver, copper, nickel, platinum, palladium, mercury, zinc and cadmium selenide [START_REF] Pomerantz | Coverage of si substrates by selfassembling monolayers and multilayers as measured by ir, wettability and x-ray di_raction[END_REF] [66] to be used for many applications, including the modification of surface properties like hydrophobicity or biocompatibility [START_REF] Lee | Surface characterization on binary nano/microdomain composed of alkyl-and amino-terminated self-assembled monolayer[END_REF]. Several analytical methods were successfully used to characterise them, like X-ray Photoelectron Spectroscopy (XPS), Reflection-absorption spectroscopy by polarisation modulation (PM-IRRAS) , X-ray diffraction (XRD), contact angle goniometry, ellipsometry, AFM, etc. [START_REF] Ariga | Self-assembly as a key player for materials nanoarchitectonics[END_REF] [69]. [75]. The thickness of the film can be controlled by varying the length of the hydrocarbon chain, and the properties of the film surface can be modified by the terminal functional group [START_REF] Herzer | Orthogonal functionalization of silicon substrates using self-assembled monolayers[END_REF].

Many types of SAMs have been widely reported in the literature. Table 1.1 shows a list of some systems that have been used for the formation of SAMs. The head group used always depends on the type of the surface. The substrates most commonly used are gold, hydroxylated (activated) surfaces of SiO2 on Si, Al2O3 on Al, and glass. For example, long chain fatty acidsbased SAMs on alumina were studied since the 1950s [START_REF] Bowden | The Friction and Lubrication of Solids. Part I[END_REF]. Other examples like thiols or sulfides on gold, and organosilanes on silicon or silica surfaces are also widely studied. In addition to metals and semiconductors, other classes of oxides surfaces like Al2O3 and TiO2 are used to deposit n-alkanoic acids and phosphonic acids [START_REF] Xiao | Chain length dependence of the frictional properties of alkylsilane molecules self-assembled on mica studied by atomic force microscopy[END_REF]. Obviously, the two most extensively studied classes of SAMs are derived from the adsorption of alkanethiols or disulfides on gold [15] [79] [START_REF] Vericat | Self-assembled monolayers of thiols and dithiols on gold: new challenges for a well-known system[END_REF] and organosilanes on a Si/SiO2 substrate (figure 1.8) [81] [82]. Although the former combination has received the most attention presumably because of the ease of preparation, silane-based monolayers on oxide surfaces such as SiO2 possess some advantageous features over thiols on gold. SAMs on gold are widely used in the literature for their ease of preparation. It is easy to deposit and pattern and it is relatively inert to oxidation under atmospheric conditions [START_REF] Nuzzo | Adsorption of bifunctional organic disulfides on gold surfaces[END_REF] . Depending on the application, SAMs on gold have some disadvantages. The significant obstacle to the implementation of SAMs in everyday applications is the poor durability of these thin films under ambient and more extreme conditions. SAMs of alkanethiols on gold are often used also to explore the interactions between cells and a material surface. However, several reports have shown that alkanethiols on gold may have limited shelf-life or deteriorate under biological conditions and are not appropriated for long-term studies [START_REF] Phillips | Electrochemical control of the thermal stability of atomically thin Ag films on Au(111)[END_REF].

SAMs based on the chemistry of the organosilanes can be integrated into silicon technology and present good chemical and physical stabilities due to their partial in plane polymerisation, allowing extensive handling and further chemical modification steps with no degradation of the monolayer. In addition, silica substrates are optically transparent and low-cost materials. The surface silanisation has shown to be also appropriated for long-term study of cellular development [78] [82]. Thus, SAMs of silanes on glass have the advantage of being compatible with fluorescence studies and are stable for a long period of time (at least one to several months). The chemical formula of organosilanes is RSiX3. R is an organic group linked to Si by a Si-C bond, which is stable toward hydrolysis and X is a hydrolysable organic group, in most cases ethoxy, methoxy or chloro. These molecules can be grafted onto many oxide surfaces, for example, SiO2, Al2O3, and TiO2 [START_REF] Wasserman | Structure and reactivity of alkylsiloxane monolayers formed by reaction of alkyltrichlorosilanes on silicon substrates[END_REF]. The following section presents a specific study of SAMs formation by the grafting of organosilanes onto silica surfaces: formation mechanism, factors affecting SAMs properties, deposition methods and characterisation methods.

SAMs formation onto SiO2 surfaces

Before attaching molecules to the surface, the precleaning of surface is a prerequisite to obtain a clean oxide layer with high density of silanol groups (Si-OH) on the surface. These silanol groups provide a highly hydrophilic surface allowing molecules to diffuse on the physisorbed ultra-thin water layer as well as anchoring sites for silanisation [3]. SiO2 surface can be activated by many methods to obtain a clean and hydroxylated surface. There are various activation methods like using acidic solution [START_REF] Arkles | Hydrophobicity, Hydrophilicity and Silanes[END_REF], UV/ozone treatment, irradiation method including plasma or finely a mix of two methods. Using acidic solutions removes organic and inorganic contaminants as well as unwanted particulates (dust, silica, silicon, metals) [85] [86] . Other chemicals can be used to remove contamination like Piranha (mixture of sulfuric acid and hydrogen peroxide) or basic medium of ammonia and hydrogen peroxide. UV radiation in an oxygen atmosphere removes contaminants by transforming organic compounds, like hydrocarbons and oils, into gases or water-soluble species such as fatty acids [87] [88]. In general those cleaning methods yield a density of ∼10 15 OH groups/cm 2 [START_REF] Petitdidier | Growth mechanism and characterization of chemical oxide films produced in peroxide mixtures on Si(100) surfaces[END_REF], and thus, in principle, the same density of organic molecules could be chemisorb at the surface. In our work we used a combination of two methods for cleaning and activating surfaces; first starting by washing the surface with water and then sonicating in chloroform, followed by UV/ozone treatment [86] [90].

SAMs based on organosilanes are attractive to functionalise silica surface. The commonly assumed mechanism for silanisation to form a monolayer on the surface can be explained in three distinct steps in figure 1.9. First the head group of the coupling agent comes into contact with the substrate, followed by a hydrolysis reaction of the SiX3 to afford trisilanol groups involving water present on the oxide's surface. These trisilanol groups interact by H-bonding with the silanol onto the surface and also with those of neighboring silanes. Finally the last step of the mechanism involves the condensation reaction to give the siloxane bond with the surface and provides the cross-linking between adjacent molecules [START_REF] Brzoska | Silanization of Solid Substrates: A Step toward Reproducibility[END_REF]. Some studies show that maximum 20% of the OH of surface are used to bond silanes which is not enough to promote anchoring of the SAMs entirely through the Si-O-Si bond with the surface. Meanwhile, a polymerised network is created between silane molecules where molecules are linked to each other. In this way, each silane is involved in a bond formation either by linking to the surface or to the neighbouring molecule [13] [81]. The presence of the cross-linked network of Si-O-Si bond explains why in some cases wafers modified with alkylsiloxane-based monolayers exhibit roughness which is similar or lower than for unmodified substrates [82] [92]. This view was later confirmed by Allara et al [START_REF] Allara | Evidence for a Unique Chain Organization in Long Chain Silane Monolayers Deposited on Two Widely Different Solid Substrates[END_REF]. The observation of a lower roughness of silanised wafers compared to unmodified wafers, as observed in X-ray reflectivity experiments by Silberzan et al., also indicated that not all molecules are individually linked to the surface, but rather form a cross-polymerised network of molecules with only a few bonds to the surface [START_REF] Silberzan | Silanation of silica surfaces. A new method of constructing pure or mixed monolayers[END_REF]. This scenario was supported by the IR studies of Tripp and Hair [START_REF] Tripp | An Infrared Study of the Reaction of Octadecyltrichlorosilane with Silica[END_REF] [94]: they found in a low-frequency IR study that the adsorbed species form few, if any, Si-O-Si bonds with the surface. This mechanism is sensitive to many conditions such as water content, temperature, the nature of the head group and more other factors. Moreover, all these factors have an impact on the quality and the properties of the resulted SAMs. In the following section, the different parameters that influence the quality of the monolayer will be described [START_REF] Li | AFM investigation of effect of absorbed water layer structure on growth mechanism of octadecyltrichlorosilane self-assembled monolayer on oxidized silicon[END_REF] [96].

Parameters influencing the SAMs formation

Controlling the molecular organisation at the nanometre scale is essential to develop the desired properties of SAMs for specific applications. Therefore, the understanding and the control of the self-assembly process is of prime importance in this field [START_REF] Barth | Molecular architectonic on metal surfaces[END_REF].To achieve this level of control over surface modification all the parameters have to be optimised for the SAMs preparation. There is a large number of parameters that influences the quality of SAMs [START_REF] Silberzan | Silanation of silica surfaces. A new method of constructing pure or mixed monolayers[END_REF] [98] [START_REF] Tian | Infrared Characterization of Interfacial Si-O Bond Formation on Silanized Flat SiO2/Si Surfaces[END_REF]. The following sections will present in detail the effect of some parameters on SAMs properties.

Deposition conditions 1.3.4.1.1. Water content and solvent polarity

The amount of water strongly affects the quality of the SAMs because incomplete monolayers are formed in the total absence of water [START_REF] Le Grange | Effects of surface hydration on the deposition of silane monolayers on silica[END_REF], whereas excess of water leads to uncontrolled polymerisation in solution yielding polysiloxane-based aggregates on the surface of interest. A small amount of water is required to form dense and homogeneous monolayer, which may be seen as a polysiloxane layer linked to the surface by Si-O-Si bonds [START_REF] Kinser | Conductive Atomic Force Microscope Nanopatterning of Hydrogen-Passivated Silicon in Inert Organic Solvents[END_REF] [102], those cases are presented in figure 1.10. Indeed, this water layer comes from exposing the surface to the humidity, so it is highly dependent on the ambient relative humidity (RH) during the preparation. For example, Mrotek et al have shown that at 18% relative humidity (RH), there is just one water monolayer on the SiO2 surface, against several ones at 45% RH. In contrast, an ordered monolayer cannot be obtained without a water layer [START_REF] Parikh | An Intrinsic Relationship between Molecular Structure in Self-Assembled n-Alkylsiloxane Monolayers and Deposition Temperature[END_REF] [93] [START_REF] Desbie | Impact of chain length, temperature, and humidity on the growth of long alkyltrichlorosilane self-assembled monolayers[END_REF]. As a consequence, grafting is usually performed in organic medium with a strict control of the water content in the solvent and adsorbed onto the surface. For example, surface modification which used toluene or benzene leads to the densest silane films in comparison with e.g. pentane, carbon tetrachloride, or cyclohexane. This is due to the fact that these solvents are able to remove a significant amount of water from the substrate surface to have a thin layer of water The nature of the solvent has an impact on the quantity of water as well as the solubility of the coupling agent. The layer of water on the surface of the substrate is necessary for the hydrolysis reaction. However, it is necessary to limit the quantity and therefore the thickness of this water layer on the surface in order to obtain a monolayer instead of a multilayer or aggregate as shown in figure 1.10. Depending on its nature, solvent will have different water extraction capacity on the surface. Solvent effect can be noticed by decreasing or increasing water solubility depending on the polarity. For example, Cheng et al. [START_REF] Cheng | Solvent Effects on Molecular Packing and Tribological Properties of Octadecyltrichlorosilane Films on Silicon[END_REF] used four solvents: hexadecane, toluene, chloroform, and dichloromethane for SAMs preparations. They obtained very smooth monolayer films when either hexadecane or toluene were used in the deposition process. They also claimed that lower polarity solvents resulted in better molecular packing and smoother OTS films. On the other hand, other study from Rozlosnik et al., [START_REF] Rozlosnik | Effect of solvents and concentration on the formation of a self-assembled monolayer of octadecylsiloxane on silicon (001)[END_REF] showed the contrast. It concluded that the deposition of OTS from dodecane solutions resulted in multi-layered films.

That gives evidence of the fact that solvent nature is a poorly understood parameter since dodecane and hexadecane, which both belong to the same family (non-polar, linear hydrocarbons), affected the quality of monolayers in a different way. This is another indication that preparation conditions are critical for SAMs formation. Consequently, grafting is usually performed in organic medium because these organic solvents are able to remove a significant amount of water from the substrate surface. In addition, most organosilanes are not water soluble which makes the grafting of organosilanes in aqueous medium difficult and preferable in organic medium [START_REF] Sugahara | 29Si-NMR study of hydrolysis and initial polycondensation processes of organoalkoxysilanes. II. Methyltriethoxysilane[END_REF].

The nature of the solvent also affects the kinetics of assembly of the monolayer. This parameter has an impact onto the dynamic equilibrium governing the adsorption of molecules on the surface. Some studies report that the role of the solvent in determining the monolayer structure depends on the solvent compatibility with the monolayer. In more details, the influence of the solvent on the bond formation would be due to solvent-surface and solvent-molecule interactions, which complicate the thermodynamics and kinetics of assembly. Solvent-substrate interactions can decrease or block the rate of adsorption of the molecules from solution [START_REF] Mali | Principles of molecular assemblies leading to molecular nanostructures[END_REF] [112]. Indeed, solvent molecules must be displaced from the surface prior to the adsorption of the molecules, which are less prevalent in solution than the solvating molecules. As a result, some studies suggest that the rate of formation of SAMs is faster in certain nonpolar solvents (heptane, hexanes) than polar solvent like ethanol. Hydrocarbon solvents may improve the kinetics of formation in some cases, but at the same time the strong solvent-molecule interactions in these solutions impede the organisation of SAMs. Molecules of the hydrocarbon solvents can incorporated themselves into the monolayer structure during formation and this interaction is facilitated by the linear structure of the solvent molecules and the strong intermolecular interactions (van der Waals attractions) between the solvent molecules and the alkyl chains of the coupling agent. Therefore, the ideal solvent for SAMs formation should not only dissolve the adsorbate molecules but also resist intercalation into the monolayer [START_REF] Peterlinz | In Situ Kinetics of Self-Assembly by Surface Plasmon Resonance Spectroscopy[END_REF] [114].

Temperature

In fact, by increasing the sample's temperature, the energy of the system increases. This energy increase can be used to realise a phase transition from a disordered phase to an ordered phase or from an organised phase to another one. Also, the increase of the kinetic energy of the system with the temperature breaks the molecular bonds between molecules (molecule-molecule interactions) allowing the molecules to diffuse easily at the surface of the sample, which facilitates the formation of new molecular bonds between active sites [115] [116].

Many studies have proved that temperature has a big influence on the deposition of the monolayer and the order of molecules in the layer. Silberzan et al [START_REF] Silberzan | Silanation of silica surfaces. A new method of constructing pure or mixed monolayers[END_REF]. have given a first clue by pointing out the importance of water traces and of the temperature of reaction. These two are linked together via a recently proposed deposition mechanism shown in figure 1.9. In particular, they have observed a monotonous increase in the quality of the layer as the temperature of reaction was decreased from 35° C to 18 °C. This temperature dependence was interpreted by these authors as due to a solubility decrease of the silane in the hexadecane, which favours their physisorption on the solid substrate. When the temperature decrease, the solubility of either water or silanes decreases which affects the kinetics of the physisorption step. But the length of the spacer is also important to be noticed. When shorter chains are used, a lower temperature becomes necessary because temperature affects the silane physisorption step. In general, Brzoska et al. [91] [117] have highlighted the existence of a critical temperature, linearly increasing with the chain length (from 10 to 22 carbon atoms), above which the complete SAM is disordered, and below which the SAM is well ordered. For example, an OTS monolayer achieves its optimal quality in 2 min at 18 °C while even a 24-h reaction time did not lead to a satisfactory result at 30 °C [START_REF] Silberzan | Silanation of silica surfaces. A new method of constructing pure or mixed monolayers[END_REF].

Coupling agent solution (Concentration and age)

The concentration of the coupling agent's solution is crucial for the SAMs growth and its final quality. As all parameters are interfering with each other, the concentration of the coupling agent is inversely related with deposition time: normally low concentrations require long immersion [118] [119]. In practice, SAMs formed by immersion for a week in solutions with low concentrations do not exhibit the same physical properties than the ones of SAMs prepared from more concentrated solutions [START_REF] Rozlosnik | Effect of solvents and concentration on the formation of a self-assembled monolayer of octadecylsiloxane on silicon (001)[END_REF]. Desbief et al., reported that increasing the concentration (from 10 -2 M to 2.5 × 10 -1 M) formed less ordered films. because too many molecules may deposit at the same time. Thus, their self-assembly in two dimensions might be difficult and promote aggregates formation [START_REF] Desbie | Impact of chain length, temperature, and humidity on the growth of long alkyltrichlorosilane self-assembled monolayers[END_REF]

Coupling agent structure

The three parts of the coupling agent affect the architecture of SAMs specially the spacer which essentially controls the self-assembly and the degree of order in the film [START_REF] Gooding | The molecular level modification of surfaces: from self-assembled monolayers to complex molecular assemblies[END_REF]. They can also affect the kinetics of self-assembling.

Head group

As previously discussed, for silica surfaces the attachment to the surface is through siloxane covalent bonds which give stability for such SAMs. The siloxane covalent bond resulted from hydrolysis and condensation reaction between the head group of the coupling agent (RSiX3)

and the hydroxyl group on the surface. All these reactions normally occur concurrently and consecutively, and each has its own kinetic parameters [124] [125]. The most commonly hydrolysable groups are methoxy, ethoxy and chloro organosilanes. The reactivity of the coupling agent strongly depends on the nature of the silyl group. Thus, trichloroorganosilanes (X = Cl) are much more reactive than trialkoxyorganosilanes (X = OR, R is alkyl) and hydrolyse very quickly in the presence of water. In fact, the hydrochloric acid released in the medium catalyses this process. For trialkoxyorganosilanes the reaction is slower, and the size of the hydrolysable group is one structural property of alkoxysilane precursor monomers that influences their hydrolysis and condensation reactions rates. Increasing the alkoxysilane hydrolysable group size leads to longer reaction time and slower kinetics, suggesting steric hindrance to the reactions [START_REF] Deetz | Reactive modeling of the initial stages of alkoxysilane polycondensation: effects of precursor molecule structure and solution composition[END_REF]. Triethoxyorganosilanes (X = OEt) are less reactive than trimethoxyorganosilanes (X = OMe) [START_REF] Naik | Effect of Leaving Group on the Structures of Alkylsilane SAMs[END_REF]. To speed up the reaction with trialkoxyorganosilanes, it is possible to use a catalyst such as the trichloroacetic acid (TCA).

The high reactivity of trichlorosilanes and their sensitivity to water may be disadvantages which limit therefore the possibility for the presence of a functional terminal group due to incompatibility and/or interaction of the chlorosilane with the terminal group. This behaviour can potentially prevent the self-assembly process and therefore the formation of a well-ordered monolayer. On the other hand, more homogeneous SAMs are usually obtained with triethoxyorganosilanes and trimethoxyorganosilanes. Alkoxyorganosilanes are more stable and easier to control under ambient conditions, while chlorosilanes are very sensitive to humidity and tends to form aggregates [START_REF] Aswal | Self assembled monolayers on silicon for molecular electronics[END_REF] [125] [START_REF] Issa | Kinetics of Alkoxysilanes and Organoalkoxysilanes Polymerization: A Review[END_REF]. The use of trimethoxysilyl coupling agents appears to be a good compromise between reactivity and ease of handling.

Indeed, grafting conditions have been optimised in our research group to form densely packed monolayers by using trimethoxysilyl derivatives. The effect of various experimental parameters was examined. First, the addition of a weak acid to the solution, e.g., acetic acid (pKa = 4.76) led to a significant grafting enhancement. Interestingly, the use of a stronger acid (trichloracetic acid, TCA), (pKa = 0.66) at a concentration of 10 mol % led to well-ordered monolayers, showing a very strong catalytic effect of TCA on the grafting process. Consequently, most of our grafting processes containing a trimethoxysilyl group were carried out using TCA as a catalyst [START_REF] Dinh | Route to Smooth Silica-Based Surfaces Decorated with Novel Self-Assembled Monolayers (SAMs) Containing Glycidyl-Terminated Very Long Hydrocarbon Chains[END_REF].

Spacer

The molecular structure of the spacer is strongly affecting the thickness, packing density and the order of molecules in the SAMs as well as their kinetics of formation. It may impact many factors such as the length of the self-assembling molecule, the angle at which it is oriented relative to the surface and the nature of intermolecular interactions. The spacer could be either pure alkyl chain, or alkyl chain incorporating other functional groups.

a) Pure alkyl chain

The intermolecular interactions depend on the nature of the spacer. In the case of pure alkyl chains, the driving force is Van der Waals interactions, and it is known to be influenced by the spacer length. For example, the ordering behaviour of n-alkyltrichlorosilane-based films depends on the alkyl chain length. In order to obtain a monolayer with alkyl chains in all transconformation, the optimum molecular length is eighteen carbons, oriented perpendicular to the surface. In contrast, silanes with a relatively very short alkyl chain of three or eight carbons or a very long one with thirty carbons lead to less ordered films [108] [130] .

b) Alkyl chain incorporating a functional group

The introduction of functional groups such as amide, urea, sulfone, aromatic or diacetylene into the linear alkyl chains may constitute an alternative way to control the molecular self-assembly of SAMs. These functional groups can interact laterally by hydrogen bonding (amide or urea), dipole interaction (sulfone), -stacking (aromatic) or covalent bonding (diacethylene), improving the stability and the mechanical properties of SAMs [START_REF] Gooding | The molecular level modification of surfaces: from self-assembled monolayers to complex molecular assemblies[END_REF] [131] [START_REF] Li | Following the Azide-Alkyne Cycloaddition at the Silica/Solvent Interface with Sum Frequency Generation[END_REF]. Among those functional groups, the amide group has been found to consistently enhance SAMs stability due to its tendency to form strong intermolecular hydrogen bonds [START_REF] Clegg | Control of Monolayer Assembly Structure by Hydrogen Bonding Rather Than by Adsorbate-Substrate Templating[END_REF].

Hydrogen bonds are not only stronger than Van der Waals, but they have interesting properties like being directional and selective. Directional means that atoms bonded prefer specific orientations in space relative to one another allowing the formation of organised supramolecular networks, while selectivity describes how an atom binds more preferentially to one atom than another. As a result, the molecular skeleton can be tailored in order to establish hydrogen bonds at a precise position of the molecular core, enabling to create organised architectures in a predictable way. In this way it is easier to adjust and tune the molecular architectures stabilised through hydrogen bonds than other bonds. Many researches demonstrate that the hydrogen bonds can be at the origin of a self-assembly process, from which organised architectures are formed [START_REF] Wang | In vitro stability study of organosilane self-assemble monolayers and multilayers[END_REF].

A study by S. Song et al. [START_REF] Song | Preparation and Tribological Behaviors of an Amide-Containing[END_REF] showed that an amide-containing stratified self-assembled film (figure 1.11) can be elaborated on a silicon surface by a simple two-step method: First, N- [3-(trimethoxylsilyl)propyl]ethylenediamine molecules are self-assembled on silicon surfaces, followed by deriving with stearoyl chloride through a surface coupling reaction. They showed that the existence of two layers of hydrogen bonds can enhance the stability of the film by double in-plane cross-linking. Urea containing SAMs in literature can be prepared by the functionalisation of isocyanate terminated SAMs by coupling with amines (figure 1.12) but it is recognized that isocyanate coupling agents are limited to use due to their difficult preparation. Moreover, no spectroscopic proof of the presence of the isocyanate at the surface has been achieved so far. Furthermore, all the strategies led to partial or total hydrolysis or side reactions of the isocyanate group when spectroscopic analyses were performed with silicas or materials [135] [136]. Since the distance between urea groups (4.5-5 Å) is larger than the distance between two Si atoms of siloxane bridge (∼3.2 Å), it may explain the disorder observed for the alkyl chains.

Indeed, the distance between the alkyl chains is not sufficient enough to establish van der Waals interactions and consequently does not favour all-trans conformations. Moreover, the terminal ester groups may generate steric hindrance, favouring also the disorder of the alkyl chains. Afterward, in 2015, the group reported another study of SAMs containing amide and urea groups possessing an epoxy-terminal group and a trimethoxysilyl anchoring group as shown in figure 1.14. They confirmed that the molecular assembly is mainly based on the intermolecular hydrogen-bonding between adjacent amide or urea groups in the monolayers which also has an impact on the grafting process. The nature of the spacer could also affect the kinetic of the head group [START_REF] Okumoto | Theoretical Study of Hydrolysis and Condensation of Silicon Alkoxides[END_REF]. A study of our research group involving a trimethoxysilyl head group compared the deposition time of SAMs containing an alkyl spacer to SAMs containing urea or amide groups within the spacer. It was noteworthy that the monolayers containing internal urea or amide groups are formed only after 12 h of immersion in the salinization solution, whereas the grafting takes at least 20 h for monolayers possessing long alkyl chains. This feature is certainly due to the fact that using hidden polar functionalities increased the solubility in organic solvent and also the affinity with hydrophilic surfaces [START_REF] Ramin | Epoxy-Terminated Self-Assembled Monolayers Containing Internal Urea or Amide Groups[END_REF].

Terminal group

The basis for the molecular design and tailoring of SAMs should be complete knowledge of the interrelationships between the molecular structures and tribological properties of SAMs, as well as a deep understanding at the molecular level. Most significantly, chemical modifications of the terminal groups via surface reactions can effectively allow to tune the surface characteristics such as conductivity, wetting properties, adhesion ability, friction, and other attributes, in a desired and controlled manner. These chemical modifications and the attachment steps are generally performed through nucleophilic substitution reactions, click chemistry, Diels-Alder reactions, C-C coupling reactions, electrostatic interactions, or supramolecular chemistry [3].

Another important property of the surface that is strongly affected by the nature of the terminal functional group is the hydrophilicity. It can be controlled depending on desires. For example, for a hydrophobic surface the commonly used terminal group is the nonpolar methyl (CH3) group or trifluoromethyl (CF3) while to produce hydrophilic surfaces, the commonly used terminal groups are alcohol (OH) or carboxyl acid (COOH) groups. Selecting the type of the terminal group depends on the application of the SAMs. Typically, terminal groups are connected to a molecular chain in which the terminal end can be functionalised (i.e. adding -OH, -NH2, -COOH, N3 or -SH groups) to vary the wetting and other interfacial properties [START_REF] Aswal | Self assembled monolayers on silicon for molecular electronics[END_REF]. SAMs with a variety of functional end groups have been prepared. However, azideterminated SAMs are becoming very valuable due to their reactivity via the Huisgen reaction ''click chemistry''.

Azide terminated SAMs

Classically, azide-terminated SAMs can be obtained by two methods of post-functionalisation In the 90ths, it has been shown for the first time by Balachander and Sukenik that SN2 reactions can be used for the conversion of the end-functional groups of monolayers [START_REF] Brzoska | Silanization of Solid Substrates: A Step toward Reproducibility[END_REF]. This first condition-set reflects a well investigated protocol for the grafting of bromoorganotrichlorosilanes. Chemical reactions or modifications on solid surfaces is different from organic reactions in liquid phase and difficult to follow. In SAMs the nucleophile must penetrate the monolayer to attack the (C-Leaving group) bond from the backside. Therefore, the nucleophile must be a small and strong nucleophile. Moreover, there is a kinetic barrier, due to the fact that the monolayer is closely packed and that the nucleophile needs to penetrate the monolayer. In addition, the success of a displacement reaction in SAMs depends strongly on the type of SAM and its defects [START_REF] Fryxell | Nucleophilic displacements in mixed selfassembled monolayers[END_REF]. Despite all those limitations, as already shown in literature by several research groups, the classical method to prepare azide-terminated SAMs is by the SN2 reaction applied on bromine-terminated SAMs (post functionalisation method described in figure 1.15a) [START_REF] Zhang | Adjusting the Surface Areal Density of Click-Reactive Azide Groups by Kinetic Control of the Azide Substitution Reaction on Bromine-Functional SAMs[END_REF].

A second post functionalisation method consists in derivatising amino-terminated monolayers via an azidification reaction with an efficiency ranging from 30% to 90% (described in figure 1.15b) [START_REF] Chen | Arylsilanated SiOx Surfaces for Mild and Simple Two-Step Click Functionalization with Small Molecules and Oligonucleotides[END_REF]. The steric effect seems to prevent a complete surface azidification. In this process, commercial 11-azidoundecyltrimethoxysilane was used directly to prepare a solution to produce azide SAMs by the classical immersion method [146] [147]. The deposition of p-azidomethylphenyltrimethoxysilane has also been studied [START_REF] Li | Following the Azide-Alkyne Cycloaddition at the Silica/Solvent Interface with Sum Frequency Generation[END_REF]. Furthermore, there are few examples of the preparation of azide-terminated SAMs onto silica surface by the direct grafting of azido-silylated compounds by chemical vapor deposition [START_REF] Vos | Chemical Vapor Deposition of Azidoalkylsilane Monolayer Films[END_REF] [149] and only one example describes the deposition of an azide-terminated monolayer by the Langmuir-Blodgett technique [START_REF] Niehaus | Patterning of Functional Compounds by Multicomponent Langmuir-Blodgett Transfer and Subsequent Chemical Modification[END_REF]. It is important to early mention that, recently, in our research laboratory, we have successfully prepared an azide-terminated SAM by using the spin coating technique in ambient atmosphere and tested its reactivity via the CuAAC reaction [START_REF] Rouvière | Hydrosilylation of Azide-Containing Olefins as a Convenient Access to Azidoorganotrialkoxysilanes for Self-Assembled Monolayer Elaboration onto Silica by Spin[END_REF]. This first preliminary trial opens the door for using the spin coating process to deposit SAMs rapidly and efficiently.

Mixed SAMs

In principle, direct regulation of the chemical composition of SAMs can be achieved by the preparation of mixed SAMs. Mixed-SAMs is a common procedure to form self-assembled monolayers from two (rarely more) different molecules in spacer chain or the functional end group. There are three main purposes in doing this [START_REF] Rissner | The Electronic Structure of Mixed Self-Assembled Monolayers[END_REF] :

-The first purpose is to increase in reactivity by spacing target-binding functional headgroups with non-reacting (diluting) end groups. Many papers have shown that having more reactive functional end groups at the surface did not necessarily lead to a greater overall reactivity. This is often attributed to steric hindrance or interactions between close-packed reactive end groups. So, in this way this dilution can enhance the overall reactivity, due to a greater availability of the reactive sites.

-The second reason of preparing mixed SAMs is to control the overall hydrophilicity of the surface by mixing different hydrophobic and hydrophilic molecules. This is particularly important in biosensing research and understanding subtleties of the biomolecular interactions with artificial materials.

-The third purpose is to provide multi-functional SAMs incorporating different reactive end groups and thus allowing to immobilise chemoselectively several different molecules of interest.

Such mixed monolayers are typically synthesised by exposing the surface to a mixture of two reactive molecules. There are two main methods to prepare mixed SAMs: the one-step deposition method and the stepwise deposition method figure 1.16 [START_REF] Böhmler | Self-Assembled Molecular Platforms for Bacteria/Material Biointerface Studies: Importance to Control Functional Group Accessibilit[END_REF]. Importantly, with the co-adsorption method (the one step deposition method), there is a control over the final molecular composition and the order of the SAMs. However, formation of coadsorbed SAMs is more complicated than single component systems. It is important to know that the surface ratio between the two molecules in the SAMs after the self-assembly may be very different from the ratio of the two molecules in solution before the self-assembly, and it is notable that phase segregation can occur leading to the existence of domains enriched with one or the other at the surface [START_REF] Smith | Patterning self-assembled monolayers[END_REF] . In some studies, alkyltrichlorosilanes with the same chain length (C8 or C11) and different terminal groups such as Br (or CF3), diluted in CH3 matrix, showed generally a nonlinear dependency of the monolayer composition with the composition of the silanisation solution [START_REF] Heisea | Mixed silane self assembled monolayers and their in situ modification[END_REF] [153] [START_REF] Giraud | Aminofunctionalized monolayers covalently grafted to silica-based substrates as a robust primer anchorage in aqueous media[END_REF] .The preferential adsorption of brominated (or fluorinated) alkylsilanes can be explained by the different polarities of the silylated compounds.

However, for the Br/CH3-terminated SAM, one study gave an opposite result with deposition solutions matching the surface composition [START_REF] Lagutchev | Self-assembly of alkylsiloxane monolayers on fused silica studied by XPS and sum frequency generation spectroscopy[END_REF]. NH2/CH3 (or NH2/N3) terminated SAM showed also a preferential adsorption of amino-terminated alkylalkoxysilane [START_REF] Heisea | Mixed silane self assembled monolayers and their in situ modification[END_REF] [153] (or phenylalkoxysilane) which can be explained by the ability of amino groups to interact with the silanols groups onto the surface through hydrogen-bonding [158] [159]. Sometimes the monolayer composition reflects the solution stoichiometry. Some mixed SAMs prepared from short and long n-alkyltrichlorosilanes (C4 + C8, C12, C16, C18) presented similar adsorption with respect to the chain length [START_REF] Offord | Kinetic control in the formation of self-assembled mixed monolayers on planar silica substrates[END_REF]. Similar results were obtained for mixed SAMs built with vinyl/CH3 or naphthyl/CH3 terminated trichlorosilanes [71] [161]. However, it is still unclear which parameters govern the formation of mixed SAMs, revealing the difficulties to control the distribution and ratio of various functional groups. To summarise, the relative composition of adsorbates in a mixed SAMs strongly depends on molecular interactions dominating the SAMs formation such as chain-chain, anchoring group-substrate, and coupling agent-solvent interactions. As a result of those variations, two types of mixed surfaces can be obtained:

homogeneous surfaces with the two silanes randomly distributed [START_REF] Martin | Liquid mechanical behavior of mixed monolayers of amino and alkyl silanes by atomic force microscopy[END_REF], and phase separated surfaces inducing nanodomains which are rich in individual components [START_REF] Wirth | Mixed Self-Assembled Monolayers in Chemical Separations[END_REF] [164] [START_REF] Desbief | Different types of phase separation in binary monolayers of long chain alkyltrichlorosilanes on silicon oxide[END_REF]. In general, the control of composition and topography of mixed SAMs is dependent on parameters such as the structure of the silane compounds and the deposition conditions. Despite these limitations, mixed SAMs have been used greatly in biosensing research.

The second method of preparing mixed SAMs is the stepwise method, by using mono component solutions in a given order resulting in pre-adsorption of a partial monolayer for the first silane. Then, the deposition of the second silane on uncovered areas of the surface completed the monolayer. Similar organisations of binary monolayers were obtained using the two deposition methods for two organosilanes bearing similar silyl groups [START_REF] Mathauer | Binary self-assembled monolayers as prepared by successive adsorption of alkyltrichlorosilanes[END_REF]. The advantage of this method is to overcome the difference of reactivity of the two compounds which could not be balanced by the deposition solution composition [START_REF] Wu | Stepwise Adsorption of a Long Trichlorosilane and a Short Aminosilane: an AFM, ATR, and Contact Angle Study[END_REF] [168] [START_REF] Feng | Construction of the homogeneously mixed SAM composed of octyltriethoxysilane and octadecyltrichlorosilane by taking advantage of the molecular steric restriction[END_REF] . To summarise, the first method (the co-adsorption) is advantageous for use with two components with similar chemical properties, and the second one is more useful when the molecules differ in their chemical properties.

Although the composition of the mixed SAMs depends strongly on the experimental conditions used for the grafting process, mixed SAMs of bromo-and alkylsilanes can be used to adjust the density of azide terminal groups during the preparation of azide terminated SAMs [START_REF] Fryxell | Nucleophilic displacements in mixed selfassembled monolayers[END_REF] [155] [START_REF] Böhmler | Self-Assembled Molecular Platforms for Bacteria/Material Biointerface Studies: Importance to Control Functional Group Accessibilit[END_REF]. A complete substitution was achieved when the bromine-terminated silane is at least one methylene unit longer than the methyl-terminated silane, suggesting that bromine end groups are less sterically hindered. In contrast, the substitution yield is 80% when the two silylated compounds have the same alkyl chain length [170] [171]. Finally, the density of azide groups on the surface can be controlled kinetically by an appropriately timed quenching of the substitution reaction of pure bromo-terminated SAMs, leading to monolayers with azide groups randomly distributed. This last method for mixed SAMs elaboration prevents preferential surface adsorption and phase separation [START_REF] Gouget-Laemmel | Functionalization of Azide-Terminated Silicon Surfaces with Glycans Using Click Chemistry: XPS and FTIR Study[END_REF].

Compared to alkyl chains, a few studies described the preparation of mixed silane-based monolayers with internal functional group like amide, carbamate, or urea groups in the alkyl chains able to self-assemble by hydrogen-bonding. As previously mentioned, the presence of hydrogen bonds gives extra stability for SAMs. Mixed nitrobenzyl ester-/methyl-terminated SAMs have been studied in our research laboratory as shown in figure 1.17. The aim was to prepare hydrogen bonding mixed SAMs and control the concentration of the reactive terminal groups in the monolayers using the one step deposition method. This study showed that the monolayer composition for a small mole fraction of the ester is identical to the molar ratio of the two organosilanes in the deposition solution revealing a non-preferential surface adsorption of one compound. By using gold nano particles as nanomarkers, they revealed a nonhomogeneous distribution of the nitrobenzyl ester-terminated coupling agent in the monolayer, leading to phase separated surfaces with nanodomains rich in individual components [START_REF] Meillan | Mixed Self-Assembled Monolayers with Internal Urea[END_REF].

This behavior could be explained by the presences of polar urea groups in the molecular structure of both organosilanes which allow them to have the same ability to self-assemble by hydrogen-bonding and to have also the same affinity with hydrophilic surfaces. In many examples, one molecule with a functional group of interest is diluted in the mixed monolayer by another molecule that does not have any specific function. In contrast, having two functional group (bifunctional surfaces) of interest is less studied due to the difficulty in characterising and controlling the distribution and ratio of various functional groups. Therefore, the development of general methods for making controlled multifunctional monolayers is needed to accelerate progress in the biological and materials systems areas. The following figure1.18 [START_REF] Md | Orthogonally reactive SAMs as a general platform for bifunctional silica surfaces[END_REF] shows an example of a mixed SAM containing two orthogonally reactive groups, azide and amine. The amine and azide functional groups can be independently reacted with acyl chlorides and terminal alkynes, respectively. A key aspect of the orthogonally reactive-SAM strategy is that the ratio of azides to amines will determine the ratio of functional groups that are subsequently attached. It is observed that, the surface azide/amine ratio was much lower than the azide/amine ratio in the self-assembly mixture, which could be explained by the ability of aminophenylsilanes to interact with the silica surface through hydrogen bonding and self-associate via p-stacking. Additionally, the ability of amines to act as base catalysts capable of cross-linking trimethoxysilanes or condensing them with surface silanol groups could also increase the incorporation of aminosilanes into the monolayer. At the end.

this study showed a control on the surface ratio by determining the self-assembly mixture composition that would afford 1:1 azide-amine mixed monolayers.

SAMs preparation methods

There are numerous methods for placing a thin layer of organic molecules onto the surface of a material. Some methods rely on bond formation between a substrate and the modifying film and some rely on the non-specific interactions. Specifically, I would like to describe methods used to produce a monolayer on a solid substrate.

Langmuir-Blodgett layers

Langmuir-Blodgett (LB) layers are a remarkable feat of nanotechnology since their development in the 1930s by Irving Langmuir and Katharine Blodgett. The technique can be used to form a monolayer or more layers. This method relies on the interactions between amphiphilic molecules and water to create an ordered layer of molecules at the liquid-gas interface. Then the oriented molecular layer is compacted by applying lateral pressure using a barrier, causing an increase in order and packing density to form a compact molecular layer at the air-water interface (figure 1.19). To form an L-B layer, the Langmuir film is transferred to a solid support simply by passing the substrate slowly through the film. The details of the layer orientation initially depend on: the substrate surface chemistry, the type of molecule that is spread on the surface of the water, the orientation of the solid, and whether the support is lowered or raised through the interface [START_REF] Edwards | Chemically Modified Electrodes[END_REF]. However, this technique is often limited by the lack of stability due to the weak physisorbed bonds of the self-assembled systems to the substrate and the considerable defect densities of the produced molecular layers [START_REF] Moridi | Langmuir-Blodgett monolayer stabilization using supramolecular clips[END_REF].

Lithography

Lithography is another known method to build a pattern on the substrate material using UV light or electron beam or the atomic force microscope (AFM) which is the most widely used and so-called dip-pen nanolithography ( figure 1.20) [START_REF] Pimpin | review on Micro-and Nanolithography Techniques and Their Applications[END_REF]. AFM probe is coated with a thin film of a chemical of interest by immersing the cantilever in a solution or by evaporation.

Chemical molecules are deposited onto a substrate surface during the contact between the coated tip and the substrate. One drawback of dip-pen technique is that its constructed area is difficult to scale up [START_REF] Shim | Hardtip, Soft-spring Lithography[END_REF].

Chemical vapour deposition method

In this method, the solid substrate is exposed to a vapour phase of silane for a sufficiently prolonged time. Sometimes the process is carried out in ultra-high vacuum conditions, but it can also be carried out in a simplistic way. One simple way is to perform this process in a Petri dish. Specifically, the SAMs solution is dispersed in a Petri dish and the substrate is attached to the cap. The molarity of the solution, type of solvent, time given for the formation of the SAMs layer, distance between the solution and substrate and other physical conditions such as temperature, humidity, and cleanliness, should be adjusted and selected very carefully. Before reaching the final stage, full optimisation of all the mentioned factors is required. Otherwise, the process can lead to the formation of a disordered and loosely bound monolayer. During the process, the SAMs molecules evaporate and interact with the substrate surface. This interaction can occur by physical adsorption, van der Waals interactions, etc. depending on the nature of the surface and the monolayers. Regarding the control of the SAMs formation, the concentration and stability are important. This process can also form good monolayers, but it is more difficult to control, and few examples mentioned in the literature. However, due to its simplicity and the fact that no expensive instruments are necessary, this method can be useful for preliminary work [START_REF] Singh | The role of self-assembled monolayers in electronic devices[END_REF].

Immersion solution method

The immersion solution method, demonstrated for the first time in 1946 by Bigelow, Pickett, and Zisma, it is known to be substantially overshadowed all other techniques. In this method the growth of organic self-assembled monolayers is spontaneously adsorbed from solution rather than from the water/air interface, onto SiO2 and metal substrates (figure 1.21). The major advantages of this method over all are the simplicity of preparation and the flexibility of design by choosing different terminal functional groups and a better layer's stability compared to SAMs prepared by other techniques. However, structural control sometimes is difficult because several factors affect the formation and packing density of monolayers such as the nature and roughness of the substrate, the solvent, the nature of the adsorbate, the temperature and the concentration of the adsorbate [START_REF] Chaki | Self-assembled monolayers as a tunable platform for biosensorapplications[END_REF] . Historically, the immersion method in a solution (figure 1.21) is one of the easier, cheapest and most popular methods for the preparation of self-assembled monolayers. In addition, this kind of method is most suitable for mass production of self-assembled monolayers. However, there are some drawbacks to this method. One serious problem is that most of SAMs formation methods used are not compatible with the immersion method, but there is limited information that can be obtained during the growth process of the SAMs. The general steps of the classical immersion method are in general: first, cleaning and pre-treatment of the substrate (activation), followed by second step which is the immersion of the clean substrate into the coupling agent's solution. Indeed, the duration of immersion (growth time) depends on many factors like the temperature, concentration, solvent polarity and water content but there is lack of information concerning the growth process [181] [15]. In the third step, SAMs modified substrates should be removed from the immersion solution and washed to remove the physiosorbed molecules by the solvent used for the immersion solution. At the end, N2 gas can be used to dry the resulted SAMs [START_REF] Woodward | In Situ Observation of Self-Assembled Monolayer Growth[END_REF].

Spin coating

Spin coating is currently the predominant technique employed to produce uniform thin films of organic materials with thickness of the order of micrometres and nanometres. The physics of spin coating can be effectively modelled by dividing the whole process into three stages sketched in figure 1.22: deposition of the coupling agent's solution, high speed rotation, and evaporation of the solvent. In many cases the coating material is polymeric and is applied in the form of a solution from which the solvent evaporates. Spin coating was first studied for coating of paint and pitch [START_REF] Sahu | Fundamental understanding and modeling of spin coating process: A review[END_REF]. The final thickness and properties of the formed layer depend on the angular speed of spinning, the concentration of the solution, the solvent, the drying rate, the temperature, etc. Thus, it is a big challenge to adjust spin coating conditions to be used for SAMs, which explains why this method is rarely used for SAMs elaboration until now [START_REF] Yilbas | Self-Cleaning of Surfaces and Water Droplet Mobility[END_REF]. Compared to the immersion method, spin coating is faster and consumes less solvent and precursor. In spin coating, deposition lifetime can be controlled in an easier way and fresh solution could be prepared and used immediately. The biggest challenge of SAMs spin coating is to obtain a monolayer in a very short time (the spinning time) since the chains might not have enough time to arrange in a dense and packed array. Previous work on SAMs prepared by spin coating is mainly focused on organophosphonic acids or polymers [START_REF] Yilbas | Self-Cleaning of Surfaces and Water Droplet Mobility[END_REF] [185],while the spin coating of organosilanes onto SiO2 surfaces are rarely investigated and remains challenging.

Some SAMs from organosilane precursors have been deposited by spin coating onto wafers.

For example, SAMs from (3-trimethoxysilylpropyl)diethylenetriamine (DETA) have been deposited by spin coating onto Si wafers using two different solvents; propylene glycol monomethyl ether acetate (PGMEA) and methanol. Both solvents have been evaluated in terms of SAMs layer quality and found that SAMs deposited from PGMEA had better quality than SAMs deposited from methanol. Spin coating of wafer in PGMEA results showed that a concentration of 0.05 mM or below resulted in partial coverage with a tilt angle of 70° and aggregation is observed for all tested concentrations and is worse for higher concentrations, so tt is possibly induced by the non-negligible presence of water in PGMEA solvents. The aggregation of SAMs could be possibly mitigated by limiting the amount of water contained in the chosen solvent while controlling the atmosphere during spin-coating [START_REF] Sun | Optimization and upscaling of spin coating with organosilane monolayers for low-k pore sealing[END_REF].

Recently, in the C2M research group, an azide-terminated SAM has been successfully prepared by using the spin coating technique in ambient atmosphere and at room temperature. In this work, first, they reported the first convenient and wide-scope synthetic methodology to prepare azidoorganotrialkoxysilanes via last step hydrosilylation of storable azide-containing olefins and compatible with various alkyl and aryl substrates featuring urea and carbamate moieties. Second, through one example, they showed that they can be subsequently grafted covalently by spin coating onto silica surfaces in ambient atmosphere to provide an azide-terminated SAM [START_REF] Rouvière | Hydrosilylation of Azide-Containing Olefins as a Convenient Access to Azidoorganotrialkoxysilanes for Self-Assembled Monolayer Elaboration onto Silica by Spin[END_REF]. Then the ability of those azide-terminated surface to immobilise molecules via the CuAAC reaction has been proven by the PM-IRRAS technique, confirming the efficiency of the spin coating method to produce SAMs for further modification.

The immersion deposition is a method widely used in the literature, because it is compatible with a large number of organosilanes whatever the nature of the grafting head or the structure of the organic part. However, this method has disadvantages such as the reaction time (several hours) and the amount of solvent used to immerse the substrates. In contrast, the spin coating process is interesting because it drastically reduces reaction times (a few minutes) and uses very little solvent (a few microliters). The challenge is to obtain a monolayer.

SAMs characterisation methods

Surface can be understood as a transition region between the outside media, or vacuum, and the uniform bulk material. The chemical and physical properties of the materials in this transition region are very different from that of the bulk. It is imperative to analyse the functionalised substrates to have a clear understanding of the properties of molecular films, such as the wettability, the thickness, the packing density, the orientation, the available functional groups, etc. In view of the small amount of the analysed material (from micro to picomol per cm 2 ) and the general complexity of the systems, a combination of highly sensitive surface analysis techniques must be usually employed to get an insight into the surface properties. In general, for SAMs analysis, electrons, photons, X-rays, neutral species, or ions as a probe beam interact with a system in one way or the other to be analysed afterwards and give information (table 1.2). Some techniques provide information by measuring changes in energy/intensity of the primary beam while others analyse secondary ejected moieties from the sample upon the excitation provided by the probe beam. Some other techniques involve mechanical contact between the probe and the supported molecular film [3] [187]. Obviously, various analytical techniques have been employed in characterising the organic interface on a surface. These techniques are used widely for determining the relationship between the microscopic/nanoscopic structures generated and the macroscopic properties related to them. In this research various analytical techniques were used for SAMs analysis. A short description of the utilised characterisation techniques is provided in the following sections [START_REF] Liu | Interface Characterization and Control of 2D Materials and Heterostructures[END_REF].

Atomic Force Microscopy (AFM)

This technique is used to give information about the surface topography and the surface structure. AFM measures the attractive and repulsive forces between a sharp tip, attached to a Si3N4 cantilever, and the surface of a sample (Figure 1 where the tip scans the sample in close contact with the surface ; ii) the non-contact mode where the tip is oscillated at the resonance frequency with the amplitude kept constant and thus is not in contact with the sample surface but moves above the adsorbed fluid layer, iii) the tapping mode between the contact and non-contact mode [START_REF] Geisse | AFM and Combined Optical Techniques[END_REF] [189] [START_REF] Giessibl | Advances in atomic force microscopy[END_REF]. The contact mode is heavily influenced by frictional and adhesive forces and can damage samples and distort image data.

However, the non-contact mode generally provides low resolution and can also be hampered by the contaminant (e.g., water) layer which can interfere with oscillation. In contrast, tapping mode imaging takes advantages of the two above. It eliminates frictional forces by intermittently contacting the surface and oscillating with sufficient amplitude to prevent the tip from being trapped by adhesive meniscus forces from the contaminant layer. In addition, this mode of imaging shows no surface alteration and better resolution [START_REF] Zhong | Fractured polymer/silica fiber surface studied by tapping mode atomic force microscopy[END_REF] which made it our choice to analyse our surfaces in this work. It includes both a contribution from a background signal and resonance peaks characteristic of the bound states of the electrons in the surface atoms [START_REF] Brundle | Encyclopedia of Materials Characterization[END_REF] . The resonance peaks above the background are the significant features in an XPS spectrum, the peaks is related to the concentration of the element on the surface of the sample. The X-rays may penetrate deep into the sample, however, the escape depth of the ejected electrons is limited. The electrons ejected from depths greater than a few nm have a low probability to leave the surface without an energy loss, thus they contribute to the background signal, rather than appear as a well-defined peak. For this reason, XPS is a surface analytical technique used to determine the surface composition of a material only [START_REF] Adamkiewicz | Self assembled monolayers on silicon : deposition and surface chemistry[END_REF] .

Polarisation-Modulation Infrared Reflection-Absorption Spectroscopy (PM-IRRAS)

Polarisation Modulation-Infrared Reflection-Absorption Spectroscopy (PM-IRRAS) is a spectroscopic technique based on the wide known Fourier Transform Infrared Spectroscopy (FTIR) and used for the characterisation of thin films or monolayers of adsorbed matter on metal surfaces. In general, an IR beam can be resolved into p-and s-polarised components in which the electric vector oscillation is parallel or perpendicular to the plane of incidence, respectively. IRRAS provides IR spectra measurement of thin films on metal substrates with high sensitivity using p-polarised light parallel to the incidence plane. The p-polarised light generates an electric field with stationary vibration which increases sensitivity (figure 1.27).

On the other hand, PM-IRRAS is a method of finding intensity difference of s-and p-polarised light (ΔI = Ip -Is) vertical and parallel to the incidence plane respectively using a Photo Elastic Modulator (PEM) (figure 1.27). As s-polarised light does not generate an electric field of stationary vibration, the absorption is much smaller than that of p-polarised light. In addition, in PM-IRRAS, the sum of s-and p-polarisation signal (Σ I = Ip + Is) is used as reference and so there is no need to measure a reference substrate [195] [196]. Unfortunately, because of the complexity of data processing and spectrum interpretation, PM-IRRAS had been little used by researcher [START_REF] Monyoncho | The utility of polarization modulation infraredreflection absorption spectroscopy (PM-IRRAS) in surface and in situ studies: new data processing and presentation approach[END_REF] . Despite the complexity of this technique, it is our first choice in this work due to the great importance of this technique in surface characterisation.

b) a)

Contact angle goniometry

The contact angle measurement has been extensively used in research as the simplest and quickest method to measure a surface's wettability. It gives a direct indication of the surface hydrophobicity or hydrophilicity and can indicate a chemical change at the terminal functional groups of a monolayer or the cleanliness of a surface. The contact angle (θ) is the angle measured at the three-phase interface where liquid, vapor, and solid intersect (figure 1.28) [START_REF] Janssen | Static solvent contact angle measurements, surface free energy and wettability determination of various self assembled monolayers on silicon dioxide[END_REF]. The setup is relatively straightforward and consists of a macro camera, a syringe, an adjustable stage and an analysis software. The camera can record the shape of the water droplet on the surface of the sample. Through the software processing, the volume of water, the surface tension of the liquid and the contact angle with respect to the surface can be calculated. The contact angle of the liquid with the solid surface is determined by the Young equation:

γSV=γSL+γLV cos(θ)……………………. Equation 1.2

The interfacial tensions -solid/vapor (γsv), solid/liquid (γsl), and liquid/vapor (γlv) -form the equilibrium contact angle of wetting, many times referred as Young contact angle, θ. Normally, hydrophilic surfaces have contact angle smaller than 90° while hydrophobic surfaces have values higher than 90°. However, some surfaces have contact angle value of 150° or greater due to either very rough surfaces or the presence of organic contaminants which prevent wetting and result in higher contact angle [62] [198]. A summary of reported water contact angle measured on monolayers with different functional head groups is presented in table 1.3.

Conclusion and scope of the thesis

Surface properties are particularly important for biomolecules immobilisation including DNA, protein, and enzymes which are of prime importance in the biomedical field. Therefore, there is a great demand for the investigation of surface properties and SAMs offer an ideal approach for producing well controlled and organised two-dimensional surfaces by an easy and simple process. The most used method for SAMs deposition is the immersion method. But this method is sensitive to a large number of parameters (spacer type and length, water content, time and temperature, etc) which affect SAMs properties. SAMs properties can be analysed by various techniques like XPS, PM-IRRAS, AFM, etc., and those techniques can be used also to investigate further modifications on the surface.

Silane-based SAMs are stable and robust which are valuable advantages for their further modification and their use in different applications, in particular for biomolecules immobilisation, which is fundamental within many areas of science. But there are two main challenges in this field.

The first one is to control the orientation of immobilised biomolecules on the surface which helps to best preserve their activity or recognition properties and to improve the performances of the final application. Thus, bioorthogonal chemistry recently became an important tool for site-selective (i.e chemoselective) biomolecules immobilisation. Consequently, SAMs terminated with functions reactive toward bioorthogonal reactions, especially azide group, attracts more attention.

The second one is to control the distribution of molecules on the surface which could be deleterious to their activity or recognition properties. It mainly depends on the density and the accessibility of the terminal groups on the surface. This issue can be solved by understanding the behaviour of molecules during the self-assembly and investigating factors that have direct effect on the terminal group reactivity, which could lead to molecular control of SAMs and the possibility to change properties by changing parameters.

In this context the general goal of the present work is to prepare molecularly engineered azideterminated SAMs for biomolecules immobilisation via CuAAC click chemistry to be used in biological applications. The CuAAC reaction was chosen over the multitude of other interfacial chemistries because it is a straightforward and robust method to attach a large variety of molecules onto a diverse array of surfaces in a controlled way (controlling the quantity and the orientation of the biomolecules). In this work, azide is chosen to be the terminal group of SAMs for its preferable properties, azide is a small group and biorthogonal, they are essentially absent from biological systems but can be easily introduced into biomolecules. Furthermore, azides can be probed relatively easily using FTIR spectroscopy and XPS. Before using the CuAAC reaction to attach biomolecules to surfaces, first we need to design surfaces prone to such kind of immobilisation. Many parameters will be studied in order to control the desired properties of the surface and to provide a comprehensive study of structure / properties relationships for azide-terminated SAMs depending on the elaboration processes and the coupling agents structures. In addition to providing a new concept of mixed SAMs as another helpful tool to enhance the biomolecules immobilisation. To summarise we have three main objectives to this dissertation:

First, we are proposing a novel approach of azide-terminated SAMs preparation based on direct grafting of azide coupling agents instead of the post functionalisation method. We have two types of azide terminated self-assembling molecules depending on the spacer nature: alkyl and urea chains. We exploit the synthesis of new azide-terminated coupling agents with different spacer lengths to investigate the behaviour of each urea compound; by changing carbon chains lengths in both sides (between the urea and the silica surface and between the urea and the azide). In addition, regarding grafting process, we will change some parameters (for both spacer types) like solvent polarity and grafting method to molecular engineer those surfaces and understand the effect of those changes on SAMs properties.

Secondly, spin coating method will be used to form SAMs, as a fast and less time and chemicals consuming technique instead of the conventional long-time immersion method. In this part the main concern is optimising grafting conditions and understanding the effect of changing the grafting process on the resulted SAMs.

Thirdly, a new concept of mixed SAMs will be studied aiming to enhance the distribution of molecules in the SAMs, and consequently enhance the reactivity and the accessibility of the azide terminal groups for the click reaction. This new concept based on mixing two types of molecules; silylated molecules without azide terminal group and non-silylated molecules terminated with azide group.

Finally, the last step of this work focuses on using developed azide-terminated SAMs for biomolecules immobilisation via click chemistry. Our target biomolecules are Glycoside Nucleoside lipids (GNLs) which are original low molecular weight gelators. Since the interactions between a biological system and a material depend strongly on the physicalchemical properties of the material, we will study the effect of changing the properties of developed SAMs on the immobilisation of the GNLs. Then this system will be used as a platform for stem cell culture for the first time.

Introduction

The surface properties of oxide surfaces are often tailored with chemisorbed monolayers presenting the desired functionality at the surface. If SAMs are terminated with a functional group, this array of reactive sites reacts as a suitable platform to attach other molecules onto the surface. For instant, SAMs can provide a robust attachment of biomolecules such as oligonucleotides, enzymes, and polypeptides onto solid surfaces [1]. The sensitivity, the selectivity and the rate of response of biosensors are directly related to the quality of the biomolecule's immobilisation in terms of the amount, the orientation and the nature of the linkage with the surface (covalent or non-covalent) [2]. Indeed, controlled surface modification As previously mentioned, our target functional group of SAMs is the azide group and in this chapter the spacer is an alkyl chain. In addition to its bioorthogonal properties, the azide group can be easily probed using Fourier Transform Infrared (FTIR) or PM-IRRAS technique and Xray photoelectron spectroscopy (XPS). These techniques can be used to monitor relative azide surface compositions [6] [7] [8] [9]. Aliphatic chains are typically used as spacers, but it is not clear how the silane molecules arrange on the substrate, in particular the orientation versus the reactivity of the terminal-functional group. Despite the fact that many authors characterised the surface properties of such monolayers in terms of roughness, surface energy, and layer thickness using different experimental techniques, the description of the monolayer at the molecular level is still lacking [10].

(
In general, the most used method to prepare azide-terminated SAMs is by a SN2 reaction applied on bromide-terminated SAMs [11] [12] [13]. In the 90ths, it has been shown for the first time by Balachander and Sukenik that SN2 reactions can be used for the conversion of functional groups of monolayers [11]. The first condition-set reflects a well investigated protocol for the grafting of bromine-terminated organotrichlorosilanes. Chemical reactions or modifications on solid surfaces is different from organic reactions in the liquid phase and difficult to follow. In the case of SAMs, the nucleophile must penetrate the monolayer to attack the (C-leaving group) bond from the backside. Therefore, the nucleophile must be a small and strong nucleophile.

Moreover, there is a kinetic barrier due to the fact that the monolayer is closely packed and thus the nucleophile must shed much or all of his solvent shell to be able to penetrate the monolayer.

In addition, the success of a displacement reaction in SAMs depends strongly on the type of SAMs and its defects [12]. Despite all those limitations, as already shown in the literature by several research groups, this is the most used method to prepare azide-terminated SAMs.

In contrast, less studies have been done on the direct grafting of azide-terminated molecules on SiO2 surfaces. In this process, commercial 11-azidoundecyltrimethoxysilane was used directly to prepare SAMs using the immersion method, or rarely by the chemical vapor deposition [14] [15] and the Langmuir-Blodgett technique [16]. Indeed, those studies of direct grafting of azido precursors to the surface have given general information about the successful grafting, film thickness, roughness, and the successful click reaction to attach molecules but never investigate the orientation of azide or its overall reactivity. Historically, the immersion method is one of the easier, cheapest, and most popular method for the preparation of SAMs as well as the most suitable for mass production. However, there are some drawbacks to this method like the limited information obtained during the growth process of the SAMs, in addition to the multiple factors that can affect the efficiency of the growth and the structure of SAMs as well as its quality and reproducibility. But this method is still the most used method for SAMs elaboration [17].

In this part of the work, we used the 11-azidoundecyltrimethoxysilane as a model aiming to present a comprehensive study of the self-assembly of azide-terminated SAMs. This model could provide a basis for studying the influence of changing some parameters during molecules self-assembly on the orientation and the reactivity of the terminal azide groups towards the CuAAC click reaction. Understanding the behaviour of the azide group in pre-functionalised surfaces allows us to improve its reactivity with biomolecules. In addition, the spin coating method will be used to form SAMs which could be a breakthrough in SAMs field, as a faster and less chemicals consuming method leads to economic advantages. To sum up, the general work scheme of this chapter can be described in figure 2.1 : In this section we will prepare azide-terminated SAMs by two strategies: (1) the common post-functionalisation strategy (PF) and ( 2) the direct grafting strategy (Dir). Both will be applied using two different methods: (1) by the classical immersion method (I) and ( 2) by the spin coating method (SC). Moreover, solvent effect will be studied by applying each method in different solvents. The chemical modifications of surfaces have been investigated using the contact angle measurements, PM-IRRAS, XPS and AFM. At the end, all methods will be compared to assess the impact of those changes on the resulting SAMs namely the azide orientation and reactivity. The reactivity of the azide group towards click chemistry will be evaluated for each surface by reacting with an alkayne probe via the CuAAC click reaction.

Synthesis of functional alkyltrimethoxysilane precursors

We have two target molecules in this part: 11-bromoundecyltrimethoxysilane and 11azidoundecyltrimethoxysilane. The commercially available 11-bromoundec-1-ene has been used to synthesise 11-azidoundec-1-ene, following the published procedure [18] , as shown in (5 eq.). The conditions of the hydrosilylation reaction have been previously optimised in our research group [18]. The reaction is applied in the presence of Karstedt's catalyst (Platinum commercial source, 2.5 mol %) using acetonitrile solvent at ambient temperature, under inert atmosphere for 3 hours. [20] [23]. Hence, it is very important to optimise hydrosilylation conditions (solvent, temperature, concentration, and reagents stoichiometric ratio) to avoid side reactions. For instant, to avoid polymerisation side products, it is preferred to carry out the hydrosilylation reaction just before the grafting process and have freshly prepared silane's solutions [24] . In our case, the conditions of the hydrosilylation reaction resulted in a quantitative yield for both bromo-and azidoorganosilanes: by using acetonitrile as a solvent at ambient temperature for 3 hours under inert atmosphere. In figure 2.5, the NMR spectra show a good ratio of the signal characteristic of the two protons of the methylene next to the silicon atom (-CH2Si) at 0.60-0.67 ppm, while no peaks are detected between 4.5-7 ppm (the region of alkene) confirming the absence of any isomerisation side reactions. changing significantly before and after the substitution of both SAMs, and the small differences could be because the spectrum of SAM-N3 is an average of many trials for each solvent ,while SAM-Br spectrum is for just one trial. As a result of the post-functionalisation, we can observe the appearance of the azide asymmetric stretching (aN3) band at 2098 cm -1 due to the asymmetric stretching vibration of the azido resonance structure (-N=N + =N -) [30]. surface hydrophilicity in this case. The contact angle was measured on different slides by different regions on each slide, then an average was calculated with a precision on the resulted value of ±2°. As described in the literature, we observed a decrease in water contact angle from 70° or SAM-Br grafted in toluene and 68° in chloroform to 63° and 65° respectively for SAM-N3 , those values are relatively lower than the literature values (SAM-N3: 77°-84°) [12] [31] due to differences in grafting conditions. As mentioned in the first chapter each small change could have high impact on SAMs properties. For example in some studies they are adding water during deposition or heating after deposition or using trichloroorganosilanes instead of trimethoxyorganosilanes which could explain the difference in the contact angle values [27] [26].

Another important analysis is the atomic force microscopy (AFM), which gives information about the surface topography like the surface roughness, as discussed before. The images obtained by the AFM technique could also investigate the homogeneity of the surface coverage and detects the presence of aggregations (material accumulation in some regions as multilayers)

or holes (lack in matter in some regions). AFM analysis was performed on wafer substrate with initial roughness of 0.21 nm) before the chemical grafting. Figure 2.11 shows the AFM images of SAM-N3, it shows a relatively smoother surface resulted from using chloroform (roughness of 0.41 nm) compared to toluene (roughness of 1.09 nm). But both surfaces are homogeneous, and no holes or aggregations are observed. Even if the information given by PM-IRRAS, AFM, and contact angle measurements are enough to confirm the modification of the surface by the bromide monolayer and then the postfunctionalisation to azide, we tried also XPS analysis to evaluate the conversion rate of the SN2 reaction. The XPS analyses of the Br(3d) have been done for SAM-Br grafted in chloroform as shown in the following figure 2.13. The problem in our case is the overlapping of the Br(3d) region with the Pt(4f) region, the latter coming from the catalysed hydrosilylation reaction to provide the 11-bromoundecyltrimethoxysilane. Nonetheless, we were able to decompose the spectra with Pt contributions at higher binding energy (BE) and Br contributions at lower BE (areas under the contributions are filled). On XPS spectrum of SAM-Br, Br(3d) represents 0.54 % of all atomic contributions, while it represents only 0.04 At% on XPS spectrum of SAM-N3.

This result confirms that most of the Br groups reacted to form the SAM-N3 (around 92%). In addition, when looking at the At % of single contribution, the At% of Br (ca 0.54 %) is coherent with the increase to 1.9 At% for N1s, considering that 1 Br atom is replaced by 3 N atoms. To summarise this part of the work, the formation of SAM-N3 using the post-functionalisation strategy by the immersion method is not sensitive to solvent polarity changes, which produces SAMs with similar properties whatever the solvent.

Using the spin coating method

The general steps of preparing SAM-N3 from SAM-Br here are similar to the previous section, starting by SAM-Br formation (by grafting 11-bromoundecyltrimethoxysilane to the surface) but instead of using the immersion method, here, we used the spin coating method according to a procedure optimised in our laboratory [18]. As shown in figure 2 in the range of intensity of the methylene asymmetric stretching band of 0.0025 [35]. Those experiments can be considered as reproducible results. The first observation from the figures below is that the quantity of material constituting the layer is slightly increasing by increasing drying time (as CH2 and N3 bands intensities increase) but this change is not significant between 30 minutes and 3 hours for both toluene and chloroform. In fact, there is likely more organic material onto the surface after spinning when chloroform is used in comparison with toluene because chloroform is more volatile. So its evaporation is faster during spinning than with toluene, which creates a more concentrated solution after spinning. In the case of toluene, more organic material is removed during spinning by discarding of the solution from the sample.

Moreover, during the drying time , the lower volatility of toluene makes the lifetime of the solvent phase on the substrate higher for toluene than chloroform, which makes the process of the hydrolysis condensation time longer allowing the addition of more molecules during the layer formation and thus slightly more increase in organic matter than chloroform. In the two mentioned cases we have a concentration phenomenon, in which the organic layer thickness depends on the initial amount of the material on the surface after spinning.

Azide-terminated alkyl SAMs prepared by the direct grafting strategy

As shown in figure 2.18, in this part, 11-azidoundecyltrimethoxysilane was directly grafted to the surface using both methods (the immersion and the spin coating). Three solvents have been used with different polarity and volatility: acetonitrile, chloroform, and toluene. Their relative polarities are 0.460, 0.259, and 0.099 respectively, and their boiling points at the atmospheric pressure are 81.6 °C, 61.2°C and 110.6°C, respectively. The azide group differentiates from bromine since it displays a dipole moment for the (-N=N + =N -) fragment. Thus, it could be more sensitive to the changes in solvent nature, subsequently producing SAM-N3 with different properties.

Figure 2.18. General scheme for SAM-N3 prepared by the direct grafting strategy using: ⅰ) the classical immersion method and ⅱ) the spin coating method

Using the immersion method

The protocol used for preparing SAM-N3 by the immersion method is same as the protocol applied for SAM-Br, but instead of bromide silylated compound, here, 11azidoundecyltrimethoxysilane has been used to prepare silanes solution for the immersion method (see the experimental part). Three different solvents (toluene, acetonitrile, and chloroform) have been tested and the three produced SAM-N3 have been analysed in PM-IRRAS and AFM in addition to contact angle measurements.

At the beginning of this section it is important to investigate more about the stability of SAM-N3 prepared by the direct grafting strategy instead of the post-functionalisation strategy. We decided to investigate carefully the stability of the azide moiety during the grafting process and then immobilised on the surface after time. First, we performed a control experiment by immersing PM-IRRAS substrates following the classical immersion process but using a solution of 11-azido-undec-1-ene in chloroform instead of the corresponding silylated coupling agent. PM-IRRAS analyses of the washed slides confirm that no organic material was grafted on the surface and thus, that the azide group does not act as a competitive anchoring group. At the end of the immersion time, the solution was concentrated under reduce pressure to recover the organic material and analyse it by 1 cm -1 and 2929 cm -1 , and the symmetric stretching modes (sCH2) are at 2857 cm -1 , 2854 cm -1 , and 2857 cm -1 for toluene, acetonitrile and chloroform, respectively. Those values indicate the formation of a disordered layer for all solvents. The azide stretching band is observed at 2099 cm -1 for toluene, 2099 cm -1 for acetonitrile and 2103 cm -1 for chloroform, which agrees with the expected azide band peaking at around 2100 cm -1 for the three solvents. Comparing the spectra of the three solvents shows that almost same intensities of CH2 stretching bands are obtained for both toluene and chloroform while more intense bands are observed for acetonitrile. That can be explained by the higher polarity of acetonitrile which increases its ability to absorb and solubilise water and thus has an impact on the polycondensation of the coupling agents resulting in higher quantity of matter than for other solvents [36]. In addition, it is known that the organic layer is grafted by pre-formed and pre-organised oligomers of the coupling agent in solution.

Some solvents favour the pre-organization in solution of the film and could fill the empty places [37]. In contrast, the non-expected result is the variability of the relative intensity of the aN3 band compared to the ones of CH2 depending on the solvent. More precisely, the intensity of the azide band compared to the ones of CH2 bands is significantly lower after grafting in polar solvents such as chloroform or acetonitrile than grafting in toluene. The more significant effect has been observed when using chloroform. Considering normalised spectra with respect to the aCH2 band (figure 2.20b) to compare the three solvents highlights this result. As detailed above, the azide's degradation during the grafting process or the SAMs aging can be ruled out.

To explain this observation, we hypothesise that the tilt angle (ϴ) of the azide group in the monolayer could lead to different orientations depending of the solvent, which could affect its visibility in PM-IRRAS according to the selection rule. In this case, if the azide group is parallel to the surface it will not be detected by PM-IRRAS, while the intensity of the band will be maximum if the azide group is perpendicular (vertical) to the surface figure 2.20c , and all azide oriented in between the parallel and perpendicular positions will be detected in PM-IRRAS with a variable intensity [38]. Thus, it will create differences in intensities of the azide band for the same organic layer quantity on the surface (the normalised spectra). In this case, it is observed from the normalised spectra that using polar solvents to prepare SAMs-N3, especially chloroform, would lead to azide groups oriented more parallel to the surface than when toluene is used. The values obtained for the direct grafting by the immersion method in toluene and acetonitrile are in the same range of the ones obtained by the post-functionalisation route using toluene (63°) and chloroform (65°), while for the direct grafting using chloroform provides a slightly lower value which could be related to the lower azide intensity on the surface. Finally, when going from toluene to acetonitrile and then chloroform, the decrease of contact angle values is concomitant with the decrease of the azide band intensity with respect to the CH2 bands (see normalised spectra on figure 2.20b) and the dipole is more and more parallel to the surface.

Moreover, AFM images (measured on wafer surfaces with an initial roughness of 0.21 nm)

show smooth and homogeneous surfaces with roughness values of 0.55 nm, 0.54 nm, 0.45 nm for toluene, acetonitrile and chloroform respectively (figure 2.22). All images show the absence of any default (holes or aggregates), revealing a uniform grafting process on the whole surfaces.

Using the spin coating method

Here, the direct grafting of 11-azidoundecyltrimethoxysilane is applied to silica surfaces using the spin coating method. The procedure of spin coating of 11-azidoundecyltrimethoxysilane is same as the procedure mentioned previously for 11-bromoundecyltrimethoxysilane. Once again, three solvents (toluene, acetonitrile and chloroform) have been used to check the impact of changing the solvent's nature on the produced SAMs. Then, several drying times have been investigated for each solvent to optimise the spin coating method and achieve SAMs with properties consistent with the monolayers described in the literature and prepared using the classical immersion method. The spin coating of 11-azidoundecyltrimethoxysilane in chloroform has been previously done in our laboratory [18] and all conditions have been optimised according to the (aCH2) band intensity consistent with literature results (around Thus, results obtained with chloroform can be used as a reference to adjust the conditions with the other solvents. PM-IRRAS results obtained using toluene are shown in figure 2.24a. First, we can observe the presence of all the characteristic bands (azide and CH2 stretching bands) which confirms the formation of SAM-N3. The antisymmetric stretching mode (aCH2) is at 2926 cm -1 and the symmetric stretching mode (sCH2) is at 2854 cm -1 for all drying times indicating the formation of a disordered layer, while the azide stretching band is observed at 2099 cm -1 for all spectra. Compared to the result obtained using chloroform, we can see that the intensity of the CH2 bands is slightly less for 30 min of drying time while slightly higher for 3 hours. We can conclude that the period of time between 30 min and 3h is the optimum drying times to obtain the similar monolayer obtained in chloroform . Another important observation is the increase of the CH2 bands intensity by increasing the drying time, which could be explained by continuing the deposition reaction and adding more molecules to the monolayer by time. To check that, we prepared a so called "no wash" sample. After spinning, no washing was applied, and the sample has been analysed by PM-IRRAS. In figure2.24a, the intensities of the CH2 and azide bands in the "no wash" sample are slightly more intense compared to the corresponding spectra after a drying time of 30 min or 3 hours. This result clearly shows that the spin coating method allows the deposition of an organic compound layer close to the monolayer. The washing process for 30 min and 3h removed the excess of organic compound located above the monolayer. Then, after few months (3-4), we washed the "no wash" sample and analysed again in PM-IRRAS (so called "after washing"). As shown in figure 2.24b, the small decrease in bands intensities before and after washing is due to removing the excess (not reacting) molecules. This sample leads to conclude that, even with very long drying time, the deposition of molecules will stop, either because the monolayer will be saturated and no more molecules could be added, or because the solvent will be completely evaporated and stop the deposition process. This experiment shows that the excess of organic material on the surface is easily removed even after several months. In addition, obtaining a homogeneous surface in AFM without aggregates shows that this spin coating process allows to have a silane orientation close to the surface during deposition in order to lead to a monolayer without defects like aggregates. At the end, the reproducibility of this process should be checked. As both 30 minutes and 3 hours drying times are good, we can choose any of them. We chose to repeat the 3 hours. As observed from figure 2.24c, the difference between the intensities of both azide and methylene bands is so small that it can be ignored, and we can say that they are superimposed. and the symmetric stretching mode (sCH2) at 2854 cm -1 of the methylene groups for all drying times, indicating the formation of a disorder layer, while the azide stretching band is observed at 2099 cm -1 for all times. With acetonitrile, it is obvious that the drying times of 30 minutes At the end, the reproducibility of this method should be checked. 30 minutes and 3 hours are very similar, and we chose the longer time, 3 hours. As observed from figure 2.27, the difference between the intensities of both azide and methylene bands is not significant and can be ignored. We can conclude that this process is reproducible. At the end, we want to investigate the impact of changing the solvent's nature on all SAM-N3 prepared by the direct grafting strategy using the spin coating. For the spin coating, the drying time of 3 hours has been chosen. The comparison depends on all obtained characterisation data from PM-IRRAS, AFM and contact angle measurements.

In contrast with our observations for the deposition of the aziodoorganosilane by the immersion method, almost no solvent effect can be observed from the PM-IRRAS data for the spin coating method (figure 2.28). Differences in the intensities of CH2 bands between the three solvents are small which means that solvent polarity does not affect significantly the organic matter's quantity deposited by spin coating. Moreover, for the spin coating method, the bands of CH2 are slightly more intense than the ones observed with the immersion method for all solvents (except for acetonitrile) due to a better packing density than the one obtained by immersion, which could be due to having less concentrated immersion solution compared to the spinning solution. To discuss the azide orientation, it is better to observe the normalised spectra of the spin coating method in the following figure. Differences between the three solvents are very small. In contrast with the immersion method, we could conclude that the influence of changing the solvent's nature has no impact on the azide orientation for the spin coating method.

According to our knowledge, the spin coating technique has not been used and studied deeply a(N3) s(CH2) a(CH2)

for SAMs formation so still there is lack in the information. But we can put some hypothesis from those results to explain the decrease of solvent polarity impact. We hypothesise that this method is more dependent of kinetic, the fast spinning time enables the reaction to happen fast and this speed decreases the effect of changing conditions, therefore, it could decrease the role of solvent polarity.

Comprehensive study of the influence of both the grafting strategy and the deposition method on the properties and the reactivity of azide-terminated SAMs

We studied both strategies to prepare SAM-N3, the traditional post-functionalisation method and the direct grafting strategy and concluded that the post-functionalisation method is less sensitive to conditions changes in both cases of the immersion and the spin coating for toluene and chloroform. In this case, we can pick any experiment to use it for a comparison to other methods. On the other hand, the direct grafting strategy is more sensitive to changes, in which the immersion method is more sensitive to changes than the spin coating method. As a result, we will compare the three methods in one solvent. In this part, for the first time according to our knowledge, we want to deeply understand the influence of the deposition method on the azide orientation and its reactivity towards the CuAAC click reaction. Understanding the behaviour of the azide group is the key to control its reactivity with biomolecules for further modifications.

The results of this part rely on a deep analysis of PM-IRRAS, AFM, contact angle measurements and the click reactions results. We have three types of SAMs:

-PF-I-SAM-N3 was prepared by the post-functionalisation strategy from SAM-Br prepared by the immersion method (same as the one resulting from the spin coating method)

-Dir-I-SAM-N3 was obtained by the direct grafting of the pre-synthesized 11azidoundecyltrimethoxysilane using the conventional immersion method -Dir-SC-SAM-N3 was prepared by the direct spin coating of 11azidoundecyltrimethoxysilane.

Evaluation of the properties of SAM-N3 prepared by three different processes by PM-IRRAS analysis

Starting the study with acetonitrile, figure 2.30 shows a comparison of the PM-IRRAS spectra resulting from the two deposition processes: the formation of SAM-N3 by the direct immersion method and the direct spin coating method. Surprisingly, the intensities of methylene bands obtained for Dir-I-SAM-N3 are superimposed to the ones obtained for Dir-SC-SAM-N3, which means that both surfaces display the same quantity of organic matter. On the other hand, the intensity of the azide band is significantly lower for the immersion method. This result can be due to the nature of the packing density which modify dipole interactions. Immersion method used diluted solution while more concentrated solution is used in the spin coating process. With spin coating the kinetic is faster than with the immersion method. This phenomenon imposes a fast assembly. Consequently, the organisation of molecules in the monolayer and dipole interactions are different and more heterogeneous than with the immersion where the kinetic of assembly is low and allows time to reach equilibrium and so dipole interactions populations are more homogeneous. In the spin coating method the azide dipoles are more vertically oriented than in the immersion method because for the last one there is more time to afford dipole-dipole interactions leading to azide groups more parallel to the surface. During the spin coating process, the self-assembly is constrain by the spinning and the concentration effect that quickly imposes a packing density which prevent dipole interactions and promotes an upward straightening of the dipole. To summarise, when using acetonitrile solvent, the process of direct grafting (either spin coating or immersion) has no effect on the quantity of the organic matter deposited on the surface, while it has an impact on the azide groups orientation due to different kinetics during SAMs deposition. Chloroform has been used also to study the impact of the deposition processes: the postfunctionalisation by immersion, the direct grafting by immersion, and the direct grafting by spin coating. As shown in figure 2.32a, there is a significant variation in methylene bands intensities for the three methods. Moreover, the normalised spectra (figure 2.32b) show also a significant variation in the azide orientation (i.e variation in the intensity of the azide band). It leads to conclude that, when chloroform is used, the deposition method has impact on both the quantity of organic matter and the azide orientation. This conclusion motivated us to choose chloroform as a grafting solvent to study the effect of changing the deposition method on SAMs properties and azide orientation and reactivity. The rest of this section will present this study in detail. The PM-IRRAS spectra (figure 2.32) show variations in the intensity of the methylene stretching modes. We discussed in the previous sections the PM-IRRAS spectra of the three azide-terminated SAMs and confirmed the functionalisation by observing in the 3100-1800 cm - 1 region, the stretching modes of the methylene groups (3000-2800 cm -1 ) and the stretching mode of the terminated azide group (around 2100 cm -1 ). The wavenumbers of the antisymmetric (aCH2) and symmetric (sCH2) stretching modes of methylene groups are sensitive to both the conformation (gauche or trans) and the order of the alkyl chains. The (aCH2) and (sCH2) bands are observed around 2929 cm -1 and 2857 cm -1 , respectively, for all the azide monolayers, revealing a disorder of the alkyl chains in the monolayers. The intensities for the antisymmetric (aCH2) and symmetric (sCH2) stretching bands of the methylene groups (i.e. 0.0027 and 0.0015, respectively) are like those measured for azide-terminated thiol monolayer (N3C12H24SH) deposited on gold substrate [35]. Thus, these intensities of the (aCH2) and suggesting less densely packed monolayers and/or much thinner monolayers. For Dir-SC-SAM-N3 the higher value can be explained by the spin coating process where two phenomena act simultaneously, the spin and the evaporation at the ambient atmosphere [44] . At the early stage of spinning the liquid drop spreads homogeneously over the whole surface and evaporation increases the concentration which promotes the fast adsorption of silanes on the wetting surface; inducing the crosslinkage of molecules to afford a highly dense monolayer. In contrast, PF-I-SAM-N3 and Dir-I-SAM-N3 are much thinner due to the solution immersion method which uses lower concentrated silane solution. The leaving group of silanes affect the rate of hydrolysis of the silylated compound, as a result, it plays an important role in the structure of the SAMs [45]. Moreover, in diluted solution, the assembly of the monolayers using anhydrous solvent requires several hours due to the lower rate of hydrolysis of the trimethoxysilyl groups compared to other groups like trichlorosilanes. The slow incorporation of the molecules inside the layer and the steric constraints limit the saturation density which induces a greater mobility and tilting of the chains responsible for a sparsely packed SAM.

Depending on the orientation of the chains, the thickness should be equal to or less than the molecular chain length (1.9 nm) of 11-azidoundecyltrimethoxysilane, which is consistent with the calculated thicknesses. As alkyl chains are disordered in all types of SAM-N3, we consider that the behaviour of alkyl chains is similar for the three grafting processes, and the corresponding PM-IRRAS spectra have been normalised with respect to the aCH2 band. The normalised PM-IRRAS spectra (figure 3.32b) revealed significant differences in the intensity of the azide band, according to the deposition methods. The intensity of the aN3 band with respect to the aCH2 band decreases in the order PF-I-SAM-N3 > Dir-SC-SAM-N3 > Dir-I-SAM-N3. This behaviour is certainly due to the different packing density of monolayers which could affect how the adjacent azide groups interact each other, inducing different orientations of the dipoles. Due to the surface selection rule of PM-IRRAS on metallic surfaces (the intensity of a band is zero for a transition moment parallel to the surface and is maximum for a transition moment perpendicular to the surface), the different intensities of azide bands may be attributed to an orientation effect of this dipole which can explain the nonlinear relationship between the azide and the methylene bands [46]. These results suggest a preferential parallel orientation of the azide group, with a decrease of the tilt angle (with respect to the normal surface) in the order [41]. By using the optical constants of azide mode (kmax = 0.261 at 2100 cm -1 ) and calculating the IRRAS intensity of this mode for different orientations of the aN3 dipole from 0° (perpendicular to the surface) to 90° (parallel to the surface) using an uniaxial orientation, we found mean orientation values of 78°, 70.5° and 63° for Dir-I-SAM-N3, Dir-SC-SAM-N3 and PF-I-SAM-N3, respectively ( figure 2.34d).

Dir-I-SAM-N3> Dir-SC-SAM-N3 > PF-I-SAM-N3

The fact that the aN3 dipoles orientate more parallelly to the surface for less densely packed monolayer is certainly related to the presence of dipole-dipole interactions within the monolayer.

Evaluation of the properties of SAM-N3 prepared by the three different methods by AFM analysis

Moving to AFM analysis, as shown in figure 2

Evaluation of the properties of SAM-N3 prepared by the three different methods by XPS analysis

To understand and confirm results obtained by AFM and PM-IRRAS analysis, more information will be obtained by analysing SAMs in XPS technique. XPS technique will confirm the presence of the azide group in SAM-N3 and gives more information regarding the azide environment and organisation. First, XPS spectrum of 11-azidoundecyltrimethoxysilane was measured in the N1s region to acquire a spectral reference of the azido group (figure 2.36). As mentioned earlier, the three nitrogen atoms in azide have different environments. One of the nitrogen atoms is neutral, the second is negatively charged, while the third nitrogen atom is positive. Consequently, the emitted electrons hold different kinetic energies as a result of different nitrogen charges in the azide group. We observed the presence of two well defined contributions : one at low binding energies (BE), centred at 401.2 ± 0.1 eV and composed of two overlapped peaks, corresponding to the nitrogen atoms linked to the alkyl chain (=N-), and

a) b) c) d)
to the electron enriched one at the end of the azido group ( -N=); the second contribution at higher BE (405.0 ± 0.1 eV) is assigned to the electron deficient nitrogen atom (=N + =) in the middle of the azido group. A ΔBE = 3.8 eV is observed, with a higher intensity of the low BE component, which is in very good agreement with what is usually observed in the literature for azido groups [2] [47].

Evaluation of the azide groups reactivity towards click reactions for SAM-N3 prepared by the three different methods

The differences in azide orientation depending on the method could affect its reactivity for further modification reactions. Therefore, it is now important to investigate the accessibility of these azide groups for further surface reactions. The reactivity of the azide groups with terminal alkynes using the CuAAC reaction was assessed using PM-IRRAS and XPS spectra of a pure SAM-N3 before and after click reaction with alkyne-terminated compounds. To explore this point, we examined the immobilisation of two types of organic probes bearing an alkyne function, 1-(but-3-yn-1-yloxy)-4-nitrobenzene and 1-heptyne via the CuAAC reaction (figure 2.38). The nitro substituent has been chosen since it is easily detected in the infrared region, from the antisymmetric (aNO2) and symmetric (sNO2) stretching modes of the NO2 moiety at 1515 and 1342 cm -1 , respectively. 1-heptyne compound has been used for XPS study to avoid having other nitrogen atoms that could interfere with the analysis of nitrogen containing species such as azide and triazole groups. The CuAAC reaction was performed using a copper sulfate / sodium ascorbate catalytic system in a mixture of water and DMSO containing the organic probe (The protocol is described in detail in the experimental part). For the XPS study, 11-azidoundecene was reacted with 1-heptyne to synthesize the triazole by the (CuAAC) reaction, using a copper sulfate / sodium ascorbate catalytic system in a mixture

Conclusion

In this chapter we studied the impact of changing grafting conditions on the properties of SAM-N3 bearing an alkyl chain spacer and how the terminal azide groups orientation could affect its reactivity towards click reaction. After deep analysis of data obtained from XPS, AFM, PM-IRRAS and contact angle measurements we concluded the following results: First: Post-functionalisation method is less sensitive to condition changes, since the structure of the coupling agent bearing a bromine terminated alkyl chain seems to be the main parameter driving the self-assembly process and the final nanostructure of films. Consequently, postfunctionalised SAM-N3 displays almost the same properties using both the immersion and the spin coating method in toluene and chloroform. All have almost the same quantity of the organic matter on the surface and the azide orientation is preferable in a perpendicular position to the surface.

Second: Spin coating method can be used for SAMs formation. We succeeded in optimising conditions to produce SAMs with properties consistent with SAM-N3 described in literature, either following a two-steps post-functionalisation method or the direct grafting of an azidoorganosilane. This process seems to be less sensitive to grafting conditions changes compared to the immersion method due to the fast deposition time. Our approach of the combination of both the direct grafting strategy and the spin coating method to produce azideterminated SAMs could be a breakthrough in the field of SAMs elaboration. This resulted in decreasing work steps, deposition time, consuming chemicals, in addition to performing the deposition at ambient temperature and open air, leading to economic benefits.

Finally, we have demonstrated that the grafting process can influence the orientation and the reactivity of azide-terminated monolayers onto silica surface. The reactivity of the azido group on the surface towards the alkyne in solution is closely related to the orientation of the azide.

Notably, the more the azide is vertically oriented the more it is accessible and reactive. The orientation of azido dipoles at the surface strongly depends on the method used to prepare the monolayer, and the post-functionalisation method provides the best orientation of the azido groups for further reaction. We demonstrate that it is possible to control the amount of reactive azides and consequently the number of molecules immobilised on the surface after the click reaction by choosing the deposition method. In addition, the concept of producing SAMs with azides having different orientations and different reactivities (figure 2.42) towards click chemistry could be an alternative to the mixed SAMs concept, which aims sometimes to decrease the number of reactive terminal groups. In this way instead of preparing mixed SAMs it is only using the direct grafting strategy by either the immersion or the spin coating method.

Introduction

The self-assembly process originates from a complex balance between molecule-molecule interactions and molecule-substrate interactions. It is helpful to modify the molecule-molecule interactions to control the molecular self-assembly of the molecules in the monolayer.

Classically, an alkyl chain spacer establishes Van der Waals interactions which are responsible for the assembly of the molecules. Compared to Van der Waals interactions, hydrogen bonds are stronger, selective, and directional, and those properties make it better to establish bonds at a precise position of the molecule, enabling to create organised architectures in a predictable way [1]. Self-assembly mediated by molecular recognition provides unique and convergent routes to realise ensembles of highly organized systems. This point inspired us in this work to use molecules able to form hydrogen bonds to obtain predictable self-assembled architectures demonstrating well-defined surfaces, in addition to provide versatile platforms for steering and monitoring the assembly of molecular nanoarchitectures in exquisite detail. Indeed, molecular architectonic on surfaces represents a versatile rationale to realise structurally complex nanosystems with specific shape, composition, and functional properties, which bear promise for technological applications [2] [3] [4] [5] Based on the surface chemical reaction and synthetic approaches, the chemical structures of SAMs can be manipulated at molecular level, and as discussed before concerning the coupling agents forming SAMs, each part has an effect on the quality and the tribological property of SAMs. It is generally believed that strong affinity of the molecules to the substrate is a basic requirement for effective boundary lubrication [6]. Regarding the spacer part, lots of works have been done to discover the origin of the chain length dependence. It was found that loosely packed and disordered structures of the SAMs are obtained from shorter alkyl chain (of 3-8 carbons), on the other hand, it is observed that for SAMs with no defects longer chain length can endure heavier applied load. This is because longer chains are more flexible to tile in response to the applied load as compared to shorter ones, the optimum molecular length is 18 carbons, while increasing carbon chains more than 18 will also lead to less ordered films [7] [8]. In addition, the spacer nature has an influence on the SAMs packing density and structure.

It is hypothesised that the functional groups interact laterally taking the form of hydrogen bonding, dipole interactions, π-stacking are able to influence the integrity, stability and tribological performances of SAMs [9]. Finally, the terminal functional groups have a significant effect on the surface nature of SAMs, such as wettability and adhesion, depending on its nature (polar terminal groups such as -OH, -NH2, N3 and -COOH and apolar terminal groups such as -CH3 and -CF3), and the spatial orientation (depending on the odd or even number of carbons within the alkyl chain spacer) [10].

Understanding the role of each part of the coupling agent will help to understand the behaviour of those molecules during the assembly of the monolayer and the resulted properties of SAMs.

For ureido coupling agents we have the trimethoxysilane anchoring group for all compounds, so we expect to have the same affinity to the surface. We have also urea in the spacer part expecting packed and more ordered layers compared to SAMs with alkyl spacer, due to the presence of hydrogen bonds. But according to our knowledge there is no study in the literature investigating the optimum number of carbon chains to form the best ordered ureido SAMs. In this work, we aim to investigate different lengths of carbon chains and evaluate the effect on the properties of ureido SAMs. The change in carbon chain length could affect ureido SAMs in different ways. For instant, changing the length could affect the organisation of the molecules in the layer, which could create changes in the order of alkyl chains, urea unit, and azide group orientation. All those changes could have impact on the packing density of SAMs as well as the topography and the wettability of the surface. Another way is that the change in length could also affect the strength of the hydrogen bonds between molecules in the SAMs, stronger hydrogen bonds supposed to have more ordered and dense layers. Consequently, both the organisation of molecules and the strength of hydrogen bonds could influence the final structure and reactivity of SAMs. It is also important to take the advantage of the observations made in chapter 2 regarding the azide group orientation and reactivity. It leads us to expect that the reactivity of the azide group on the terminal side could be affected by any change in ureido SAMs. This is not the first study of urea containing SAMs in our research laboratory. A study in 2011 [11] used PM-IRRAS in cooperation with other characterisation techniques to investigate urea containing SAMs grafted onto SiO2/Au substrate, it found that the carbonyls of the urea groups are preferentially parallel to the substrate surface favouring intermolecular hydrogen bonding and consequently a close packing of the molecules attached to the surface. By contrast, the alkyl chains present a large number of gauche conformations and are poorly oriented. Moreover, parallel oriented carbonyls to the surface allow hydrogen bonding between the molecules. This feature as well as covalent bonding with the substrate through the siloxane groups improve the stability of the SAMs and produce close packing of the molecules attached to the surface. Those observations have been confirmed again by a recent study in 2015 in our laboratory, which reported a study of SAMs containing amide and urea groups possessing an epoxy-terminal group and a trimethoxysilyl-anchoring group. This study confirmed the previous conclusion: the molecular assembly is mainly based on the intermolecular hydrogen-bonding between adjacent amide or urea groups in the monolayers [12]. So far, there is no ureido SAMs with an azide terminal group on silica surface reported in the literature, but only one report of amide containing azidoalkylthiol SAMs and their in-situ functionalisation (figure 3.1) [13]. This is mainly because the synthesis of such linkers bearing azide, amide and thiol groups are complicated, and they are inherently unstable as the thiol group can reduce the azide groups and catalyse the hydrolysis of the amide groups. Secondly, ureido SAMs will be prepared by the immersion method in three different solvents (toluene, acetonitrile, and chloroform) to optimise grafting conditions. Since this new family of urea compounds is not described in the literature for both preparation and grafting, except one molecule ((CH3O)3Si-(CH2)10-NH-CO-NH-(CH2)8-N3) [18] which has been prepared and grafted in our research group, it was optimised in term of synthesis and grafting by the immersion method [18] [19]. For the new ureido coupling agents, adjusting immersion conditions and understanding the behaviour of each molecule in the monolayers during the assembly process is a great challenge. Moreover, spin coating method will be used to form SAMs, which is another challenge to be optimised in this work. Finally, all SAMs will be tested by click chemistry to evaluate the azide reactivity. In general, the approach of this work is to investigate the influence of hydrogen bonds on the molecular order of ureido SAMs with terminal azide groups, and to understand the influence of the length of the alkyl spacer from both sides of urea on the properties of the resulted SAMs and terminal azide reactivity towards click reactions.

Synthesis of a new family of ureido coupling agents

This section presents a novel synthesis of ureido coupling agents bearing an azide-terminal group with different length of alkyl chain in both urea sides and trimethoxysilane as a head group. Despite the relatively long synthesis protocol and the sensitivity of some reagents, we succeeded in optimising the synthesis protocol for each coupling agent with a satisfying yield and high purity. Figure 3.4 shows the new family of ureido coupling agents with the abbreviation that will be used for each one. Typically, an efficient synthesis of the urea functional group is achieved through the condensation of an amine with an isocyanate [20] [21] or the coupling of amines with phosgene or a phosgene equivalent [22] [23]. The first method is preferable despite the limited number of commercially available isocyanates because of the toxicities related to the use of phosgene in their synthesis, leading to some health and environmental concerns [24] [25] . Moreover, isocyanates sensitivity due to hydrolysis with water producing carbamic acid could create some difficulties during their synthesis and storage [26]. Previously, in our research group, we described for the first time an efficient synthetic methodology to prepare a coupling agent bearing urea spacer, an azide-terminal group from one side, and an alkene-terminal group on the other side which can be further hydrosilylated to add the anchoring group [19]. Due to the poor stability of organotrialkoxysilanes, it would be preferable to implement synthetic routes involving the introduction of the silylated group during the last step of the synthesis and just prior to the SAMs formation process (figure 3.5) [19].

Synthesis of the ureido coupling agents (group a)

In this group, we have a long carbon chain between urea and silicon (long tail of 10 carbon atoms), and different lengths on the other side between urea and azide; a1 with an eight-carbon chain, a2 with a three-carbon chain, and a3 with a two-carbon chain. We will use the typical efficient synthesis of the urea functional group through the condensation of an amine with an isocyanate. The common part of all compounds in this group is the side ten-carbon chain with the alkene-terminal side which comes from the isocyanate reagent [27]. Previously, in our laboratory, we described a simple way to prepare the isocyanate reagent with a good yield using an acyl chloride, by the classical Curtius reaction via the acyl azide intermediate (figure 3.7)

(The detailed protocol is described in the experimental part).

Synthesis of the ureido coupling agents (group b)

In this group, we have a short carbon chain between urea and silicon (short tail), and different lengths on the other side between urea and azide; b1 with an eight-carbon chain and b2 with a three-carbon chain. The synthesis of those compounds follows the same protocol than the one applied for the synthesis of the group a compounds, by coupling an isocyanate with an amine.

Here, the common part of those compounds is the side chain of three carbon atoms with an alkene end group which comes from the commercially available 3-isocyanatoprop-1-ene.

Synthesis of compound b1:

This compound is produced by coupling 8-azidooctylamine (compound 4) with 3-isocyanatoprop-1-ene. The synthesis of compound 4 has been described previously in the protocol of compound a1. As shown in figure 3.12, at the end, compound 4 is subjected to the condensation reaction with the commercially available 3isocyanatoprop-1-ene resulted in the production of compound b1 precursor, followed by the classical hydrosilylation to afford compound b1: Si-C3-U-C8-N3. (The detailed protocol is described in the experimental part).

Deposition of azide-terminated ureido SAMs using the immersion method

In this part, urea coupling agents have been used to form SAMs. Both immersion and spin coating methods have been investigated since azide-terminated ureido silylated coupling agents have not been used previously to prepare SAMs in the literature (except compound a1 [18]).

This section consists of three parts: The first part presents the PM-IRRAS simulated spectra, to have a theoretical model for each monolayer. The second part presents detailed SAMs formation using the immersion method, in which many parameters have been investigated to optimise the conditions and to study the behaviour of coupling agents with some changes.

Finally, the last part presents SAMs formation using the spin coating method and the conditions optimisation for each compound. Moreover, for both the second and the third parts, the reactivity of the azide end groups has been evaluated to check their accessibility by the click reaction.

Infrared Reflection-Absorption Spectroscopy (IRRAS) developed in the 60s defining the experimental conditions allow to improve the detectivity of the measurements for monomolecular films deposited on metal surfaces. The computer program used to calculate the IRRAS spectra of urea compounds deposited onto SiO2/Au substrates is based on the Abeles' matrix formalism [29], which has been generalised for anisotropic layers [30]. Several parameters must be fixed in the program such as the number of layers, the angle of incidence (set to 75°), and the polarization of the infrared beam. The anisotropic optical constants (refractive index and extinction coefficient in the three space directions) of each compound must be determined beforehand (vide infra), because the grafting of SAMs does not induce orientation in the xy plane (substrate surface), uniaxial symmetry of molecular orientation can be assumed for the calculations (nx = ny = nxy and kx = ky = kxy). The optical constants of each compound have been determined from polarized ATR spectra, using the interdependence of n(ῡ) and k(ῡ) by the Kramers-Kronig relations. We have recently used this methodology to determine the isotropic optical constants of urea containing compounds. To summarise, from the optical constants determined by ATR, it is possible to calculate the IRRAS (1-Rp(d) /Rp(0)) spectra of a thin layer deposited on a substrate using a computing program developed in the Molecular Spectroscopy Group [31] [32] based on the matrix formalism of Abeles [29] adapted to calculate the optical properties (transmittance, reflectance, etc.) of anisotropic multilayer systems [30]. It allowed us to estimate the surface coverage of ureido SAMs from calculating the IRRAS spectrum of a compact monolayer of each urea coupling agent deposited on SiO2/Au substrate. The simulated isotropic spectrum of each urea compound is shown in the following figure 3.13. Those data can be used as a reference to determine the best conditions to obtain a compact monolayer. To make it easier, all simulated data are summarised in table 3.1, it shows the estimated intensities of the azide and methylene bands as well as amide I and amide II bands. The intensity of the azide band will be used to investigate the behaviour of the azide group on the surface in term of dipole orientation [11]. Considering the selection rules in PM-IRRAS, the intensity of the azide band is maximum when the azide dipole is vertically oriented from the surface, while the intensity is zero when the azide dipole is parallel to the surface. Due to having differences in the monolayer's intensities between the experimental and the simulated data, we prefer to use the value (R =I(ѵN3)/I(ѵaCH2)) to evaluate azide orientation. We can estimate azide orientation by comparing the value of R in any experiment (Rexp) to the value of R by simulation (Rth): if (Rexp) is lower than (Rth), it means that azide is in a position more parallel to the surface than in the simulated model, while if (Rexp) is higher than (Rth) it means that dipole azide is in a position more vertical to the surface.

To summarise, the intensity of the methylene bands will be used as a reference of the intensity range to form a monolayer, while the intensity of the azide band with respect to the methylene band (R) could give information about azide orientation. We can evaluate results by comparing the experimental data to the simulated data. Those data will be used as references for the grafting process to optimise conditions and to check the effect of changing some grafting parameters on the ureido compounds behaviour during SAMs formation for both the immersion and the spin coating methods.

Deposition of azide-terminated ureido SAMs using the immersion method in different solvents

Azide-terminated ureido SAMs have been prepared by the immersion method using solvents with different polarities. Thus, we have to find the best polarity that enables the proper balance between molecule-solvent, molecule-molecule, and molecule-surface interactions. High polarity solvents will likely provide preferable molecule-solvent interactions and less molecules on the surface. In contrast, low polarity solvents will likely provide more molecule-molecule and molecule-surface interactions, which could create some aggregations. Depending on those hypotheses, we chose to deposit azide-terminated ureido SAMs in three different solvents:

acetonitrile, chloroform, and toluene of relative polarity: 0.460, 0.259, and 0.099 respectively [33], and compare results to choose the best solvent that produces SAMs with satisfying properties.

The deposition of compound a1 has been described and optimised previously in our research group [18]. It follows the same protocol than the one implemented for the alkyl azide described in chapter 2. At the beginning, slides should be cleaned and preactivated, then immersed in a solution of the urea coupling agent containing TCA (10 mol%) for 14h at 20°C under inert atmosphere. At the end of the grafting, slides should be removed from the reactor and washed, dried, and analysed using the PM-IRRAS technique, AFM, and contact angle measurements, (the detailed procedure is described in the experimental part). This immersion protocol has been applied to all urea compounds by using the three solvents (toluene, acetonitrile, and chloroform). Then results have been compared to choose the best solvent. The results of this part rely on a deep analysis of PM-IRRAS, AFM, contact angle measurements and the click reactions results. In this section we have five SAMs:

-I-SAM-a1 prepared by grafting compound a1 using the immersion method -I-SAM-a2 prepared by grafting compound a2 using the immersion method -I-SAM-a3 prepared by grafting compound a3 using the immersion method -I-SAM-b1 prepared by grafting compound b1 using the immersion method -I-SAM-b2 prepared by grafting compound b2 using the immersion method

In this section, we have first a general overview of all PM-IRRAS, AFM and contact angle data. Second, we will discuss the effect of each solvent on: the intensity of the organic matter for each SAMs by (PM-IRRAS data), the topography of the surface (by AFM data), and the hydrophilicity (by contact angle data). Finally, we will compare the effect of the immersing solvent on the molecular orientation in SAMs, in particular on the azide orientation.

In this part, the aim is to have a general idea about the behaviour of ureido compounds with changing solvent polarity. There are significant differences in the quantity of the organic matter on the surface by changing the solvent for each compound, which can be evaluated from peaks intensities. To have more information and connect results together, AFM results

and contact angle values are shown in table 3.2. Moreover, we can have a general overview of the PM-IRRAS results, figure 3.14 shows the spectra of all azide-terminated ureido SAMs in the three different solvents (toluene, acetonitrile, and chloroform). We can observe all distinguished bands: in the 3100-2700 cm -1 region characteristic of the methylene vibrations, in the 2100 cm -1 region characteristic of N3 vibrations and in the 1800-1500 cm -1 region characteristic for urea groups vibrations as the amide I and amide II bands. The presence of those bands confirms the successful deposition of urea compounds on the surface.

Deposition of azide-terminated ureido SAMs using the immersion method in acetonitrile

Starting by acetonitrile, comparing the PM-IRRAS spectrum of each SAMs deposited in acetonitrile to the spectra of other solvents shows clearly having less organic matter on the surface (figure 3.13). Moreover, the intensity of the νaCH2 band is less in acetonitrile than the simulated spectra (table 3.3), which can be explained by either a non-complete layer on the surface or having a monolayer with poor packing density. of carbon atoms in the alkyl segments. Compound a2 showed the higher value (66°) which could be explained by the high roughness of the surface. Indeed, as shown in table 3.2, contact angle values for all solvents are less than the values of azide-terminated alkyl SAMs (11 carbons) in literature (77°-84°) [34] [35]. It could be due to the presence of the urea unit decreasing the hydrophobicity of the surface. According to our knowledge there is one study of azide-terminated SAMs containing amide embedded in the alkyl chain in the literature [13].

The total number of carbons in those SAMs are 4, 6, and 10, formed on gold surfaces prepared using the immersion method in ethanol. The contact angle of such SAMs are: 65° ± 1.7, 70° ± 1.5 and 84° ± 1.6, respectively. This study concluded that the contact angle values indicate an increase in the hydrophobicity when the number of carbon atoms increased. Those conclusions are consistent with our results, we have lower contact angle than for alkyl azide (of 11 carbons) and this value is less by decreasing the number of carbons in azide-terminated ureido SAMs. decreasing the kinetic of self-assembly and finely limit the condensation reaction (both molecule-molecule and molecule-substrate), leading to a low quantity of the organic matter on the surface. Another explanation for the low matter density is the high polarity of acetonitrile which could remove the water layer from the surfaces. These water molecules are needed for the hydrolysis and condensation reaction to form the monolayer (figure 3.16), and if the water quantity is not enough on the substrate it results in having less organic matter on the surface. In general, SAMs in this solvent also show smoother surfaces with the lowest roughness as well as the lowest contact angle values compared to other solvents. To have more information regarding the saturation of the monolayer, we tried to increase the immersion time. We increased the immersion time for compound a2 from 14h to 20h and 72h as shown in figure 3.17. We observed that the saturation of the monolayer is at 14h, as the intensities of all peaks in the PM-IRRAS spectra are not significantly changed by increasing time to 20h or to 72h. In addition, the values of roughness slightly increased with increasing the immersion time. AFM analysis shows the absence of holes or aggregations for those samples. Meanwhile, very small change in the contact angle values is observed. All those data indicate the saturation of the organic layer at 14 hours immersion time. To conclude, using acetonitrile for the deposition of the ureido silylated compounds by the immersion method will produce SAMs with low packing density and very smooth surfaces compared to other solvents.

Deposition of azide-terminated ureido SAMs by the immersion method in toluene

Compared to the previous solvents, as shown in figure 3.14, toluene gives intermediate results between acetonitrile and chloroform in term of organic matter density, except for SAMs formed from compound a3 (toluene gives same results as chloroform) and compound b2 (toluene gives same results as acetonitrile). But compared to the simulated data (table 3.5), it is the solvent with the best intensities of νaCH2, leading to consider toluene as the optimum solvent that produces SAMs in the range of the simulated monolayer values. The range of contact angle values are slightly lower (55° to 65°) than other solvents. However, the topography of the surface (from AFM results in table 3.2) shows a roughness between 0.6 nm and 1 nm while the roughness of the initial surface is 0.21, those values are intermediary of chloroform and acetonitrile. As shown in figure 3.19, all azide-terminated ureido SAMs formed by the immersion method in toluene are homogenous without any defects, without any presence of aggregations or holes, and the smoothness of the surface increases by decreasing the carbon number.

)

Overview of the impact of the immersing solvent on the molecular orientation of the azide-terminated ureido SAMs

It is important to investigate solvent effect on the orientation of both azide and urea. It is previously mentioned that amide I which correspond to the carbonyl of urea group adopt a preferential orientation parallel to the surface [12], and this is the case here for all solvents. It's interesting to note that the difference in the relative intensity between the bands amide I and amide II are more pronounced for compounds a1 and b1 in acetonitrile (table 3.6) due to having the carbonyl group (C=O) more parallel to the surface. For azide orientation comparison, we prefer to analyse a normalised spectrum on the methylene antisymmetric band for one compound in the three different solvents (figure 3.20). For all the compounds except b2 the intensities of amide II are similar in all the solvents due to a lesssensitivity to orientation of this band. However, it is observed that solvent effect is not the same on azide orientation for all molecules. In the normalised spectra, the intensities of methylene bands are the same, then any differences in the intensities of azide bands is due to different orientations. More intense azide band indicates more vertical azide to the surface (due to the selection rule of PM-IRRAS).

Starting by compound a1, chloroform and toluene have the same effect on azide orientation, and both solvents affect azide to be more in vertical to the surface than acetonitrile. On the other hand, compound a2 shows the opposite, more vertical azide to the surface is observed in acetonitrile than chloroform and toluene. Both chloroform and toluene have the same effect on azide orientation to the surface.

Compound a3 shows different behaviour, there is variation between the three solvents, acetonitrile produced the most vertical azide to the surface, followed by toluene then chloroform. Surprisingly, compound b1 follows exactly the same behaviour as compound a1.

Finally, the spectrum of compound b2 reveals the presence of methyl band at 1960 cm -1 relative to pollutant and render difficult to discuss the normalised spectrum on methylene band. This compound possesses only six carbon atoms which seems to make it more sensitive to pollution.

For compound b2 the normalised spectrum on amide II is more consistent because this band is less sensitive to contamination and present no orientation effect. Figure 3.20f shows similar intensity for azide band in toluene and chloroform and slightly less intense for acetonitrile.

These results show no significative solvent effect on the orientation of the azide group for compound b2.

)

The effect of each solvent on azide orientation for each molecule can be also observed by using the simulated data, by plotting the experimental (nRexp = n(I(ѵN3)/I(ѵaCH2))) value for each solvent and compare it to the theoretical value (nRth) obtained from the simulated data (figure 3.21). It is obvious that all experimental values (nRexp) are less than the theoretical (nRth)

showing an azide dipole systematically less vertical. For compound b2 in CHCl3, nR is similar to nRth which may be attribute to the higher amount of materials on the surface resulting of high roughness (1.81 nm). There is big variation in the azide orientation by changing the solvent polarity and the molecular structure. We can conclude that the change in the molecular structure of urea molecules has great influence on the azide orientation. Meanwhile, changing solvent polarity could significantly have a different effect for each molecule on the azide orientation. Thus, it is better to choose one solvent to investigate the effect of changing the structure of ureido compounds on the azide orientation and SAMs properties. In addition to all mentioned advantages of toluene, SAMs grafted in toluene by the immersion method presented a good reproducibility. Figure 3.22 shows two trials superimposed with no significative differences which highlight the reproducibility.

)

For azide-terminated ureido SAMs, the toluene is chosen as the best grafting solvent for the immersion method. To understand the self-assembly behaviour of urea coupling agents, we will deeply study the influence of the number of carbon atoms in the alkyl segments on the resulted azide-terminated ureido SAMs prepared in toluene.

Comparison between all azide-terminated ureido SAMs prepared by the immersion method in toluene

This comparison will show the effect of changing the carbon chain length on the properties of the produced SAMs. For PM-IRRAS data (figure 3.23), we will discuss deeply the three distinguished region for azide-terminated ureido SAMs ; the region in 3200-2600 cm -1 displays the characteristic bands of the methylene stretching vibrations, the region at 2100 cm -1 is characteristic of the asymmetric stretching vibration νaN3 of the azide group, and the 1700-1400 cm -1 region is characteristic of the amide I and II modes related to the urea part.

We will also compare AFM and contact angle measurements, for that we present table 3.7

summarising the data of peaks positions in PM-IRRAS spectra, AFM and contact angle measurements. As shown in figure 3.23, the region in 3200-2600 cm -1 displays the characteristic bands of the methylene stretching vibrations. The methylene stretching region provides specific information about the conformation and van der Waals interactions of the alkyl chains. Normally, increasing the carbon chain length results in higher intensity in this region, which is clearly observed in interactions and increase the order of the molecules in the organic layer, but the presence of the urea part makes the hydrogen bond network the driving force, as discussed previously [12]. The nR ratio indicates the relative intensity of the azide band compared to the antisymmetric methylene band normalised by the number of carbon atoms (n) in the compound. The higher this ratio is, the more the azide dipole has a vertical orientation. Thus, we can use here this ratio to compare the azide orientation in all azide-terminated ureide SAMs as shown in figure 3.24.

Surprisingly, it is observed that compound b2 (the shortest compound) has the highest intensity of azide in a vertical position to the surface, followed by compound a1 (the longest) as well as compound a2 and b1 of intermediate length. Thus, the compound a3 provides an azide dipole more parallel to the surface. It has been proven that the distance between the alkyl chains is (4.5-5 Å) which is too large to enable van der Waals interactions due to the presence of urea groups. So the main concern will be for the region of the amide I (carbonyl stretching, ν C=O) and amide II (in plane N-H bending, ν N-H) modes in the 1700-1400 cm -1 region (table 3.7). This region could also give information about H-bonding interactions. Hutchison proposed that any shift of the amide II peaks to higher energies indicate an increasing restriction imposed on the N-H bending motion due to the intermolecular H-bonding interactions within the monolayer [11], which explains the differences in the position of amide I and amide II due to having different interactions strength.

In addition, according to the selection rule, the peak intensity increases when the dipole moment of the absorber is perpendicular to the reflective surface plane and decreases when the dipole moment of the absorber is parallel to the reflective surface plane. As previously discussed, the carbonyl group in urea is preferable in a parallel orientation with respect to the surface. The differences in the intensities variation of amide I and II are not significant for all compounds (table 3.6), leading to conclude that the differences in carbonyl orientation of the urea group are not very different by changing the carbon chain length. This region is not only giving information about the urea orientation, but the frequencies difference between amide I and amide II (Δν) reveals the strength of the hydrogen bonds in the monolayer. For a cumulative number of carbon atoms between the two alkyl chain segments, the hydrogen bonding interaction is in the same range (Δν = 74-80 cm -1 ) up to 12 carbon atoms. From 13 carbon atoms SAMs with the longer alkyl chain a1 and a2 shown the best Δν with 56 cm -1 and 66 cm -1 respectively, while H-bonding self-assembly decreases significantly as the alkyl chain decreases. The contact angle measurements give information on the hydrophobic and hydrophilic properties of the surface. The values are higher (64°) for SAMs with at least 13 carbon atoms. For alkyl spacer, it is mentioned in the literature that the optimum number is 18 carbons to obtain a well-ordered surface, and SAMs with shorter carbon chains produce less ordered monolayers [36] [37]. This point motivated us to compare SAMs with a relatively long chain to a short one. In this section, the aim is to investigate if a spacer containing a urea function prone to hydrogen bonding will have the same behaviour. We will compare I-SAM-a1 having a spacer made from a urea group and an alkyl chain of 18 carbons with I-SAM-b2 having a spacer made from a urea and an alkyl chain of 6 carbons.

In the PM-IRRAS spectra in figure 3.26, the difference in the intensities of the methylene bands is easily explained by the different number of carbon atoms in each compound. Both molecules have a single urea function which is consistent with similar intensities for the amide bands of the two monolayers. The region of the methylene bands showed disordered monolayer for both SAMs. The antisymmetric (νaCH2) and symmetric (νsCH2) stretching vibrations of the methylene groups are observed at 2929 cm -1 and 2857 cm -1 , respectively for I-SAM-a1, while for I-SAM-b2 the (νaCH2) band is at 2929 cm -1 and the symmetric (νsCH2) stretching vibration is at 2858 cm -1 , indicating for the both SAMs a similar disorder for the alkyl chain. All PM-IRRAS data lead to conclude that the driving forces of the self-assembly in I-SAM-a1

is the hydrogen bonds, while for I-SAM-b2 this force is less. On the other hand, the short alkyl chain seems to be more favourable to have upright azide dipole than the long alkyl chain.

The wettability and the topography of both SAMs should be compared also. The AFM results previously showed smoother surface for I-SAM-b2 with a roughness of 0.6 nm than I-SAM-a1 with a roughness of 0.98 nm instead of 0.21 nm for the initial wafer. Moreover, I-SAM-b2 is more hydrophilic (contact angle of 53°) than I-SAM-a1 (contact angle of 64°). Those observations could be explained by having different packing density of SAMs and differences in the organisation of the alkyl chains in each SAMs as previously explained for PM-IRRAS data. Furthermore, higher roughness for I-SAM-a1 could be a reason to have higher contact angle by having higher azide population on the surface.

To summarise, both SAMs (I-SAM-a1 and I-SAM-b2) have interesting properties in terms of hydrophilicity, topography, and azide intensity on the surface. But azide reactivity should be tested towards the click reaction for all azide-terminated ureido SAMs, to check the effect of changing azide-terminated ureido SAMs properties on the terminal azide reactivity.

Evaluation of the reactivity of the azide in azide-terminated ureido SAMs prepared using the immersion method toluene

To evaluate the reactivity of the azide group towards the click reaction, we used the same nitro probe with a terminal alkyne used in chapter 2. This nitro probe compound is easily detected by the PM-IRRAS technique. The general scheme of the CuAAC reaction performed with azide-terminated ureido SAMs is described in figure 3.27 (the detailed protocol is described in the experimental part). The click chemistry protocol that was used is the one from chapter 2, SAMs has been immersed in a solution of the nitro probe in a petri dish, then the solutions of CuSO4.5H2O (a source of Cu(Ӏ) catalyst) and of sodium ascorbate ( the reducing agent to reduce Cu (Ⅱ) to Cu(Ӏ)) have been added respectively to the SAMs in the petri dish and the reaction medium has been shacked for 1 night in the dark. At the end of the reaction, SAMs have been removed, washed, and analysed using the PM-IRRAS technique and contact angle measurements. related to the nitrophenyl moiety. However, for all azide-terminated ureido SAMs, the disappearance of the azide band is not complete, but its intensity significantly decreased compared to before click which is consistent with the covalent immobilisation of the nitro probe by the triazole formation. In all cases, no degradation of the monolayer was observed after the click reaction because there is no significant variation in the intensities of the methylene bands.

The presence of non-reactive azide groups on the surface could be due to their non-accessibility. This result shows that a minimum of 11 carbon atoms are needed to have a good accessibility of the terminal azide group in the monolayer. On the other hand, six carbon atoms are detrimental for the accessibility and so the reactivity.

Deposition of azide-terminated ureido SAMs using the spin coating method

It is observed in chapter 2 that the spin coating method is less sensitive to changing conditions than the classical immersion method. This observation led us to use the same spin coating protocol for urea coupling agents as a first trial. Then depending on results, we could change conditions to optimise results. In general, each urea coupling agent has been spin coated in toluene following the same protocol as the one implemented for the alkyl azide. Different I. SC-SAM-a1 prepared by grafting compound a1 using the spin coating method II. SC-SAM-a2 prepared by grafting compound a2 using the spin coating method III. SC-SAM-a3 prepared by grafting compound a3 using the spin coating method IV. SC-SAM-b1 prepared by grafting compound b1 using the spin coating method V. SC-SAM-b2 prepared by grafting compound b2 using the spin coating method

Deposition of SC-SAM-a1 using the spin coating method

Toluene was the optimum solvent for ureido SAMs prepared by the immersion method, which make it our first choice as the spin coating solvent. Different drying times have been investigated; 30 minutes, 3 hours, 5 hours, and 24 hours. All analysis data for SC-SAM-a1 deposition using the spin coating method are shown in figure 3.30. It is observed from the PM-IRRAS spectra the presence of all significant bands related to the presence of compound a1 on the surface: in the 3100-2700 cm -1 region characteristic of the methylene vibrations, the 2000-2100 cm -1 region characteristic of N3 vibrations and the 1700-1500 cm -1 region characteristic of the amide I and amide II bands. Consequently, the presence of those bands confirms the successful deposition of urea compounds on the silica surface by the spin coating method. The second observation is that there is no significant change in bands intensities with increasing the drying time which indicates no increase in the quantity of the organic matter on the surface.

Moreover, the organic matter is much less than the quantity on the surface obtained by the immersion method suggesting less densely packed monolayers or incomplete monolayers. The intensity of (aCH2) by spin coating is not more than 0.002 for all drying times, this value is almost half the value obtained by the immersion method in toluene (0.0047), which means having less organic material on the surface. It leads to conclude that, for SC-SAM-a1, whatever the drying time the amount of material remains similar, which could be explained by the low affinity of this molecule to the polar surface and its preference to remain in the solvent phase. immersion method in toluene. On the other hand, the contact angle for this trial is 74°, which is significantly higher than the value obtained by the immersion method (63°), indicating big changes in the wettability. As shown in the AFM image figure 3.32, the surface is smooth with no aggregations or holes and with a roughness of 1.29 nm (the initial surface is SiO2/Au with a roughness value of 0.90 nm). To conclude, increasing the concentration of the spinning solution has a small impact on the organic matter on the surface (slight increase) and less impact on the topography of the surface. But still this concentration doesn't produce SAMs of the intensity of aCH2 obtained by the immersion method in toluene. Thus, it is not the optimum conditions for the spin coating of compound a1. the intensity of the aCH2 band is 0.0040 at 3 hours and slightly more for 5 hours with a value of 0.0046 which is very close to the immersion method (0.0047). The contact angle is 62° and 65° for 3 and 5 hours, respectively in acetonitrile, while it is 66° and 68° for 3 and 5 hours, respectively in chloroform. The contact angle is in the same range for all solvents, which is expected because of having small differences in the surface topography. The initial surface is SiO2/Au with a roughness value of 0.90 nm while for 3 and 5 hours in acetonitrile the roughness is the same with 1.95 nm and in chloroform the values are 1.18 nm and 1.21 nm, for 3 and 5 hours respectively. AFM images are shown in figure 3.34, it is observed that all surfaces are smooth, and there is no holes and aggregations. The results of contact angle and AFM lead to conclude that changing the solvent of spin coating has no significant impact on the topography and the wettability of the surface, but great impact on the quantity of the organic matter. Chloroform is the optimum solvent for the spin coating of compound a1 at 5 hours drying time to have intensity of a monolayer consistent with the immersion method in toluene. vibration bands are very close to the immersion method which is 0.0038 for aCH2. Strikingly, the intensity of the azide band is higher for spin coating than for the immersion method, which could be due to an orientation effect with the azide group obtained more upright by the spin )

coating process than by the immersion method. As noticed before, for a shorter drying time less matter is obtained on the surface which can be observed clearly for the 3h drying time compared to 5 h. As previously discussed, this increase in the organic matter by increasing the drying time could be due to having the solvent on the surface for longer time, which enable continuing the process of hydrolysis-condensation reaction under room atmosphere and promote the packing density. However, the contact angle values are 62° for 3 hours and slightly higher (68°) for the 24 hours but both are in the same range of the immersion method (65°), indicating no big difference in surface the wettability by changing the grafting method. AFM analysis is done for the 24 hours drying time as it showed better results in PM-IRRAS. As shown in figure 3.35, the surface is relatively smooth with a roughness of 1.40 nm (the roughness of the initial PM-IRRAS surface is 0.9 nm) and the surface has no defects (holes or aggregations). To summarise, the best conditions for the spin coating of compound a2 and producing SAMs with properties in the range of the immersion method in toluene, is by using toluene as spinning solvent for 24h drying time . Moving to the grafting method effect, it is concluded that 24 hours drying time is optimal due to having quantity of matter in the same range as the immersion method in toluene. As shown in the normalised spectra, azide is more in a vertical orientation by the spin coating method. To conclude, both the grafting method and drying time have an influence on the azide orientation for SC-SAM-a2. surface. In addition, it is observed from the PM-IRRAS spectra that for the drying time of 24 hours, the intensities of CH2 and both amides bands are very similar to the ones obtained by the immersion method. The intensity of the aCH2 band is 0.0037 for spin coating at 24 hours and 0.040 for the immersion method. As observed for compound a2, compound a3 presents the same behaviour with the higher intensity of the azide band for the spin coating than the immersion method, indicating more vertical azide by the spin coating method (PM-IRRAS selection rule). The drying time of 3 hours produced less organic matter on the surface as all band's intensities are less than the intensities of the 24 hours, which could be explained by an incomplete monolayer or less packing density of the monolayer. The contact angle shows small differences from 62° for 3 hours to 67° for 24 hours drying time, which is not far from the value of the immersion method (65°). As the 24 hours drying time is the optimum time for SC-SAM-a3, AFM analysis is done for this surface. As shown in figure 3.37, the roughness is 1.62 nm (the initial surface is SiO2/Au with a roughness value of 0.90 nm). It is observed from the AFM hight image that the surface is not homogeneous and has some defaults. To conclude; SC-SAM-a3 is successfully prepared by the spin coating method in toluene, and the optimum drying time is 24 hours, but the surface is not very smooth as the immersion method. Again, as toluene is the optimum solvent of both methods (immersion and spin coating) for the preparation of monolayer of compound a3, it is interesting to investigate the effect of changing the grafting method on the azide orientation as well as the drying time, by using the normalised spectra in figure 3.38. We have the same behaviour as the one observed for SC-SAM-a2.

Increasing the drying time in the spin coating leads to reorganise the azide group to be more As shown in figure 3.39, the surface is smooth with no defects and the roughness is 0.97 nm (the initial surface is SiO2/Au with a roughness value of 0.90 nm). Moreover, the use of both chloroform and toluene at 3 hours drying time provides almost superimposed spectra, having similar methylene and azide bands intensities. It leads to conclude that for 3 hour drying time, using chloroform and toluene will produce the same quantity of the organic matter on the surface, and they have the same effect on the azide orientation. While for 24 hours drying time, the behaviour is different, the intensity of aCH2 band is higher for chloroform (0.0032) than toluene (0.0022). The value for chloroform is very close to the value of the immersion method in toluene (0.0037). As a result, chloroform is better for the spin coating of compound b1 and the optimum drying time is 24 hours. In chloroform, the contact angle for SC-SAM-b1 is 64° for 3hour and 67° for 24 hour drying time, which is slightly more than the value of the immersion method (60°). However, the AFM analysis in figure 3.41 shows smooth and homogenous surfaces with roughness values of 0.99 nm for the 3 hours and 0.93 nm for the 24 hours (the initial surface is SiO2/Au with a roughness value of 0.90 nm). It can be also observed from AFM images that there is no presence of any holes and aggregations. To summarise, compound b1 can successfully produce SAMs by the spin coating method in chloroform at 24 hours drying time and these conditions provide a smooth surface with no defaults.

Deposition of SC-SAM-b2 using the spin coating method

This compound has been spin coated in toluene at 3 and 24 hours drying times, as previously done to study the best conditions of spin coating, following the same protocol. The results of PM-IRRAS for SC-SAM-b2 are shown in figure 3.42. As usual the first check is for the presence of all significant bands related to compound b2 to confirm the successful of the spin coating for SAMs deposition. It is observed that all the vibration bands of CH2, azide and amides correspondent to compound b2 are present, confirming the successful formation of SC-SAM-b2. Then checking the quantity of the organic matter shows that the intensity of the aCH2 band is 0.001 for the 3 hours drying time, while it is more for the 19 hours (0.0012), and the last value is similar to the value obtained from the immersion method (0.0012). On the other hand, the vibration bands of both azide and urea (amide Ӏ and amide Ⅱ) are much higher for 19 hours than for 3 hours despite the small difference in the aCH2 band intensities. This increase in the intensities of azide and urea bands is due to the increase in the quantity of the organic matter on the surface, but the short length of alkyl chain implies a lower sensitivity in PM-IRRAS. The contact angle is slightly changed from 55° for the immersion method to 65° and 63°, respectively for 3 and 19 hours drying time. Finally, the AFM image of 19 hours drying time (figure 3.42) indicates a smooth and homogeneous surface with a roughness of 1.52 nm (the initial surface is SiO2/Au with a roughness value of 0.90 nm). At the end, it is concluded that compound b2 can be spin coated successfully to produce SAMs which are as good as the SAMs prepared by the immersion method, by using toluene at drying time of 19 hours.

Comprehensive study of all azide-terminated ureido SAMs prepared by spin coating method

In this section, the aim is to have more information about ureido SAMs prepared by the spin coating method, in particular, SAMs with the optimum properties that are consistent with the SAMs prepared by the immersion method in toluene. Table 3.9 summarises more details about the data obtained by PM-IRRAS (figure 3.43), AFM and contact angle measurements. This section will show the effect of changing the carbon chain length on the properties of the produced SAMs by the spin coating process. SC-SAM-a1 and SC-SAM-b1 show best results in chloroform, which means changing another parameter (the solvent) in addition to the molecular structure. Thus, this section is more a deep investigation than a comparison. antisymmetric (νaCH2) and symmetric (νsCH2) stretching vibrations of the methylene groups are observed at 2925-2930 cm -1 and 2850-2859 cm -1 , respectively, which was the same for the immersion method. The alkyl chain in all ureido SAMs are disordered whatever the deposition method. Furthermore, using nRexp could be a way to compare the azide orientation depending on the number of carbon atoms in the molecular structure. As shown in figure 3.44, The compounds b1 (C11) and b2 (C6) show a more vertical azide dipole than the compounds with a longer alkyl chain. From compound b1, the azide dipole is less vertical as the number of carbon atoms in the molecular structure increases. Moving to the region of the amide I (carbonyl stretching, ν C=O) and amide II (in plane N-H bending, ν N-H) modes in the1700-1400 cm -1 region, this region could also give information about H-bonding interactions and urea orientation. As previously discussed, the carbonyl group in urea is preferable in a parallel orientation with respect to the surface [38]. The differences in the intensities of amide I and II are not significant for all compounds, leading to conclude that changing the number of carbon atoms is not affecting urea orientation for those SAMs when they are elaborated by spin coating. This was the case also by the immersion method in toluene. This region is not only giving information about urea orientation, but the frequencies difference between amide I and amide II (Δν) reveals the strength of the hydrogen bonds in the monolayer.

By changing the accumulative number of carbon atoms between the two alkyl chain segments, the hydrogen bonding interaction is changing. H-bonding self-assembly is higher when the size of the alkyl chain decreases. This situation is inverse to that observed for the immersion method. In addition, there seems to be an odd even effect for the H-bonding self-assembly as a function of carbon number. The even compounds a1 and a3 have Δν values of 68 cm -1 and 71 cm -1 respectively while for the odd compounds a2 and b1 the values are lower at 62 cm -1 and 59 cm -1 respectively showing a much stronger self-assembly by H-bond.

It can be concluded that, in the spin coating method, the driving force of SAMs formation is the intermolecular hydrogen bonding for all ureido SAMs, but the strength of this force increases by decreasing the number of carbon atoms. In addition, there seems to be no correlation between the contact angle measurements and the length of the alkyl chain in contrast with the immersion method. To conclude, for both methods the assembly of the monolayer is done by the establishment of intermolecular hydrogen bonding. However, for the same compound the quality of this self-assembly will depend on the deposition method. By immersion, the strength of the hydrogen bond increases with the number of carbon while for the spin coating process it is the opposite. In general, for both methods the compounds with the lowest number of carbon (C6) seems to be more favourable to have a vertical azide dipole.

Evaluation of the reactivity of the azide group in azide-terminated ureido SAMs prepared by the spin coating method

The nitro probe with a terminal alkyne used in chapter 2 will be used also for those SAMs to evaluate the reactivity of the azide group towards the click reaction. As previously mentioned, this nitro probe compound is easily detected by the PM-IRRAS technique. The general scheme of the CuAAC reaction performed with azide-terminated ureido SAMs is described in figure 3.45 (the detailed protocol is described in the experimental part). The click chemistry protocol that was used is the one from chapter 2 and for azide-terminated ureido SAMs prepared by the immersion method. As a preliminary work, this reaction has been applied for two types of SAMs: SC-SAM-a2 and SC-SAM-a3 and analysed using the PM-IRRAS technique and contact angle measurements. (C=C), 1515 (aNO2) and 1342 cm -1 (sNO2) related to the nitrophenyl moiety. However, the azide band is not completely disappeared, but significantly decreased compared to before click which is consistent with the covalent immobilisation of the nitro probe by the triazole formation. In both cases, no degradation of the monolayer was observed after the click reaction, because there is no significant variation in the intensities of the methylene bands. The presence of non-reactive azide groups on the surface could be due to their non-accessibility because of the high intensity of azide population on the surface.

Conclusion

In this chapter, we presented a new protocol to synthesise coupling agents bearing both an azide terminal group and a urea spacer with different lengths. The described protocols afford highly pure compounds with satisfying yields. Those ureido coupling agents were successfully used directly for SAMs formation by using the immersion and the spin coating methods. The immersion method was applied in three different solvents (toluene, acetonitrile and chloroform), and we found that the best solvent is toluene compared to the simulated data obtained by the PM-IRRAS, and to other properties studied by the AFM and contact angle measurements. Moreover, the azide-terminated ureido SAMs prepared by the immersion method in toluene were successfully clicked using an alkyne terminated nitro probe, but the azides in those SAMs were not completely consumed after click. The formation of two-components SAMs is more complicated than single component systems, and the surface ratio between the two molecules in the SAMs after self-assembly may be very different from the ratio in the solution before self-assembly. In addition, segregation can occur leading to the existence of domains enriched with one or the other at the surface [2]. Indeed, the control of the composition and the topography of the mixed SAMs is dependent on the structure of the silylated compounds and the deposition conditions or other parameters. As previously discussed in chapter 1, mixed SAMs can be prepared by two main methods: the onestep deposition method and the stepwise deposition method [1], the first method is preferred for two components with similar chemical properties and the second one is more useful when the molecules differ in their chemical properties [3] [4] [5] As previously discussed in chapter 1, compared to alkyl chains, less studies described the preparation of mixed silane-based monolayers using internal functions able to self-assemble by hydrogen-bonding, which could give extra stability for SAMs. Our research group studied this SAM-a1-50%), which represents our new concept. The PM-IRRAS spectra are presented in the figure 4.3b. It shows that the intensities of amide I and amide II are similar in the two mixed SAMs which implies the same amount of materials onto the surface. Interestingly, the intensity of the azide band at 2097 cm-1 is 30% greater in the case of free I-SAM-a1-50%. This may be due to a slightly higher incorporation of the silane within the SAM or to a slightly more favourable orientation of the terminal azides for observation in PM-IRRAS. Those observations could prove that the dibutylurea is well incorporated within the monolayer.

Synthesis of the mixing compounds

As previously discussed, the typical protocol to synthesise the urea functional group is by the condensation of an amine with an isocyanate [8] [9]. Both compounds c1 and c2 have been previously prepared in our research group [10] in this region. In all cases the amide I and amide II positions are identical for both SAMs at 1636 cm -1 and 1581 cm -1 , respectively. However, the intensities of amide bands are too high to be compatible with the monolayer, which explains the high roughness (2.02 nm) of this surface (I-SAM-c1-100%) as shown in figure 4.6c, while the roughness of the initial wafer surface is 0.21 nm. The contact angle measurements also show no change for both SAMs, the value is 85° for I-SAM-c1-100% and 86° for I-SAM-c1-75%. These results indicate that the non-silylated compound is not incorporated in the layer therefore, the mixed SAM is not realised due to the preferential adsorption of the compound c1 onto the surface. This situation can come from an insufficient quantity of compound d2 or the molecular structure is too different to ensure a mixed self-assembly close to the surface. Increasing the length of the alkyl chain between the silicon and the urea could allow the compound d2 to be inserted inside the monolayer.

Formation of the mixed SAMs by the spin coating method

In this part, mixed SAMs will be formed by using the spin coating method. The protocol is same as the protocol followed in chapter 2 and 3. We used the silane solution of the classical concentration of 4 x10 -3 mol. L -1 . The two compounds (c1 and d2) are mixed by the define percentages prior to the hydrosilylation process to have final spinning solution of a concentration of 4 x10 -3 mol.L -1 in urea compounds, and toluene was used as the spinning solvent. After spinning, different drying times (30 minutes, 3 and 24 hours) were checked to see if it affects the formation of the mixed SAMs. Then SAMs were washed, dried and analysed in PM-IRRAS. We prepared SC-SAM-c1-100% and mixed SC-SAM-c1-50%. The analysis of SAMs in PM-IRRAS is shown in figure 4.8. First, it is proven that the spin coating method can be used also to form SAMs from compound c1, as all distinguished bands of compound c1 can be observed, the methylene vibrations in the 3100-2700 cm -1 region and the amide I and amide II bands in the 3100-2700 cm -1 region. There are differences in the amide bands intensity between the immersion and the spin coating method. By the spin coating process, the intensities of amide bands are lower and compatible with the monolayer. We can confirm the successful use of the spin coating method to prepare SC-SAM-c1-100% but not the mixed SAMs as there is no azide band in any of SC-SAM-c1-50% whatever the drying times. Another important observation is that the amount of silylated compound in the deposition solution influences the amount of material on the surface. Comparing SC-SAM-c1-50% to SC-SAM-c1-100% in any drying time spectra in figure 4.8, we can see that the intensity of the amides and methylene bands are less for SC-SAM-c1-50%. Then, the drying time effect is also observed by comparing the intensity of the methylene and amides bands of SC-SAM-c1-50% in 30 min, 3 and 24 hours. We can observe the increase of bands intensity due to the increase in the organic matter on the surface. The same result can be observed for SC-SAM-c1-100% in 30 min, 3 and 24 hours. But the main concern here is the formation of mixed SAMs which is still not achieved.

The reason could be that the non-silylated compound has no affinity to the surface despite the hydrogen bonding interactions with the silylated molecules. It motivated us to test this hypothesis in the following section.

Testing the affinity of the non-silylated compound d2 to the surface by the spin coating method

The aim of this section is to investigate the affinity of compound d2 to the surface. We chose to use the spin coating method for this part because it is faster to give preliminary results for this study. First, we applied the spin coating protocol to compound d2 and analysed SAMs in PM-IRRAS. As observed from figure 4.9, there is a very small quantity of the organic matter on the surface with bands in the methylene vibrations region (3100-2700 cm -1 ) and the amide I and amide II bands in the 3100-2700 cm -1 region, but the shape of amides bands is not clear and obviously no azide band at 2100 cm -1 . The absence of the azide band confirms the absence of compound d2 from the surface, and the presence of the mentioned peaks is due to contamination, because the surface is very hydrophilic with hydroxyl groups, and after spinning, if compound d2 has no affinity to the surface, other molecules from the air could be adsorbed on the surface, which creates some peaks in the PM-IRRAS. To have more information about compounds d2 affinity, we tried to apply the spin coating of a mixture of silylated and non-silylated compounds (50% mixed) but without washing the surfaces after spinning. After few days (4-7), surfaces have been analysed in PM-IRRAS, then washed, dried and analysed again in PM-IRRAS. This trial is applied for both mixed SAMs of compound c1 and c2 and results are shown in figure 4.10. For the samples without washing, we can observe for both compounds all the characteristic bands of the methylene and urea groups. Moreover, the appearance of the azide band for both mixed SAMs in the "no wash sample'' which could be due to the incorporation of compound d2 into the SAMs. It is interesting to note that the intensities of amide bands are compatible with the monolayer. After washing, the azide band disappeared and both the methylene and the amides bands decreased.

It is consistent with the removal of compound d2 from the surface, confirming that the strength of hydrogen bond interactions of this compound d2 with the silane is not sufficient to withstand the washing protocol. On the other hand, this trial ensures again the efficiency of the washing process to remove non reacted (excess) molecules from the surface. To summarise, the new concept of mixed SAMs by having a mixture of silylated and non-silylated molecules cannot be applied due to removing the non-silylated molecule from the surface by washing. However, these results show that it is possible to incorporate the non-silylated compound into the monolayer using the spin coating process whereas this is not possible by the classical immersion method. Then after washing compound d2 was removed from the SAMs due to the weak hydrogen bonding interactions with the silylated compound. coating process allowed to obtain a mixed SAMs with the incorporation of the non-silylated azide derivative. However, the self-assembly by hydrogen bonding remains too weak to withstand the washing protocol leading to the total loss of the non-silylated compound. In addition, AFM analysis seems to show that there is no phase segregation between the two types of molecules ensuring a homogeneous distribution of both of them in the monolayer.

Nevertheless, these results open new possibilities since non-silylated urea compounds could be used as a template to control the distance between organosilanes carrying the terminal function of interest. In order to maintain the non-silylated compound in the monolayer, a perspective would be to integrate in the molecular structures of the two components of the mixed SAMs a unit allowing a lateral polymerisation such as diacetylenes.

Chapter 5

Immobilisation of GlycoNucleoLipids (GNLs) to azide-terminated SAMs for cell culture applications

Introduction

In the development of biomedical devices, artificial organs and biosensors, it is crucial to gain a clear understanding of the interaction of these biomaterials with the biological tissues at the cellular level. Cell adhesion to artificial materials is affected by their surface properties such as wettability, roughness, surface topography, and chemical functionalities [1] [2]. Thereby, the use of suitable substrates and their surface modification techniques are essential to achieve a desired cellular response in stem cell-based therapies [3]. Therefore, surface modified nano biomaterials could further contribute to stem cell research with great efficiency. Briefly, the attachment of molecules or substances through physical, chemical or a combination of both methods, on the surface of materials could enhance their properties and help in controlling stem cell fate and functions.

Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favourable artificial environment. Culture conditions vary widely for each cell type, but the artificial environment in which the cells are cultured invariably consists of a suitable vessel containing a substrate or medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and gases (O2, CO2), and regulates the physicochemical environment (pH, osmotic pressure, temperature). Most cells are anchorage dependent and must be cultured while attached to a solid or semi-solid substrate (adherent or monolayer culture), while others can be grown floating in the culture medium (suspension culture) [4]. Cell culture is one of the major tools used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells (e.g., metabolic studies, aging), the effects of drugs and toxic compounds on the cells, and mutagenesis and carcinogenesis. It is also used in drug screening and development, and large-scale manufacturing of biological compounds (e.g., vaccines, therapeutic proteins). The major advantage of using cell culture for any of these applications is the consistency and reproducibility of results that can be obtained from using a batch of clonal cells [5].

The development of biocompatible artificial matrixes that can be used for the culture of stem cell remains a great challenge in tissue engineering. Moreover, the choice of biomaterial and its subsequent treatments play a critical role in this process, but there are many approaches to modifying biomaterials, and each has its advantages and disadvantages for their intended tissue.

actuators, and other molecular devices [14]. Moreover, GNLs are non-cytotoxic bioinspired compounds, but the modification of the chemical structures of GNLs is crucial for obtaining molecular structures that exhibit properties useful for a wide range of advanced applications [15]. In our work, considering the aforementioned impact of the surface modified nanobiomaterials in the field of stem cell culture, GNLs featuring an alkyne terminal group will be covalently linked with azide terminal SAMs by a 1,2,3-triazole bridge via the CuAAC reaction. Then the behaviour of SAMs bearing GNLs will be investigated as a scaffold for stem cell culture.

The first section of this chapter provides the first account of the orthogonal, covalent immobilisation of GNLs to azide-terminated SAMs via copper(I)-catalysed azide-alkyne "click" cycloaddition (CuAAC). Azide-terminated SAMs of both alkyl and urea spacers will be used to investigate this immobilisation. The resulted SAMs will be analysed by PM-IRRAS and contact angle measurements to follow changes after immobilisation. This step provides a new scaffold for cell culture. In the second section, the new scaffold will be used to grow specific type of stem cells. At this stage, we will check the toxicity and the influence of the surfaces on all steps of the cell growth (adhesion, differentiation, and proliferation).

Immobilisation of GNLs to azide-terminated SAMs

In this section, the approach of GNLs immobilisation to azide-terminated SAMs followed the same protocol of the CuAAC reaction than the one used previously to click alkyne terminated probes in both chapters 2 and 3, by immersing azide-terminated SAMs in a solution of GNLs and a copper sulfate / sodium ascorbate catalytic system in a mixture of water and DMSO, under inert atmosphere in the dark. Importantly, this click approach is likely to be applicable to any biomolecule combinations that can be modified to include an alkyne terminal side and may therefore be broadly applicable for any biomolecule immobilisation. However, it is important to investigate whether the attachment to SAM-N3 is specific (covalent) as a result of CuAAC reaction or non-specific (Van der Waals or any dipole-dipole interactions). We performed a control by immersing SAM-N3 in a solution of GNLs under inert atmosphere in the dark but without adding the copper sulfate / sodium ascorbate catalytic system. The resulted surface was then analysed in PM-IRRAS. As shown in figure 5.2, we can clearly see that there is no change in the PM-IRRAS spectrum of SAM-N3 before and after the test, and no new bands of GNLs like (OH or amides). The small increase in the methylene band is likely due to the presence of some contamination. Thus, it leads to conclude that GNLs are not able to be immobilised to SAM-N3 by the nonspecific interactions.

Immobilisation of GNLs to azide-terminated SAMs bearing an alkyl spacer

In this category, we have different types of SAM-N3 bearing an alkyl spacer prepared using different strategies and processes. We found that the post-functionalisation method provides the best orientation of the azido groups (best accessibility) for the CuAAC reaction. Thus, we chose those SAMs from this category to investigate GNLs click, and particularly PF-I-SAM-N3. Before showing the results of the click reaction, it is important to show the IR spectrum of GNLs and know the signature of each group. Figure 5.3 shows the IR spectrum of the GNLs.

We can observe all the distinguished bands: the presence of the broad band at 3395 cm -1 which is characteristic of the hydroxyl groups, the antisymmetric (νaCH2) and symmetric (νsCH2) stretching vibrations bands characteristic of methylene in the 3100-2700 cm -1 region, the amide I and amide II bands in the 1700-1500 cm -1 region, the symmetric bending of CH2 at 1466 cm -1 , the C-O-C stretching band at 1259 cm -1 and the stretching band of C-OH at 1096 cm -1 . for the resulted surface after the immobilisation. Surprisingly, the contact angle decreased just from 65° (for PF-I-SAM-N3) to 63° for the surface after the immobilisation, which indicates a small increase in the surface hydrophophilicity but this change is not very significant.

Considering that GNLs have five hydroxyl groups providing hydrophilicity, we hypothesise that the orientation of GNLs on the surface is in a position in which the hydroxyl groups are not on the top of the surface but on the opposite site to the inside. This issue should be investigated more in the future but at this time no proof. At the end, it can be concluded from the PM-IRRAS spectra that GNLs is successfully immobilised covalently to the PF-I-SAM-N3 via the CuAAC reaction.

Immobilisation of GNLs to azide-terminated ureido SAMs

Among urea family SAMs, we started by two SAMs prepared by the immersion method in toluene because the spin coating method still needs more investigation. We chose the longest urea molecules I-SAM-a1 (a1 is Si-C10-U-C8-N3) and I-SAM-b1 (b1 is Si-C3-U-C8-N3). The PM-IRRAS spectra is shown in the following figure 5.5 for SAMs before and after GNLs click.

All the significant bands of the GNLs appeared after click for both SAMs and we observed the slight increase in the methylene bands intensities due to having more methylene groups from GNLs after click. Moreover, it is observed that there is a small change in the region of carbonyl groups after click. Amide I band (1600-1700 cm -1 ), which corresponds to C=O stretching vibrations directly related to the backbone conformations, appears at different values depending of the type of structure [16]. Urea molecules and GNLs bearing carbonyl groups with different natures resulted in those differences. For the GNLs the position of amide I is at 1721 cm -1 and the one of amide II band is at 1636 cm -1 , while amide I bands are at 1630 and 1645 cm -1 and amide II bands at 1574 and 1565 cm -1 for I-SAM-a1 and I-SAM-b1, respectively. In addition, we observed the disappearance of the azide band (but not completely) for I-SAM-a1 after GNLs click, while for I-SAM-b1 there is a significant decrease in the azide band intensity with a small leftover of non reacted azides after click. This behaviour was the same when those SAMs were previously investigated by clicking the nitro probe on them. We conclude that the density of azides on the surface leads to crowded azide groups or steric hindrance which could decrease the accessibility of the azide groups. However, the contact angle slightly increased for I-SAM-a1 from a value of 64° before click to 66° after GNLs click. Surprisingly, the change for I-SAM-b1 is from 58° before click to 65°, which could be due to the orientation of GNLs molecules on the surface. To summarise, GNLs immobilisation to both I-SAM-a1 and I-SAM-b1 via the CuAAC reaction is successfully confirmed by the PM-IRRAS analysis. and what is clear is that the extracellular microenvironment is critical to this process [19].

Depending on that, our main concern will be the extracellular microenvironment, which is in our case the study of the effect of clicked GNLs SAMs on the adhesion, proliferation and differentiation of these cells.

According to our knowledge, SAMs modified with GNLs molecules will be used for the first time as stem cells culture, which makes it important to try also SAM-N3 before clicking GNLs and the native glass as platforms to compare results, because glass is commonly used as a conventional culture support for cells [20]. At the end of the cell growth test, cells on the solid support will be observed by the microscopic technique. For this purpose, the solid support should be transparent. Normally, we apply the surface functionalisation to wafer or SiO2/Au GNLs. Future studies could focus on creating a library of SAM-N3 of different azide intensities clicked with GNLs. The differences in azide intensities will create differences in GNLs density on the surface, which could have an influence on some or all cells growth stages. Furthermore, the presence of hydrogen bonds in the azide-terminated ureido SAMs could be another factor to be studied, since the presence of hydrogen bonds could affect GNLs orientation or molecular interactions on the surface, it could also affect cells proliferation as well as other cell behaviours such as cell differentiation or adhesion.

The library of SAM-N3 of different azide intensities can be achieved by changing the grafting methods or conditions since we previously presented SAMs with different azide intensities in chapter 2 and 3. Moreover, all observations and conclusions regarding azide orientation, reactivity, SAMs hydrophilicity and topography could be used as a guide to control the preferred SAMs properties for the bio applications.

groups is complete. This study clearly demonstrates that it is possible to control the amount of reactive azides and, consequently, the amount of molecules immobilised on the surface after the click reaction by choosing the deposition method.

A new family of molecules coupling agents bearing both a urea spacer and an azide terminal group has been synthesised with different lengths to investigate the role of changing the chain length for such SAMs. Those SAMs have been deposited successfully by the immersion method as well as spin coating method in different solvents: acetonitrile, toluene, and chloroform. The resulted SAMs have been compared to a theoretical model created using PM-IRRAS measurements. We found that toluene is the best solvent for the immersion method, while the spin coating method was better in toluene for some molecules and in chloroform for others.

Indeed, for each compound the quality of this self-assembly depends on the deposition method.

By immersion the strength of the hydrogen bond increases with the number of carbon atoms, while for the spin coating process it is the opposite. In general, for both methods the compounds with the lowest number of carbon (C6) seem to be more favourable to have a vertical azide dipole. The reactivity of azide towards biomolecules immobilisation onto these azideterminated ureido SAMs using the Cu(I)-catalysed 1,3-dipolar azide-alkyne cycloaddition (CuAAC) has been successfully achieved, and both methods (immersion and spin coating process) lead to the same reactivity of the terminal azide group around 80% for all the tested azide-terminated ureido SAMs except for the lowest number of carbon (C6) prepared by the immersion method, which has a percentage of only 50%, showing that the minimum of 11 carbon atoms is needed to have a good accessibility of the terminal azide group in the monolayer. On the other hand, six carbon atoms are detrimental for the accessibility and so the reactivity.

The new concept of mixed SAMs by having a mixture of silylated and non-silylated molecules cannot be applied by the classical immersion method. But it is possible to incorporate the nonsilylated compound into the monolayer using the spin coating process. However, the selfassembly by hydrogen bonding remains too weak to withstand the washing protocol applied after the spin coating, leading to the total loss of the non-silylated compound. In addition, AFM analysis seems to show that there is no phase segregation between the two types of molecules ensuring a homogeneous distribution of both molecules in the monolayer.

Both types of pure azide-terminated SAMs (bearing alkyl and urea) have been successfully used to immobilise GNLs. The quantity of the GNLs varies on the surface depending on the nature of SAMs and the quantity of azide groups, which could affect the further use in stem cells culture. At the end, a system of GNLs immobilised on azide-terminated SAMs bearing an alkyl spacer has been evaluated as stem cells culture platform for two types of stem cells. The preliminary results showed that the sterilisation process of surfaces is efficient and that functionalised surfaces are non-toxic to cells. We also observed enhancements in adhesion as well as proliferation and maintenance of the stemness phenotype. All these results are promising for further investigation.

At the end, this study presented a deep investigation of parameters enabling SAMs engineering with the desired properties, as a helpful tool to control biomolecules immobilisation and thus the desired bioapplication. The perspectives of this work are:

1) The spin coating method is a breakthrough that should attract more attention to be used instead of the time-consuming immersion method. The optimisation of spin coating conditions to be consistent with the immersion method results make it ideal for a commercial use for any field.

2) The new concept of mixed SAMs could be modified by trying non-silylated molecules with higher affinity to the surface, or by introducing a unit allowing a lateral polymerisation such as diacetylenes.

3) The preliminary results of using GNLs for the stem cells culture encourage to perform more studies on such surfaces for stem cells, by creating systems with different GNLs density on the surface to see the effect of this change on cells growth.

4) Finally, this thesis finished up with a relatively big list of azide-terminated SAMs and those SAMs are well studied and analysed, which makes them good candidates for various applications in the field of biology or other fields. As our main concern is the biological fields, those SAMs could be tested to immobilise a variety of biomolecules (such DNA, enzymes, proteins, and peptides) as well as polymers and functionalised nanoparticles or other species terminated with a suitable functional group to react with azide.

General Information

Materials: Chemicals were obtained from commercial sources (Sigma-Aldrich, Alfa Aesar, Acros, TCI and Fisher Scientific) and were used as received without further modification. All reactions were performed under an inert atmosphere using oven-dried glassware. Dry solvents and triethyl amine reagent were dried according to standard procedures over dehydrating agents Instrumentation: All NMR spectra were acquired in deuterochloroform. 1 H, 13 C and 29 Si NMR spectra were recorded on a Bruker Avance 300 spectrometer at 300 MHz, 75 MHz and 59 MHz respectively or on a Bruker Avance II 400 spectrometer at 400 MHz, 100 MHz and 79

MHz respectively or on a Bruker Avance III 600 spectrometer at 600 MHz, 150 MHz and 118

MHz respectively. The chemical shift data for each signal are given as δ in units of parts per million (ppm)with respect to solvent residual peak and coupling constant (J) are given in Hertz.

The multiplicity of each signal is indicated by s (singlet); bs (broad singlet); d (doublet); t (triplet); dd (doublet of doublets); dddd (doublet of doublet of doublet of doublets); q (quartet); m (multiplet).

ATR FTIR spectra were recorded on a Thermo-Scientific Nicolet iS10 FT-IR spectrometer.

High-resolution Mass spectra were performed by the CESAMO (Bordeaux, France).

ESI spectra were recorded on a QStar Elite mass spectrometer (Applied Biosystems). The instrument is equipped with an ESI source and spectra were recorded in the positive mode. The electrospray needle was maintained at 4500 V and operated at room temperature. Samples were introduced by injection through a 20 mL sample loop into a 400 mL/min flow of methanol from the LC pump. DCI measurements were carried out on a TOF mass spectrometer AccuTOF GCv (JEOL) using a direct exposure probe. One to two microliters solution of the compound is deposited on a platinum filament. In the full-scan mode, positive chemical ionization (PCI) mass spectra were recorded with CH4 as reagent gas.

Synthesis of 8-azidooctan-1-ammonium trifluoroacetate (compound 4)

This compound was synthesised following the protocol in reference [6].

To a solution of compound 3 (0.14 g, 0.50 mmol) in anhydrous dichloromethane (12 mL) at 0°C was added dropwise trifluoroacetic acid (0.45 mL, 6 mmol, 12 eq). The mixture was stirred for 12 hours at ambient temperature, then concentrated under reduced pressure. TFA traces were removed by co-evaporation under reduced pressure with toluene (3x 10 mL) to afford pale yellow oil (0.13 g, 100% yield). 1 H NMR (CDCl3, 300 MHz) δ (ppm): 7.82 (s, 3H, NH3) 3.27 (t, J = 6.9 Hz, 2H); 2.87 (t, J = 6.7 Hz, 2H) ; 1.63 (t, J = 6.9 Hz, 2H) ; 1.52 (t, J = 6.8 Hz, 2H) ;

1.30 (s, 8H). 13 This compound was synthesised following the protocol in reference [2].

Sodium azide (1.73 g, 26.6 mmol, 2 eq.) was dispersed in 20 mL of anhydrous toluene at 0°C under inert atmosphere followed by adding 10-undecenoyl chloride (2.69 g, 13.3 mmol, 1eq.).

After 3h of stirring, sodium azide (0.86g, 1 eq. -13.3 mmol) was added to the reaction mixture and stirred for 12h at ambient temperature. The resulted solution was cannulated to a flask containing 200 mL of toluene at 75°C under inert atmosphere and stirred for 1 night at 75°C.

At the end, the reaction mixture was let to go for room temperature, filtered over celite, condensed under reduced pressure to afford a white solid (1.80g 76% yield). 1

Synthesis of 3-azidopropan-1-amine (compound 6)

This compound was synthesised following the protocol in reference [7].

Synthesis of 2-azidoethan-1-amine (compound 7)

This compound was synthesised following the protocol in reference [8] A solution of sodium azide (1.12 g, 17 mmol, 2 eq.) was added to a solution of 2chlorpropylamine hydrochloride (1.0 g, 8.5 mmol, 1 eq.) in 20 mL of water and heated for 75 °C for 3 nights under reflux. Then the reaction mixture was let to go for ambient temperature and basified by sodium hydroxide (pH>10) and extracted with ether (5 x 30 mL). The organic extracts were dried over MgSO4, filtered and condensed under reduced pressure to afford pale yellow volatile oil (0.6 g, 82% yield). This compound was synthesised following the protocol in reference [6].

Synthesis of the self-assembling molecules precursors

Compound 4 (0.17 g, 1.0 mmol, 1 eq.) was dissolved in anhydrous 10 mL DMF under inert atmosphere. Distilled triethylamine (0.38 mL, 2.46 mmol, 1.5 eq) was added and the mixture was stirred for 1 hour at ambient temperature. A solution of 10-isocyanoto-1-decene (compound 5) (0.51 g, 2.92 mmol, 1.1 eq) in anhydrous DMF (10 mL) was then added and the mixture was stirred for 12 hours at ambient temperature. The mixture was concentrated under reduced

Figure 1 .Chapitre 1 :Chapitre 3 :

 113 Figure 1. Représentation schématique de la stratégie conduisant aux différentes plateformes développées dans cette thèse afin d'adapter les propriétés des surfaces de SiO2 pour l'immobilisation covalente des biomolécules Cette stratégie comporte trois étapes principales. Premièrement, la synthèse d'agents de couplage à terminaison azoture possédant des espaceurs alkyle ou urée sera réalisée. Dans cette partie, l'incorporation d'une unité urée facilitera la synthèse et les capacités d'auto-assemblage de ces molécules [14]. Deuxièmement, nous étudierons l'élaboration de monocouches à partir de ces organosilanes avec des densités d'azoture ajustables en utilisant la méthode classique d'immersion et la méthode de dépôt par centrifugation (spin coating). De plus, un nouveau concept de SAM mixtes sera étudié dans ce travail pour la famille des composés urée. En effet, ces SAM mixtes seront faites de deux types de molécules : une molécule silylée sans groupe terminal fonctionnel et une molécule non silylée avec un groupe fonctionnel azoture. Ce concept vise à ajuster la distribution des molécules et à espacer les groupes fonctionnels dans les SAMs en s'appuyant sur les liaisons hydrogène comme force motrice de la formation des SAMs. Toutes les SAMs résultantes seront caractérisées par diverses techniques, principalement la microscopie à force atomique (AFM), la spectroscopie photoélectronique à rayons X (XPS) et la spectroscopie PM-IRRAS (spectroscopie de réflexion-absorption par modulation de polarisation). Ensuite, les SAMs à terminaison azoture réagiront avec les espèces d'intérêt à terminaison alcyne par la cycloaddition azoture-alcyne catalysée par le Cu(I) (CuAAC). Cette étape doit permettre d'évaluer la réactivité de l'azoture et sera utilisée en particulier pour l'immobilisation des glyconucléolipides (GNL) afin de créer une nouvelle plateforme pour la culture de cellules souches. Ces travaux ont été menés à l'Institut des Sciences Moléculaires de Bordeaux (ISM, UMR 5255), d'octobre 2017 à octobre 2020, au sein du groupe Chimie Moléculaire et Matériaux

Figure 2 .

 2 Figure 2. Représentation schématique de l'orientation des azotures et de leur réactivité

Figure 3 .

 3 Figure 3. Famille d'agents de couplage portant un espaceur urée et un groupe terminal azoture

Figure 4 .

 4 Figure 4. Représentation schématique du nouveau concept de SAM mixte

Figure 5 .

 5 Figure 5. Spectres PM-IRRAS des SAMs après immobilisation du GNL par réaction CuAAC sur PF-I-SAM-N3, I-SAM-a1 et I-SAM-b1

  chemistry for biomolecules immobilisation to create a new platform for bioapplications. The general approach used in this work is shown schematically in figure 0.1.

Figure 0 . 1

 01 Figure 0.1 schematic representation of the strategy providing the platforms developed in this thesis to tailor the properties of SiO2 surfaces for the covalent attachment of biomolecules

Chapter 1 :Chapter 2 :Chapter 3 :Chapter 4 :Chapter 5 :

 12345 This work was carried out at the Institute of Molecular Sciences of Bordeaux (ISM, UMR 5255), from October 2017 to December 2020, within the Molecular Chemistry and Materials (C2M) group, under the direction of Dr. Luc Vellutini and Dr. Emilie Genin. The analyses of SAMs were carried out in collaboration with the Molecular Spectroscopy Group (GSM) of the ISM, by Dr. Thierry Buffeteau for the PM-IRRAS analyses and Julien Hunel for the characterisations by AFM. XPs analysis was performed in collaboration with Dr. Vincent Humblot in FEMTO-ST Institute, UMR CNRS 6174, Université Bourgogne Franche-Comté.This manuscript is divided into five chapters: This first chapter presents an introduction to the chemistry and strategies of biomolecules immobilisation to SAMs. Then it gives an overview of SAMs chemistry (definition, formation, properties, characterisation, and applications). Finally, it presents a study of azide-terminated SAMs preparation and their chemistry as those are the SAMs of interest in our work. This second chapter describes a comprehensive study of azide-terminated SAMs bearing an alkyl spacer by different spectroscopic techniques and discusses the effect of changing preparation methods and strategies on the orientation and the reactivity of the azide terminal group towards the CuAAC reaction. This third chapter presents the multistep organic synthesis of a new family of azideterminated coupling agents bearing a urea unit and their use for SAMs deposition. The effects of changes in the length of spacer on the produced SAMs is deeply studied. It also presents the use of the spin coating method, a breakthrough in this field for those compounds. This fourth chapter provides a new concept of mixed SAMs resulting from mixing silylated and non-silylated molecules. This chapter describes the synthesis of functional nonsilylated ureido compounds and the trials for mixed SAMs deposition by both the immersion and the spin coating methods. This fifth chapter describes the use of the elaborated SAMs to immobilise the bioinspired GNLs molecules (a new low molecular weight gelator) and provides SAM-GNLs, which will be used as stem cells culture platforms. At this stage, the growth of the stem cells depends on the physical-chemical properties of the produced system.

Chapter 1 .Contents 1 . 1 . 1 . 2 .

 11112 Introduction to biomolecules immobilisation and Self-Assembled Monolayers Introduction .................................................................................................................................. Immobilisation of biomolecules to SAMs ..................................................................................

Figure 1 . 1 .Figure 1 . 2 .

 1112 Figure 1.1. Schematic representation of the attachment of BSA on a carboxylate terminated SAM[17]

Figure 1 .

 1 3 shows an example of a biotinylated silicon surface (a welldefined silicon surface functionalised with biotin through a protected amino-1-alkene anchorage to the silicon external atoms). Streptavidin capture by the biotinylated surface has been demonstrated using; fluorescence microscopy, AFM and XPS. This efficient functionalisation of the surface allows it to be implemented in biochemical sensors to directly detect biomolecules and the use of silicon surfaces can be adapted to functionalise AFM cantilevers[25] .

Figure 1 . 3 .

 13 Figure 1.3. Schematic representation of a streptavidin Texas red-coated surface immobilised on a biotinylated surface [25]

Figure 1 . 5 .

 15 Figure 1.5.A selection of reactions that match the click chemistry criteria[28]

Figure 1 . 6 .

 16 Figure 1.6. Illustration of 1) azide-Staudinger ligation , 2) thermal azide-alkyne cycloaddition then 3) copper catalysed azide-alkyne cycloaddition (CuAAC), 4) Strain-promoted azide-alkyne cycloaddition (SPAAC) ) [37] The azide-Staudinger ligation (figure 1.6) is known as the non-traceless azide-phosphine ligation and achieves high yields in water, proceeding readily at pH 7. It forms an amide bond between one nitrogen atom of the azide and an ester group situated ortho to phosphorous of a triarylphosphine, via the generation of an iminophosphorane intermediate. Most importantly, the Staudinger ligation meets most of the molecules criteria required in a biologically compatible environment: rapid reactions inside the cell, no toxic byproducts and stability in

Figure 1 . 7 .

 17 Figure 1.7. Schematic description of the structure of SAMs prepared from an organic coupling agent

Figure 1 . 8 .

 18 Figure 1.8. schematic representation of a) alkanethiols on gold and b) trichloro-or alkoxysilanes on Si/SiO2

Figure 1 . 9 .

 19 Figure 1.9. Schematic representation of steps involved in the mechanism of SAMs formation on a hydrated silicon surface

[ 105 ]

 105 [START_REF] Choi | Formation and characterization of monodisperse, spherical organo-silica powders from organo-alkoxysilane-water system[END_REF] [START_REF] Greenwood | Aqueous silane modified silica sols: theory and preparation[END_REF].

Figure 1 . 10 .

 110 Figure 1.10. Schematic representation of the structure of organotrialkoxysilane layers for different situation of water contents in the grafting medium

Figure 1 . 11 .

 111 Figure 1.11. Schematic representation of the amide-containing stratified film on silicon[START_REF] Song | Preparation and Tribological Behaviors of an Amide-Containing[END_REF]

Figure 1 . 12 .

 112 Figure 1.12. Preparation of SAMs containing urea by functionalisation of isocyanate terminated SAMs with an amine terminated reagent [137] In contrast, our research group developed since several years [138] silylated coupling agents possessing urea or amide groups in the linear alkyl chains. The presence of urea or amide groups improves the solubility of coupling agents in organic solvents, and urea or amide moieties, are easily generated by several synthetic methods. It is a noticeable advantage over long-alkyl chain derivatives for which the synthetic routes are time-consuming and give relatively weak yields with poorly soluble organosilanes. To characterise in Polarisation modulation infrared reflection adsorption spectroscopy (PM-IRRAS) the hydrogen-bonding SAMs with urea or amide groups, a close collaboration was establish with Dr. Thierry Buffeteau of GSM research group of ISM [138]. Information about the orientation of the alkyl chains and the urea groups were obtained from the PM-IRRAS study of SAMs containing a urea group in the linear alkyl chains and terminated by an ester group, grafted onto SiO2/Au substrates (figure 1.13). Thus, they found that the carbonyls of the urea groups are preferentially parallel to the substrate

Figure 1 . 13 .

 113 Figure 1.13. Schematic representation of the molecular orientation within SAMs containing urea groupin the linear alkyl chains and terminated by an ester group[START_REF] Ramin | PM-IRRAS Investigation of Self-Assembled Monolayers Grafted onto SiO2/Au Substrates[END_REF]

Figure 1 . 14 .

 114 Figure 1.14. Schematic representation of a) the epoxy-terminated amido SAMs and b) epoxyterminated ureido SAMs [140].

described in figure 1 . 15 .

 115 The most used method consists in the nucleophilic displacement of bromine from bromine-terminated SAMs by azide from NaN3 the salt (figure1.16a) with conversion yields of 80 to 100%, depending on the quality of the monolayers[START_REF] Zhang | Adjusting the Surface Areal Density of Click-Reactive Azide Groups by Kinetic Control of the Azide Substitution Reaction on Bromine-Functional SAMs[END_REF] [142][START_REF] Sukenik | Monolayer transformation by nucleophilic substitution: Applications to the creation of new monolayer assemblies[END_REF].

Figure 1 . 15 .

 115 Figure 1.15. Schematic representation of post functionalisation methods to obtain azide terminated SAMs from; a) bromide terminated SAMs and b) amino terminated SAMs In contrast, the way to obtain pure azide-terminated SAMs is by the direct grafting of the presynthesised azido silylated coupling agent. Compared to post functionalisation methods, less studies have been done on the direct grafting of azide-terminated molecules on SiO2 surfaces.

Figure 1 . 16 .

 116 Figure 1.16. Schematic diagram of two methods used in preparing mixed SAM: (a) the one-step deposition method and (b) the stepwise deposition method [153]

Figure 1 . 17 .

 117 Figure 1.17. Schematic representation of mixed nitrobenzyl ester-/methyl-terminated ureido SAMs

Figure 1 . 18 .

 118 Figure 1.18. Generating bifunctional surfaces from orthogonally reactive SAMs[START_REF] Md | Orthogonally reactive SAMs as a general platform for bifunctional silica surfaces[END_REF]

Figure 1 . 19 .

 119 Figure 1.19. Schematic of the process used to create an L-B layer. (a) Langmuir film formation at the air-water interface. (b) L-B film formation at the surface of the substrate [176]

Figure 1 . 20 .

 120 Figure 1.20. Schematic illustration of dip-pen nanolithography[START_REF] Pimpin | review on Micro-and Nanolithography Techniques and Their Applications[END_REF]

Figure 1 . 21 .

 121 Figure 1.21. The main steps of SAMs preparation by the immersion solution method

Figure 1 . 22 .

 122 Figure 1.22. General steps of spin coating process; a) solution deposition on the substrate, b) high speed rotation to spread the fluid, c) drying to remove solvent [183]

  .23). The tip is brought into atomic contact with the surface. At the basic level, AFM offers imaging data by measuring the deflection of the cantilever due to Van der Waals interactions with the tip and the sample's surface. In response to these forces, the cantilever deflects. The top of the cantilever is illuminated by a laser beam and the deflection changes the position of the reflected laser beam which is detected by a photodiode. The laser position on the photodetector is used in the feedback loop to track the surface for imaging and measuring. Different modes of the AFM allow it to measure surfaces topography and additional feedback such as magnetic fields, ferroelectric domains and charge density. As a result, AFM gives real 3-D images of the sample with a vertical resolution of 0.1 nm and lateral resolution of 1 nm[START_REF] Geisse | AFM and Combined Optical Techniques[END_REF].

Figure 1 .

 1 Figure 1.23. Basic principle of atomic force microscopy

Figure 1 . 24 .

 124 Figure 1.24. The three different modes of the AFM: a) contact mode b) non-contact c) tapping mode

Figure 1 . 25 .

 125 Figure 1.25. Representation of a photoelectron emission process When an atom or molecule absorbs an X-ray photon, an electron can be ejected. The kinetic energy (EK) of the electron depends upon the photon energy (hν) and the binding energy (EB)of the electron (i.e., the energy required to remove the electron from the surface). By measuring the kinetic energy of the emitted electrons, it is possible to determine which elements are near a material's surface, their chemical states, and the binding energy of the electron. Based on the measured kinetic energy (EK) of an ejected electron and knowing the energy of the incident Xray radiation (hv), the binding energy (EB) can be determined (Equation 1.1).EK = hv -EB ……………………. Equation 1.1Specific peaks are obtained for each element due to the characteristic of the electron configuration for each element. A spectrum for a SiOx/Si substrate, coated with an organic film is shown in figure1.26. It represents the number of electrons recorded at a sequence of energies.

Figure 1 .

 1 Figure 1.26. The XPS spectrum of a SiOx/Si substrate coated with an organic molecule

Figure 1 .

 1 Figure 1.27. a) Schematic illustration of the p and s polarisation radiation. b) PM IRRAS layout

Figure 1 .

 1 Figure 1.28. a) Picture of a water droplet on a surface; b) schematic representation of the liquid contact angle θ and the respective interfacial energies

  quantitatively or orientationally) becomes a key aspect in the design of a functionalised biointerface for specific applications. Click reaction is one of the best examples of the covalent chemical modification of surfaces, which allows the modification of surfaces with stable bonds in a selective and efficient way. The regioselective and bioorthogonal copper-catalysed alkyneazide cycloaddition reaction (CuAAC) constitutes an interesting way to covalently immobilise, via a specific ligation site, biomolecules and thus control their orientation onto the surface. Moreover, the CuAAC is quantitative, rapid, chemoselective and pH independent [1] [3] [4] [5].

Figure 2 . 1 .

 21 Figure 2.1. Preparation of azide-terminated SAMs by a) two different strategies; post-functionalisation (PF) and direct grafting (Dir), b) two different methods of deposition; the classical immersion (I) and the spin coating (SC)

figure 2 . 2 (

 22 figure 2.2 (the full procedure is described in the experimental part). Both 11-bromoundec-1ene and 11-azidoundec-1-ene were quantitatively hydrosilylated to 11bromoundecyltrimethoxysilane and 11-azidoundecyltrimethoxysilane using trimethoxysilane

Figure 2 . 2 .

 22 Figure 2.2. Schematic synthesis of 11-bromoundecyltrimethoxysilane and 11azidoundecyltrimethoxysilane Karstedt's catalyst has been established as the benchmark compared to other hydrosilylation catalysts in industrial hydrosilylation processes. In 1973, Karstedt developed the platinum (0) complex containing vinyl-siloxane ligands (Karstedt's catalyst, figure 2.3a), which exhibits an extremely improved activity and selectivity as well as a high solubility in polysiloxane compositions. Figure 2.3b shows the Chalk-Harrod (CH) mechanism for the platinumcatalysed hydrosilylation of alkenes; starting by the oxidative addition of HSiR3 (ICH); followed by the coordination of olefin (IICH), then the insertion of the olefin into the Pt-H bond (IIICH) and finished by the reductive elimination of the alkylsilane (IVCH). Notably, all the mechanism steps are believed to be reversible [19] [20] [21]. Indeed, hydrosilylation of terminal alkenes may give an α or β-isomer (figure 2.4). Whilst the formation of the latter one, known as anti-Markovnikov regioselectivity, is the most common. Karstedt catalysed hydrosilylation of ordinary terminal alkenes results in the formation of 1-silylalkanes (β-isomer) and is often highly selective which make it a widespread choice over the years.

Figure 2 .

 2 Figure 2.3. a) the structure of the Karstedt's catalyst [20] b) the Chalk-Harrod (CH) mechanism for the platinum-catalysed hydrosilylation of alkenes [21]Although Karstedt catalyst catalyses the formation of the desired hydrosilylation product (βisomer, its use may also result in several undesirable side reactions such as dehydrogenative silylation, hydrogenation or isomerisation of olefins, olefin oligomerisation and redistribution of hydrosilanes as shown in figure2.4. Consequently, those side reactions produce side products as impurities (not easy to be separated) in silanes solution and have direct impact on the purity of SAMs. Karstedt's catalyst often provides the alkyl silane in only 78% yield with isomerised or reduced products in 20%[20] [22]. Another disadvantage of the Karstedt catalyst is the formation of platinum black during the hydrosilylation reaction, which is responsible for some of the side products and leads to a contamination of the hydrosilylation product[20] [23][22]

Figure 2 . 4 .

 24 Figure 2.4. Reactions of the olefin with the hydrosilane in the presence of a catalyst [24].

Figure 2 . 5 . 1 H

 251 Figure 2.5. 1 H NMR spectra of11-bromoundecyltrimethoxysilane (top) and 11azidoundecyltrimethoxysilane (bottom)

Figure 2 . 7 .

 27 Figure 2.7. The general scheme of the main steps of the classical immersion method used to prepare SAM-Br The measurements of the water contact angle were done first onto surfaces after the activation step and found to have a value of almost 0°, due to the presence of the hydroxyl groups making the surface very hydrophilic. After grafting, water contact angle measurements were applied on several slides with 2-6 measurements for each slide. The average values of SAM-Br were found to be 70° for SAMs grafted in toluene and 68 ° for SAMs in chloroform, which confirms the change of the surface hydrophilicity due to covering the surface with the bromide-terminated organic layer. Moreover, all contact angle measurements are very similar on the same surface, which indicates the homogeneity of the samples. Those values are not very far from literature

Figure 2 . 8 .

 28 Figure 2.8. PM-IRRAS spectra of SAM-Br grafted in toluene and chloroform using the immersion method

Figure 2 . 9 .

 29 Figure 2.9. PM-IRRAS spectra of SAM-N3 post-functionalised from SAM-Br grafted using the immersion method in a) toluene and b) chloroform The figure 2.10 below shows a comparison between the PM-IRRAS spectra of both azideterminated SAMs obtained by the post-functionalisation of SAM-Br in chloroform and toluene. It is clearly observed that the intensities of CH2 and azide bands are very close for both solvents, which is consistent with the observation of almost the same intensities of CH2 in SAM-Br before post-functionalisation and the stability of those layers (without any degradation) during the substitution step. We can conclude that changing the solvent polarity has no influence on the resulted SAM-Br prepared by the immersion method and consequently no influence on the postfunctionalised SAM-N3.

Figure 2 . 10 .

 210 Figure 2.10. Comparison between SAM-N3 post-functionalised from SAM-Br grafted using the immersion method in toluene and chloroform Water contact angle measurements could give more information about surface modification since changing the terminal group of the surface from Br to N3 is supposed to increase the

Figure 2 .

 2 Figure 2.11. AFM images (5µm ⅹ 5µm) of a) the initial wafer surface. SAM-N3 prepared on wafer by post-functionalisation of SAM-Br prepared by the classical immersion method using: b) toluene and c) chloroform

Figure 2 .

 2 Figure 2.12. A characteristic XPS high resolution spectrum of a) a bromide surface and b) an azidesurface[33]

Figure 2 .

 2 Figure 2.13.XPS high resolution spectrum of SAM-Br (prepared by the immersion method in chloroform) and SAM-N3

  .14 , a drop ( 40 µL , 4ⅹ10-3 mol/L) of 11-bromoundecyltrimethoxysilane solution was deposited on the cleaned and preactivated surface using the spin coater (6000 rpm for 40 seconds), then the coated surface was washed after different periods of drying time to find the optimum drying time consistent with the monolayer properties. For this study, two different drying times where tested by washing slides after either 30 minutes or 3 hours. Solvent evaporation is ultimately responsible for the final thickness of the organic layer on the surface. During most of the process, flow and evaporation have the same order of magnitude, but flow only dominates for a short period at the start of the process, and evaporation only becomes the leading order process at the very end when solute concentration is high.[34] From this point, toluene and chloroform could affect the spin coating method since they have different polarity and volatility: chloroform (boiling point = 61.2°C) is more volatile and more polar than toluene (boiling point = 110.6°C). The second step of converting SAM-Br to SAM-N3 followed the same protocol used for the substitution of SAM-Br elaborated by the immersion method.

Figure 2 . 14 .

 214 Figure 2.14. Representation scheme of the main steps of the spin coating method

Figure 2 .

 2 Figure 2.15. PM-IRRAS spectra of SAM-N3 prepared by the post-functionalisation of SAM-Br prepared by the spin coating of 11-bromoundecyltrimethoxysilane in chloroform (top) and in toluene (bottom) with a drying time of 30 min (a and c) and 3 hours (b and d). The reproducibility of this process has been investigated by performing experiments twice. As shown in figure 2.16, the intensities of CH2 and azide bands are very close for both solvents at drying time of 30 min. while for 3 hours, we can observe small differences in both methylene and azide intensities. This variation in the intensities of CH2 is more important for toluene, which could be due to the less volatility of toluene leading to increasing the time of the deposition reaction, which makes the increase in layer intensity is higher for toluene than chloroform. As spin coating is applied in the open air and at room temperature that could change either the water content (due to having different humidity from time to time) or the deposition reaction temperature. Strikingly, those differences are not very significant and all experiments provide monolayers with good intensities in PM-IRRAS compared to the literature, which are

Figure 2 .

 2 Figure 2.16. PM-IRRAS data for two trials of SAM-N3 provided by post-functionalisation of SAM-Br prepared by spin coating of 11-bromoundecyltrimethoxysilane in: a) chloroform and b) toluene AFM analysis for this part was performed on SiO2/Au (initial surface roughness is 0.90 nm) before and after SAMs formation. Figure 2.17 shows the AFM images of both SAM-N3 provided from the post-functionalisation of SAM-Br prepared by spin coating in chloroform and toluene. No holes and aggregation are observed for both SAM-N3, and both surfaces are smooth with a roughness of 1.21 nm for toluene and 1.29 nm for chloroform indicating the same topography for both surfaces. Meanwhile, the contact angle of SAM-N3 provided in

Figure 2 .

 2 Figure 2.17. a) AFM images (5µm ⅹ 5µm) of a) the initial SiO2/Au surface. SAM-N3 prepared on PM-IRRAS substrate by post-functionalisation of SAM-Br prepared by the spin coating method in: b) toluene and c) chloroformThe general conclusion of the post-functionalisation strategy is that SAMs properties are driven by the behaviour of 11-bromoundecyltrimethoxysilane. Indeed, this strategy starts first by grafting bromide-terminated silanes to form SAM-Br and this step appeared to be not sensitive to solvent or method changes, which resulted in producing SAM-Br with the same properties for both methods (immersion and spin coating) by using both solvents chloroform and toluene.Then, the second step is the substitution of SAM-Br to SAM-N3. At this step, if SAM-Br have the same properties, consequently the produced SAM-N3 would also have the same properties for both methods and solvents.

  H NMR and IR. Both analyses confirm the integrity of the 11-azido-undec-1-ene. Moreover, another experiment has been applied by directly grafting the silylated molecule 11-azidoundecyltrimethoxysilane in chloroform and analysing the produced SAMs by PM-IRRAS. The formation of SAM-N3 is successful as all characteristic bands are present (azide and CH2 stretching bands): the antisymmetric stretching mode (aCH2)is 2929 cm -1 and the symmetric stretching mode (sCH2) is 2857 cm -1 , while the azide stretching band is observed at 2103 cm -1 . Then the PM-IRRAS analyses have been performed on SAM-N3 after one year of aging, and as can be observed from the following figure 2.19, there is no significant modifications in the spectra confirming the stability of the corresponding SAMs using the direct grafting strategy.

Figure 2 . 19 .

 219 Figure 2.19. Comparison between the PM-IRRAS spectrum of a fresh sample of SAM-N3 directly grafted using the immersion method in chloroform and the one of the same sample after one year In addition to chloroform, more SAM-N3 have been also directly produced in toluene and acetonitrile using the immersion method. As shown in figure 2.20a, PM-IRRAS data confirm the surface modification by the azide monolayer as all characteristic bands are present (azide and CH2 stretching bands). The antisymmetric stretching modes (aCH2) are at 2929 cm -1 , 2930

Figure 2 .Figure 2 .

 22 Figure 2.20. a) PM-IRRAS spectra for SAM-N3 directly grafted using the immersion method in toluene, acetonitrile, and chloroform. b) the normalised spectra. c) Representation of the orientation of the azide group with respect to the surface To confirm the results of each solvent which could confirm the influence of the solvent on the formed monolayer, we checked the reproducibility. As shown in figure 2.21, PM-IRRAS spectra of two trials are all superimposed for each solvent and very similar regarding the intensities of both azide and methylene bands.

Figure 2 .

 2 Figure 2.22. a) The initial wafer surface of 0.21 nm roughness. AFM images (5µm ⅹ 5µm) of SAM-N3 prepared by the direct grafting strategy in b) toluene c) acetonitrile d) chloroform using the immersion method

  )[35]. It was found that the best drying time to produce a monolayer is 30 minutes as shown in the PM-IRRAS spectrum in figure 2.23.

Figure 2 .

 2 Figure 2.23. PM-IRRAS spectra of SAM-N3 directly grafted using the spin coating method in chloroform.

Figure 2 .

 2 Figure 2.24. PM-IRRAS spectra of SAM-N3 directly grafted using the spin coating method in toluene; a) at three different drying times, b) before and after washing in a period of few months, c) comparison between two trials.

Figure 2 .

 2 Figure 2.25 shows the PM-IRRAS results obtained using acetonitrile solvent, the most polar and second in volatility among the three solvents. Once again, the spin coating of the organic layer is confirmed by the presence of the antisymmetric stretching mode (aCH2) at 2926 cm -1

and 3

 3 hours lead to very close intensities, with intensities for the methylene bands consistent with the monolayer. We could conclude that both 30 minutes and 3 hours drying times are in the range of the optimum monolayer PM-IRRAS data.

Figure 2 .

 2 Figure 2.25. PM-IRRAS spectra of SAM-N3 directly grafted in acetonitrile using the spin coating method at three different drying times As observed for toluene, the washing process after spin coating removes the excess material above the monolayer. It is interesting to note from figure 2.25 that, whatever the drying time of 30 min or 3 hours, the intensities of the methylene bands are similar, showing saturation as early as the 30 min. After several months of drying, the washing process leads to a decrease in the intensity of the azide band to reach the intensity obtained after 30 min of drying (figure 2.26) This observation confirms that excess of organic material can be removed by washing and that a monolayer is formed by spin coating after 30 min of drying. After several months of storage, the surface was probably polluted, explaining why the intensity of the methylene bands did not decrease.

Figure 2 .

 2 Figure 2.26. PM-IRRAS spectra of SAM-N3 directly grafted in acetonitrile using the spin coating method: before and after washing in a period of few months

Figure 2 .

 2 Figure 2.27. PM-IRRAS spectra of two trials of SAM-N3 directly grafted in acetonitrile using the spin coating method

Figure 2 .Figure 2 .

 22 Figure 2.28. a) PM-IRRAS spectra of SAM-N3 prepared by the spin coating method in three different solvents. b) Normalised spectra with respect to the νaCH2 band Not only PM-IRRAS spectra have been used to compare the chosen spin coated samples (3 hours drying time in three solvents), but also contact angle and AFM data. Starting by contact angle, it shows values in the same range (73° for toluene, 68° for acetonitrile and 69° for chloroform), which indicates that the three SAM-N3 produced using the three solvents have the same wettability. AFM images (figure 2.29) show smooth surfaces for all samples. The roughness value of the initial surface (SiO2/Au) is 0.90 nm, while the roughness of the three surfaces are 1.32 nm for toluene, 1.25 nm for acetonitrile and 1.10 nm for chloroform. Also, AFM results confirm having almost the same topography for all solvents. It confirms the conclusion that the spin coating method is less affected by solvent polarity (regarding the studied solvents).

Figure 2 .

 2 Figure 2.30. PM-IRRAS spectra of SAM-N3 directly grafted in acetonitrile by using two deposition methods: the immersion method and the spin coating method Moving to the study to toluene, we compared the three processes: the post-functionalisation by immersion, the direct grafting by immersion, and the direct grafting by spin coating. As shown in the following PM-IRRAS spectra (figure 2.31a), there is a variation in both the intensities of the methylene and azide groups bands. The Dir-SC-SAM-N3 provides the higher quantity of organic matter on the surface in comparison with both Dir-I-SAM-N3 and PF-I-SAM-N3. In this case, it is better to use the normalised spectra in figure 2.31b to have a better understanding

Figure 2 .

 2 Figure 2. 31. a) Non normalised and b) normalised PM-IRRAS spectra (with respect to the νaCH2 band) of SAM-N3 grafted in toluene by using different deposition methods

Figure 2 .Figure 2 . 3 ÅFigure 2 .

 2232 Figure 2.32. a) Non normalised and b) normalised PM-IRRAS spectra (with respect to the νaCH2 band) of SAM-N3 grafted in chloroform by using different deposition methods At the beginning, PM-IRRAS data have been collected to present more statistics on peak locations (particularly for the methylene peaks) and areas (particularly for the azide peaks) in order to understand how different those surfaces actually are. For PF-I-SAM-N3, we had the average of data of 5 experiments at the beginning. Then more work has been applied and reached 15 experiments, while for Dir-I-SAM-N3, we had the average of 3 experiments first, then one more experiment has been applied and we had a new average of 4 samples. However, for the Dir-SC-SAM-N3, we directly used the average spectrum for 2 experiments. As previously mentioned, chloroform is the grafting solvent for all processes here. The frequencies of the methylene and azide peaks measured on the different PM-IRRAS spectra as well as the average frequencies and the standard deviation for PF-I-SAM-N3 and Dir-I-SAM-N3 are reported in the following table 2.1:

  bands observed in figure 2.32a are consistent with the formation of a single monolayer on the surface. For PF-I-SAM-N3 and Dir-I-SAM-N3 the intensities of the methylene bands are weaker than for Dir-SC-SAM-N3, which corresponds to less matter on the surface,

Figure 2 . 35 .

 235 Figure 2.35. AFM height images of a) The initial substrate (SiO2/Au), b) PF-I-SAM-N3, c) Dir-I-SAM-N3, d) Dir-SC-SAM-N3

Figure 2 .

 2 36 presents XPS spectra of Dir-SC-SAM-N3 surfaces in the N1s region for the three different methods of deposition. The XPS spectra of PF-I-SAM-N3 is consistent with the theoretical expectation and literature data[32] but is quite different than those of Dir-I-SAM-N3 and Dir-SC-SAM-N3, despites the fact that the three SAMs compositions are identical. The theoretical data shows of azido groups have three contributions:the two contributions at low binding energy (BE) are assigned to the electron enriched (-N=) and neutral (=N-) nitrogen atoms, while the contribution at higher BE is assigned to the electron deficient (=N + =) nitrogen atom. In figure2.36, corresponding to the post-functionalised PF-I-SAM-N3, the N1s profile is identical to the one observed for the azido compound in (figure2.36 left) with two contributions separated by around 4 eV, and with a higher intensity of the low BE component. In contrast, the XPS spectra obtained for both direct grafting of SAMs (Dir-I-SAM-N3 and Dir-SC-SAM-N3), presented in figure2.36, reveal a different N1s profile: first, the high BE peak at around 405 eV is weaker than for PF-I-SAM-N3, and the contribution at lower BE is broader and could be fitted with at least three contributions, in opposition of the two observed for PF-I-SAM-N3. The extra contribution appears at 1 eV higher than the two other ones and is likely due to the central nitrogen atom (=N + =) in dipole-dipole interactions with neighboring azido groups. Such interactions involve antiparallel orientation of azido groups with sharing electrons, resulting in the energy shifts observed in figures 2.36.

Figure 2 . 36 .

 236 Figure 2.36. High resolution XPS spectra of the N1s region for: left) the theoretical results of 11azidoundecyltrimethoxysilane (azido groups, right) three types of SAMs: a) PF-I-SAM-N3, b) Dir-I-SAM-N3 and c) Dir-SC-SAM-N3. The PM-IRRAS and XPS results show that azide groups are present at the surfaces with different orientations and arrangements (isolated dipoles and antiparallel dipole-dipole interactions) depending on the deposition methods. It is important to note that if antiparallel dipole-dipole interactions occur, the most favourable orientation for the involved azide groups is certainly parallel to the surface, as shown in figure 2.37. Consequently, due to the surface selection rule of the PM-IRRAS, these azide groups do not contribute to the intensity of the aN3 band.

Figure 2 .

 2 Figure 2.37. illustration of the two expected orientations of azide groups in a) PF-I-SAM-N3 b) Dir-SC-SAM-N3 and Dir-I-SAM-N3

Figure 2 .

 2 Figure 2.38. Left) Schematic representation of the molecular organization inside the SAM-N3 depending on the deposition methods: a) by post-functionalisation from SAM-Br (PF-I-SAM-N3 ) and b) by the direct grafting of 11-azidoundecyltrimethoxysilane by immersion (Dir-I-SAM-N3) and spin coating (Dir-SC-SAM-N3) processes. Right) Schematic representation of the corresponding SAMs after click reaction with the nitro probe (-SAM-NO2) and the heptyne compound (-SAM-H). After the covalent immobilisation of 1-(but-3-yn-1-yloxy)-4-nitrobenzene, the chemically modified surfaces (SAM-NO2) have first been characterised by PM-IRRAS. We observed in figure 2.39a the complete disappearance of the aN3 band at 2100 cm -1 on the PF-I-SAM-NO2 spectrum associated with the appearance of new bands at 1608 (C=C), 1594 (C=C), 1499 (C=C), 1515 (aNO2) and 1342 cm -1 (sNO2), related to the nitrophenyl moiety. The total disappearance of the aN3 band in the PM-IRRAS spectra clearly demonstrated the complete reaction of the azide groups on the surface with the acetylene compound and indirectly the successful formation of 1,2,3-triazole moiety on the monolayer. In addition, the region of methylene stretching modes does not reveal any degradation of the monolayer during the click reaction since the bands remained comparable in position and in intensity. The similar observations were obtained for Dir-I-SAM -NO2 and Dir-SC-SAM -NO2 revealing again the complete reactivity of the azide groups. However, the PM-IRRAS normalised spectra to the (aCH2) in figure 2.39b show that the intensity of the characteristic bands of the organic probe

Figure 2 .

 2 Figure 2.39. a) Non normalised PM-IRRAS spectra before and after the click reaction with the nitro probe for PF-I-SAM-N3. b) Normalised PM-IRRAS spectra of SAMs after click reaction for the three methods of deposition. c) Intensity of the νaNO2 band of SAM-NO2 plotted as a function of the intensity of the νaN3 band of the corresponding SAM-N3.

  DMSO (figure 2.40). Afterward the resulted triazole compound has been hydrosilylated and analysed in XPS as a molecular compound. This silylated compound was also grafted by the classical immersion method to provide a reference SAMs incorporating the molecule obtained after click. It is analysed by XPS to be used as a reference for the click reaction of SAM-N3 and 1-heptyne (all the experimental details are in the experimental part).

Figure 2 . 40 .Figure 2 .

 2402 Figure 2.40. Synthesis of the silylated triazole compound

Figure 2 .

 2 Figure 2.41. left: High resolution XPS spectra of the N1s region for a) 11azidoundecyltrimethoxysilane and b) the silylated triazole compound. Right: High resolution XPS spectra of the N1s region for triazole SAMs obtained either directly or after click reaction of SAM-N3 with heptyne: a) SAM-H, b) PF-I-SAM-H, c) Dir-I-SAM-H, and d) Dir-SC-SAM-H

  b) PF-I-SAM-H c) Dir-I-SAM-H d) Dir-SC-SAM-H a) SAM-H

Figure 2 . 42 . 3 . 1 . 3 . 2 . 3 . 2 . 1 . 3 . 3 . 3 . 3 . 1 . 3 . 4 . 3 . 5 .

 2423132321333313435 Figure 2.42. Schematic representation of azide orientation and reactivity

Figure 3 . 1 .

 31 Figure 3.1. The schematic representation of amide containing azidoalkylthiol SAMs[13]

Figure 3 . 2 .

 32 Figure 3.2. PM-IRRAS spectrum of azide-terminated SAMs containing a urea group

Figure 3 . 3 .

 33 Figure 3.3. Schematic representation of main work steps in this chapter; 1) synthesis of urea coupling agents with different lengths, 2) SAMs formation, 3) Evaluation of azide reactivity by click chemistry

Figure 3 . 4 .

 34 Figure 3.4. Schematic representation of a new family of ureido coupling agents of different lengths: group a with a long tail, and group b with a short tail

Figure 3 . 5 .

 35 Figure 3.5. Schematic description of the general protocol for ureido coupling agent's synthesis

Figure 3 . 6 .

 36 Figure 3.6. Schematic description of the two ureido groups; group a of three compounds (a1, a2, a3) having a long carbon chain between urea and silicon (long tail), and group b of two compounds (b1 and b2) having a short carbon chain between urea and silicon (short tail)

Figure 3 . 7 .

 37 Figure 3.7. The general scheme of the synthesis of 10-isocyanatodec-1-ene

Figure 3 . 9 .

 39 Figure 3.9. The schematic synthetic route for the synthesis of compound a1

Figure 3 . 10 .

 310 Figure 3.10. The schematic synthetic route for the synthesis of compound a2

Figure 3 . 11 .

 311 Figure 3.11. The schematic synthetic route for the synthesis of compound a3

Figure 3 . 12 .

 312 Figure 3.12. The schematic synthetic route for the synthesis of compound b1

Figure 3 . 12 .

 312 Figure 3.12. The schematic synthetic route for the synthesis of compound b2

Figure 3 . 13 .

 313 Figure 3.13. The simulated PM-IRRAS spectra for all ureido SAMs

Figure 3 .

 3 Figure 3.14. PM-IRRAS spectra of all azide-terminated ureido SAMs in three different solvents (toluene, acetonitrile, and chloroform)

  homogeneous surfaces without holes or aggregations (figure 3.15), with a roughness less than 0.6 nm which support the hypothesis of having less packing density in the monolayers. The contact angle values vary from 53° (C6) to 66° (C13) depending of the number

Figure 3 .

 3 Figure 3.15. AFM height images (5µm ⅹ 5µm) of a) I-SAM-a1 b) I-SAM-a2 c) I-SAM-a3, d) I-SAM-b1 e) I-SAM-b2The high polarity of acetonitrile could induce strong interactions between silylated molecules and the solvent. It could thus affect the accessibility of the molecules to the surface by

Figure 3 . 16 .

 316 Figure 3.16. The mechanism of the organic layer formation on the substrate

  terminated ureido SAMs formed in chloroform are relatively higher in roughness compared to other solvents, the values are in the range of 1.4 nm to 4.85 nm while the roughness of the initial surface is 0.21 nm. This observation can be explained by either the formation of a multilayer or the presence of aggregations. AFM images ( figure3.18) show some differences in the topography of all SAMs; some aggregations are observed for I-SAM-a3, I-SAM-b1 and slightly less aggregations for I-SAM-a2, I-SAM-a1, I-SAM-b2, showing the heterogeneity of the surface prepared in chloroform, but those results are consistent with the high intensity of methylene bands observed in PM-RRAS data. In contrast, the contact angle values are in the range of 48° to 64° which is slightly higher than acetonitrile, but those values are in the range of those obtained with other solvents, which indicates not having big difference in the hydrophilicity of the surface. To summarise, azide-terminated ureido SAMs formed by the immersion method in chloroform could produce high quantity of the organic matter on the surface and relatively with higher roughness.

Figure 3 .

 3 Figure 3.18. AFM height images (5µm ⅹ 5µm) of a) I-SAM-a2 b) I-SAM-a3, c) I-SAM-b1 d) I-SAM-b2 grafted by the immersion method in chloroform (the initial surface is wafer)

Figure 3 .

 3 Figure 3.19. AFM images (5µm ⅹ 5µm) of, a) I-SAM-a1 b) I-SAM-a2 c) I-SAM-a3 d) I-SAM-b1 e) I-SAM-b2 deposited on wafer by the immersion method in toluene

Figure 3 .

 3 Figure 3.20. (a-e): PM-IRRAS spectra normalised to νaCH2 of all azide-terminated ureido SAMs in three different solvents (toluene, acetonitrile, and chloroform). f) PM-IRRAS spectra normalised to amide Ⅱ band for compound b2 in three different solvents (toluene, acetonitrile, and chloroform)

Figure 3 .

 3 Figure 3.21. A plot of the theoretical (nRth) values compared to the experimental (nRexp) values for all azide-terminated ureido SAMs prepared by the immersion method in toluene, acetonitrile, and chloroform To conclude about the best solvent for azide-terminated ureido SAMs deposition using the immersion method, comparing the intensity of νaCH2 of experimental spectra with respect to the corresponding simulated spectra of each SAM, toluene seems to be more appropriated to give a monolayer. The values of νaCH2 intensities in toluene are the closest to the estimated values among all solvents. Moreover, AFM results showed smooth surfaces without

Figure 3 .

 3 Figure 3.22. PM-IRRAS spectra of all azide-terminated ureido SAMs prepared (twice) in toluene by the immersion method

Figure 3 .

 3 Figure 3.23. PM-IRRAS spectra of all azide-terminated ureido SAMs prepared by the immersion method in toluene.

figure 3 . 23 .

 323 figure 3.23. The highest intensity of the methylene stretching vibration bands are for compound a1 (18 carbons), the intensities are getting less and less by decreasing the chain length to give the least intensities for compound b2 of 6 carbons. For the alkyl chains conformation, all azideterminated ureido SAMs show disordered alkyl chains, as the position of the antisymmetric (νaCH2) and symmetric (νsCH2) stretching vibrations of the methylene groups is observed at 2929 cm -1 and 2855-2858 cm -1 , respectively indicating disordered alkyl chains in the monolayer. Indeed, increasing the carbon chain length should lead to have more van der Waals

  ) a2 (C13) a3 (C12) b1 (C11) b2 (C6) Toluene nRth nRexp SAMs the hydrogen bond strength increases significantly (Δν = 66 cm -1 ) to reach a maximum (Δν = 56 cm -1 ) for 18 carbon atoms. The last value is consistent with the Δν value of 56 cm -1 observed for ureido SAMs with 17 carbon atoms which confirms the presence of strong intermolecular hydrogen bonding [11] [27]. From this result and considering the disorder of the alkyl chains, the major driving forces operating in the molecular assembly are mainly based on the intermolecular hydrogen bonding between urea groups in the monolayers. To have more information, table 3.8 shows the values of Δν calculated for all ureido compounds in the molecular state (solid powder) before SAMs formation, and those values were compared to the values of Δν calculated for SAMs. Compounds a1 and a2 shown clearly the lowest Δν with 48 cm -1 and 45 cm -1 , respectively. When the number of carbon atoms decreased the Δν increases significantly to reach 70 cm -1 for the compound b2. Therefore, self-assembly by hydrogenbonding is stronger as the carbon number increases. We observed the same trend in monolayers.

From 12 carbon

 12 atoms the hydrophilicity increases when the number of carbon atoms decreased to reach 53°. The graph in figure3.25 highlights the cooperative effect of hydrogen-bonding interactions and hydrophobic interactions to control the self-assembly of the molecule in the monolayer. From 13 carbon atoms upwards, self-assembly is achieved by the establishment of strong hydrogen bonding and increases significantly with the number of carbon atoms. On the other hand, between 6 and 12 carbon atoms self-assembly is similar with a much weaker hydrogen bond interaction. Therefore, a variation in the length of the alkyl chain below 12 carbons has little impact on the intermolecular hydrogen bonding network. Only hydrophilicity increases with decreasing carbon number.

Figure 3 . 3 . 3 . 1 . 6 .

 33316 Figure 3.25. A plot of the contact angle and Δν for all azide-terminated ureido SAMs prepared by the immersion method in toluene

Figure 3 .

 3 Figure 3.26. PM-IRRAS spectra of I-SAM-a1 and I-SAM-b2

Figure 3 . 27 .

 327 Figure 3.27. The general scheme of clicking the nitro probe and azide-terminated ureido SAMs prepared by the immersion method in toluene It is expected to see a disappearance of the azide vibration band (aN3) around 2100 cm -1 and the appearance of new bands at 1608 cm -1 , 1594 cm -1 , 1499 (C=C), 1515 (aNO2) and 1342 cm -1 (sNO2) related to the nitrophenyl moiety. But the bands of (C=C) and (aNO2) are not observed in the PM-IRRAS because they are in the same range as amide I and amide II which screen them. Figure 3.28 shows all azide-terminated ureido SAMs before and after the click reaction of the nitro probe. For all clicked SAMs, we clearly observed the appearance of new bands at 1608 (C=C), 1594 (C=C), 1499 (C=C), 1515 (aNO2) and 1342 cm -1 (sNO2)

Figure 3 .

 3 Figure 3.28. The PM-IRRAS spectra of all azide-terminated ureido SAMs (prepared by the immersion method in toluene) before and after clicking the alkyne terminated nitro probe

Figure 3 .

 3 Figure 3.29. A histogram of the percentages of the reacted azide for the clicked azide-terminated ureido SAMs

  been tested to find the best conditions consistent with the optimum results of the immersion method (the exact protocol described in the experimental part). All azideterminated ureido SAMs have been characterised by PM-IRRAS, contact angle measurements and AFM. We have five SAMs:

Figure 3 .

 3 Figure 3.30. PM-IRRAS spectra of SC-SAM-a1 prepared in toluene by the spin coating method at different drying times However, the contact angle values are in the range of 65°-68° for all spin coating trials of SC-SAM-a1 and not vary from the value obtained by the immersion method (63°), which indicates no big change in the hydrophilicity of those SAMs. Also, AFM analysis shows that the topography of the surface is not different; the roughness of the initial surface (SiO2/Au) is 0.90 nm, while the roughness of SC-SAM-a1 is 1.34 nm for 3 hours, 1.29 nm for the 5 hours and 1.19 nm for the 24-hour, showing smooth surfaces with no aggregations or holes (figure 3.31).

Figure 3 .

 3 Figure 3.31. AFM images (5µm ⅹ 5µm) of SC-SAM-a1 deposited in toluene by the spin coating method at different drying times; a) 3h b) 5h and c) 24hTo optimise spin coating conditions for SC-SAM-a1 and have more information about the behaviour of compound a1 in this process, we tried to change some parameters. We have two choices, either changing the concentration of the silanes solution or changing the solvent of the spin coating process.

Figure 3 .

 3 Figure 3.32. Left: PM-IRRAS spectra of SC-SAM-a1 prepared in toluene by the spin coating method using concentrated spinning solution (4 x10-3 mol.L -1 ). Right: The AFM image (5µm ⅹ 5µm) of SC-SAM-a1 deposited in toluene by a concentrated spinning solution ⅱ) Effect of the nature of the solvent: The second parameter changed to enhance the spin coating of compound a1 is trying other solvents like acetonitrile and chloroform. As shown in figure 3.33 by the PM-IRRAS spectra, both toluene and acetonitrile show the same behaviour, leading to low organic matter on the surface and no big change by increasing the drying time from 3 hours to 5 hours. In contrast, compared to other solvents, chloroform gives slightly better results in terms of the organic matter intensity. Chloroform gives more material on the surface;

Figure 3 .

 3 Figure 3.33. PM-IRRAS spectra of SC-SAM-a1 prepared by the spin coating method in three different solvents at different drying times compared to SC-SAM-a1 prepared by the immersion method in toluene

Figure 3 . 34 .

 334 Figure 3.34. AFM images (5µm ⅹ 5µm) of SC-SAM-a1 deposited by the spin coating method in a) acetonitrile 3h b) acetonitrile 5h c) chloroform 3h and d) chloroform 5h

Figure 3 .

 3 Figure 3.35. Left: PM-IRRAS spectra of SC-SAM-a2 prepared in toluene by the spin coating method at two different drying times and I-SAM-a2. Right: AFM images (5µm ⅹ 5µm) of SC-SAM-a2 prepared by the spin coating method in toluene for 24h drying timeHaving toluene as the optimum solvent for the grafting of compounds a2 by both spin coating and immersion method encouraged us to investigate the azide orientation. Not only the effect of changing the grafting method on the azide orientation can be observed but also the effect of increasing the drying time during the SAMs formation by spin coating. For this purpose, it is better to use the normalised spectra of PM-IRRAS on the antisymmetric methylene band as shown in figure3.36. Starting by the impact of changing the drying time, it can be seen that the

Figure 3 . 3 . 4 . 3 .

 3343 Figure 3.36. PM-IRRAS spectra normalised (to νaCH2 band) for SC-SAM-a2 prepared in toluene using the spin coating method at two different drying times compared to I-SAM-a2

Figure 3 .

 3 Figure 3.37. PM-IRRAS spectra of compound SC-SAM-a3 prepared by the spin coating method in toluene at two different drying times. b): AFM images (5µm ⅹ 5µm) of SC-SAM-a3 prepared by the spin coating method in toluene of 24h drying time

Figure 3 .

 3 Figure 3.39. Left: Figure: PM-IRRAS spectra of compound SC-SAM-b1 prepared by the spin coating method in toluene at two different drying times. Right: AFM images (5µm ⅹ 5µm) of SC-SAM-b1 prepared by the spin coating method in toluene of 24 hours drying time To optimise spin coating conditions for compound b1, and as chloroform showed good results for the spin coating of compound a1, chloroform has been also chosen to investigate the spin coating of compound b1. First, we can observe from the PM-IRRAS figure 3.40 all the significant bands of compound b1 confirming the successful deposition of this compound.

Figure 3 .

 3 Figure 3.40. PM-IRRAS spectra of SC-SAM-b1 prepared by the spin coating method in toluene and chloroform at drying time 3 hours and 24 hours

Figure 3 .

 3 Figure 3.41. AFM images (5µm ⅹ 5µm) of SC-SAM-b1 prepared by the spin coating method in chloroform at drying time of a) 3 hours and b) 24 hours

Figure 3 .

 3 Figure 3.42. Left: PM-IRRAS of compound SC-SAM-b2 prepared in toluene by the spin coating method at different drying times. Right: AFM images (5µm ⅹ 5µm) of SC-SAM-b2 prepared by the spin coating method in toluene at 19 hours drying time

Figure 3 .

 3 Figure 3.43. PM-IRRAS spectra of all azide-terminated ureido SAMs prepared by the spin coating method

Figure 3 .

 3 Figure 3.44. A plot of the (nRexp) values compared to the (nRth) values for all azide-terminated ureido SAMs prepared by the spin coating method

Figure 3 . 45 .

 345 Figure 3.45. The general scheme of clicking the nitro probe and azide-terminated ureido SAMs prepared by the spin coating method The PM-IRRAS spectra of the click reaction is shown in figure 3.46. It is expected to see a disappearance of the azide vibration band (aN3) around 2100 cm -1 and the appearance of new bands at 1608 cm -1 , 1594 cm -1 , 1499 (C=C), 1515 (aNO2) and 1342 cm -1 (sNO2) related to the nitrophenyl moiety. But the bands of (C=C) and (aNO2) are not observed in the PM-IRRAS because they are in the same range as amide I and amide II.Figure 3.46 shows all azide-

Figure 3 .

 3 46 shows all azideterminated ureido SAMs before and after the click reaction of the nitro probe. For both clicked SAMs, we clearly observed the appearance of new bands at 1608 (C=C), 1594 (C=C), 1499

Figure 3 .

 3 Figure 3.46. The PM-IRRAS spectra a) SC-SAM-a2 and b) SC-SAM-a3 before and after clicking the alkyne terminated nitro probe To have more details, figure 3.47 shows a histogram for the percentages of reacted azide after click. Compounds a2 and a3 show the same reactivity with percentages close to 80%. These two compounds differ only by one methylene leading to the same accessibility of the azide terminal group.

Figure 3 .Figure 3 .Figure 3 .

 333 Figure 3.47. A histogram of the percentages reacted azide for clicked SC-SAM-a2 and SC-SAM-a3 Figure 3.48 shows a comparison between the percentages of reacted azide after clicking SAM-a2 and SAM-a3 prepared using the immersion and the spin coating methods. It is clear that both methods lead to the same reactivity around 80% for compounds a2 and a3.

4. 1 .

 1 IntroductionMixed-SAMs is a common procedure to form SAMs containing two (rarely more) different molecules in the spacer chain or the functional end group. The classical concept of mixed SAMs is shown in figure4.1, these mixed SAMs contain two types of self-assembling molecules and each molecule has the three main units: the head group, the spacer and the terminal functional group. The only difference between those molecules is the nature of the terminal functional group, or in some cases the length of the spacer also. The purpose of mixing could be to increase the terminal group reactivity by spacing the target-binding functional groups with non-reacting terminal groups, because the limitations in terms of reactivity are often attributed to crowdedness and steric hindrance in the close-packed reactive terminal groups. Another purpose of mixed SAMs is to provide multi-functional SAMs incorporating different reactive terminal groups and thus allowing chemoselective reactions on the functional surface. Finally, it could be a way to control the overall hydrophilicity of the surface[1].

Figure 4 . 1 .

 41 Figure 4.1. Schematic representation of the classical mixed SAMs concept, x, y = 0,1, 2, 3, 4, etc.

Figure 4 .Figure 4 . 4 .

 444 Figure 4.3. a) Schematic representation of the two models of mixed SAMs: the classical concept of mixed I-SAM-a1-50% and the new concept of free I-SAM-a1-50%. b) The PM-IRRAS analysis of mixed I-SAM-a1-50% and free I-SAM-a1-50%

  but the family of compounds d is new. The synthesis of compounds d family follows the same protocol than the one used in chapter 3, by coupling two parts: the isocyanate reagent and an amine derivative as shown in figure 4.5. The multistep synthesis of compounds (1-4) has already been described in chapter 3. Then, compound 4 was condensed with the commercially available isocyanates in the presence of a) b)

Figure 4 .Figure 4

 44 Figure 4.6. a) Schematic representation of the mixed SAMs prepared by compound c1 and compound d2. b) PM-IRRAS spectra of I-SAM-c1-100% and I-SAM-c1-75% prepared by the immersion method in toluene. c) AFM height image (5µm X 5µm) of I-SAM-c1-100%

Figure 4 . 8 .

 48 Figure 4.8. PM-IRRAS spectra of SC-SAM-c1-100% and SC-SAM-c1-50% prepared by the spin coating method at different drying times: a) 30 min b) 3 hours c) 24 hours

Figure 4 . 9 .

 49 Figure 4.9. PM-IRRAS spectrum of the spin coating of compound d2 in toluene for 3 hours drying time

Figure 4 .

 4 Figure 4.10. PM-IRRAS spectra of the spin coating of 50% mixed SAMs in toluene after and before washing: a) SC-SAM-c1-50% and SC-SAM-c2-50% AFM analysis has also been applied to SAMs after washing to see if removing compound d2 could create some holes. The initial surface is SiO2/Au of 0.90 nm roughness, while the roughness of SC-SAM-c1-50% is 1.19 nm and SC-SAM-c2-50% is 0.88 nm. As observed in figure 4.11, Both surfaces are smooth without any defaults such as holes. The absence of holes leads to conclude that there is no molecules segregation during SAMs formation, and

Figure 4 .

 4 Figure 4.11. AFM height images of a) SC-SAM-c1-50% (500 nm image) b) SC-SAM-c1-50% (2 µm image) c) SC-SAM-c2-50% (500 nm image) d) SC-SAM-c2-50% (2 µm image)

Figure 5 . 2 .

 52 Figure 5.2. PM-IRRAS spectra of the PF-I-SAM-N3 tested to the immobilisation of GNLs without the copper sulfate / sodium ascorbate catalytic system

Figure 5 . 3 .

 53 Figure 5.3. The IR spectrum of GNLs

Figure 5 . 4 .

 54 Figure 5.4. PM-IRRAS spectra of the immobilisation of GNLs to PF-I-SAM-N3 via the CuAAC reaction

Figure 5 . 5 .Figure 5 .Figure 5 . 6 .

 55556 Figure 5.5. PM-IRRAS spectra of the immobilisation of GNLs via the CuAAC reaction onto a) I-SAM-a1 and b) I-SAM-b1 Figure 5.6 presents a comparison of the three types of SAMs clicked by GNLs. The differences could be originally resulted from different azide intensities and orientations in the three SAM-N3 before click, leading to have differences in GNLs quantity on the surfaces, and those differences create different nature of molecular interactions for GNLs. Based on amide I ofGNLs at 1698 cm -1 , the quantity of GNL immobilised is higher when using I-SAM-b1 compared to I-SAM-a1. While PF-I-SAM-N3 leads to the smallest quantity of immobilised GNL onto the surface. Those results are consistent with the previous results concerning the previous click reaction results by using the reagents (nitro probe and heptyne), the higher intensity of azide on the surface leads to more clicked biomolecules to the surface.

Figure 5 . 7 .

 57 Figure 5.7. Stem cells growth stages: adhesion, proliferation, and differentiation

  surfaces which are not transparent, and all SAMs discussed previously in this chapter are SAMs formed on SiO2/Au or wafer. But for this part, we applied the surface functionalisation of transparent cover glass (squares) in parallel to each SAMs formed on SiO2/Au to enable cells observations by the microscopy. Thus, we have three platforms: transparent cover glass (squares), PF-I-SAM-N3 on transparent glass, and PF-I-SAM-N3 on transparent glass clicked with GNLs. These tests have been done by Dr. Hélène Boeuf (INSERM, Bordeaux). She provided two types of stem cells to be used in these tests [21] [22] [23] : 1) mESC: murine embryonic stem cells (a paradigm of naive pluripotent stem cells)2) hiPSC: human iPSC (induced pluripotent stem cells) requirements to investigate further the properties of these new chemical surfaces (sterility, no toxicity, good cell adhesion and efficient proliferation) were properly obtained for both stem cell types. This test has been applied just once due to time limitations, but it important in the future to check again the reproducibility of those data.

Figure 5 . 9 .

 59 Figure 5.9. Photos of mESC and hiPSC after one-week growth on three surfaces: glass, PF-I-SAM-N3, and PF-I-SAM-N3 clicked with GNLs

(

  CaH2 for triethyl amine reagent, CH2Cl2, and CHCl3) and distilled under an inert atmosphere immediately before use. Karstedt's catalyst was purchased from Aldrich as a solution in xylene Pt~ 2% (ref: 479519). Trimethoxysilane was distilled under an inert atmosphere immediately before use. Reactions were monitored by thin-layer chromatography carried out on silica gel aluminium sheets (60F-254) using UV light as a visualizing agent or a solution of potassium permanganate and heat as developing agent. Column chromatography was performed on VWR silica gel 60 (40-63 μm).

  C NMR (CDCl3, 75 MHz) δ (ppm): 51.4, 40.3, 28.7-26.1 (6C) Synthesis of 10-isocyanatodec-1-ene (compound 5)

  H NMR (CDCl3, 300 MHz) δ (ppm): 5.87-5.72 (m, 1H, CH=) ; 5.03-4.88 (m, 2H, CH2=) ; 3.27 (t, J = 6.7 Hz, 2H, CH2N) ; 2.04 (q, J = 7.0 Hz, 2H, CH2) ; 1.67-1.54 (m, 2H, CH2) ; 1.45-1.23 (m, 10H, 5 CH2). 13 C NMR (CDCl3, 75 MHz) δ (ppm): 139.2, 121.8 (NCO) 114.3, 43.1, 33.9 , 31.4-26.7 (6C).

3 -

 3 Chlorpropylamine hydrochloride (2.0 g, 15.4 mmol, 1 eq.) was added to a solution of sodium azide (4.0 g, 61.6 mmol, 4 eq.) in 30 mL of water and heated for 75 °C for 1 night. Then the reaction mixture was let to go for ambient temperature and basified by potassium hydroxide (pH>10) and extracted with ether (1x 40 mL, 2 x 30 mL, 2 x 20 mL). The organic extracts were dried over MgSO4, filtered and condensed under reduced pressure to afford pale yellow volatile oil (0.87 g, 57% yield).1 H NMR (CDCl3, 300 MHz) δ (ppm): 3.26 (t, J = 6.7 Hz, 2H, CH2N3), 2.85-2.75 (m, 2H, CH2NH),1.78-1.66 (m, 2H, CH2), 1.5 (s, 2H, NH2).13 C NMR (75.4 MHz, CDCl3) δ (ppm): 48.6 (CH2N3), 38.8 (CH2NH), 31.9 (CH2).

  1 H NMR (CDCl3, 300 MHz) δ (ppm): 3.19 (t, J = 5.7 Hz 2H, CH2N3), 2.70 (t, J = 5.7 Hz, 2H, CH2NH), 1.33 (s, 2H, NH2).13 C NMR (75.4 MHz, CDCl3) δ (ppm): 54.3 (CH2N3) ,41.0 (CH2NH).

Synthesis of 1 -( 8 -

 18 azidooctyl)-3-(dec-9-en-1-yl) urea (a1 precursor).

  1.2.1. Non-covalent immobilisation .................................................................................................. 1.2.1.1. Physical adsorption................................................................................................................... 1.2.1.2. Affinity recognition .................................................................................................................. 1.2.2. Covalent immobilisation ......................................................................................................... 1.2.2.1. Using native functions ..............................................................................................................

1.2.2.2. Using non-native functions ...................................................................................................... 1.3. Self-Assembled Monolayers ......................................................................................................... 1.3.1. Self-Assembled Monolayers definition ................................................................................... 1.3.2. Coupling agents and SAMs types ........................................................................................... 1.3.3. SAMs formation onto SiO2 surfaces ....................................................................................... 1.3.4. Parameters influencing the SAMs formation .......................................................................... 1.3.4.1. Deposition conditions ......................................................................................................... 1.3.4.1.1. Water content and solvent polarity ........................................................................................ 1.3.4.1.2. Temperature........................................................................................................................... 1.3.4.1.3. Coupling agent solution (Concentration and age) ................................................................. 1.3.4.2. Coupling agent structure .................................................................................................... 1.3.4.2.1. Head group ............................................................................................................................ 1.3.4.2.2. Spacer .................................................................................................................................... 1.3.4.2.3. Terminal group ...................................................................................................................... 1.3.5. Azide terminated SAMs .......................................................................................................... 1.3.6. Mixed SAMs ........................................................................................................................... 1.4. SAMs preparation methods ......................................................................................................... 1.4.1. Langmuir-Blodgett layers ....................................................................................................... 1.4.2. Lithography ............................................................................................................................ 1.4.3. Chemical Vapour deposition method ...................................................................................... 1.4.4. Immersion solution method ..................................................................................................... 1.4.5. Spin coating ............................................................................................................................. 1.5. SAMs characterisation methods ................................................................................................. 1.5.1. Atomic Force Microscopy (AFM)........................................................................................... 1.5.2. X-ray Photoelectron Spectroscopy (XPS) ............................................................................... 1.5.3. Polarisation-Modulation Infrared Reflection-Absorption Spectroscopy (PM-IRRAS) ..........

1.3.2. Coupling agents and SAMs types

	Molecules able to form SAMs are called coupling agents. They have a specific structure made
	up of three parts as shown in figure 1.8: the head group (called also the anchoring group) , the
	spacer, and the terminal-functional group [13] [70]. The head-group links the coupling agent to
	the substrate's surface through a strong bond [71] [72]. The second part of the coupling agents
	is the spacer, which ensures the self-assembly phenomenon to control the packing/ordering of
	the molecules on the surface by different types of non-covalent interactions like Van-der-Waals,
	H-bonding and - interactions. The most common spacer used is an alkyl chain which
	provides the stability of the monolayer through van der Waals interactions, but other spacers
	have been reported to be used to enhance monolayers stability like amide, urea, sulfone,
	aromatic ring, diacetylene [72] [73]. Finally, the terminal group determines the
	functionalisation of the surface. It confers specific properties to the surface (hydrophilic vs.
	hydrophobic) and can also be used for further modification of the surface by grafting different
	molecules such as biomolecules or nanostructures by weak interactions or covalent bonds [74]

Table 1 .

 1 1. Examples of substrates and precursors commonly used for SAMs

formation Substrate Nature of the anchoring group Binding with substrate

	Au	R-SH (thiol)	RS-Au
	Au	Ar-SH (thiol)	ArS-Au
	Au	RS-SR (disulfide)	RS-Au
	Au	R-SR (sulfide)	RS-Au
	Si/SiO2, glass, Al2O3, and TiO2	R-SiX3 (organosilanes)	RSi-OSi (siloxane)
	Metal oxides (e.g., Al2O3, SnO2, TiO2)	RCOOH (carboxyl)	RCOO-…. Metal oxides
	Metal oxides and silicon	R-PO(OH)2	RP-O-Metal

R represents aliphatic or aromatic hydrocarbons with various active head groups X is (Cl, OCH3, OCH2CH3)

Table 1 .

 1 2. Most common surface analysis techniques[3]

	Surface characterisation techniques	Provided information
	Contact Angle Goniometry	Wetting properties
	ATR-IR Spectroscopy	Functional groups, orientation
	Polarisation modulation-infrared reflection-
		Functional groups, orientation
	adsorption spectroscopy (PM-IRRAS)
	Ellipsometry	Film thickness; refraction index
	X-ray Photoelectron Spectroscopy (XPS)	Elemental composition
	Ion Scattering Spectroscopy (ISS)	Elemental composition
	Secondary Ion Mass Spectrometry (SIMS)	Molecular composition
	Atomic Force Microscopy (AFM)	Morphology, structure, packing
	Scanning Tunneling Microscopy (STM)	Morphology, packing, structure

Table 1 .

 1 3. Water contact angle measured on different functional surface group of SAMs[START_REF] Aswal | Self assembled monolayers on silicon for molecular electronics[END_REF]

	Functional group	Contact angle (•)	Head group	Contact angle (•)
	CH3 ــ (CH2) n (n > 10)	110-117	ــ SO2	50
	CH3 ــ (CH2) n (n < 10)	97-108	ــ SO3H	30
	CH2 =CH ــ (CH2) n (20	95-105	ــ SCN	73-75
	> n > 10)
	ــ thiophene; ــ phenyl	90-92	ــ OH; B(OH)2; ــCONH2;	<15
			ــ CO2H
	ــ pyridyl	43	ــ NH2	36
	ــ CO2CH3; ــ	73-75	ــ NH3 +	42
	CO2CH2CH3; ــ CO2-
	benzyle; ــ CO2-
	thiophene; ــ CO2-
	anthracene;
	ــ X (Cl, Br, I)	80-89	ــ CN	68-74
	ــ SH; ــ S; ــ S ــ S	65-71	ــ N3	75-79

Table 3 .

 3 1. Summary of data (estimated intensities of bands) obtained by PM-IRRAS simulated spectra for all azide-terminated ureido SAMs n is the sum of carbon atoms in the spacer chain of both urea sides

	Coupling agent	n *	ѵaCH2	ѵN3	Rth	IAI *	IAII *
					(I(ѵN3)/I(ѵaCH2)) *
	a1: Si-C10-U-C8-N3	18	0.0062	0.0033	0.53	0.0076	0.0042
	a2: Si-C10-U-C3-N3	13	0.0039	0.0026	0.66	0.0031	0.0030
	a3: Si-C10-U-C2-N3	12	0.0035	0.0025	0.71	0.0029	0.0030
	b1: Si-C3-U-C8-N3	11	0.0034	0.0028	0.82	0.0028	0.0027
	b2: Si-C3-U-C3-N3	6	0.0014	0.0021	1.50	0.0022	0.0022

*

* I(ѵN3)/I(ѵaCH2) is the intensity of ѵN3 divided by the intensity of ѵaCH2 * IAI is the intensity of amide I and IAII is the intensity of amide II

Table 3 .

 3 2. Summary of data obtained by AFM, and contact angle measurements of all ureido SAMs prepared by the immersion method in toluene, acetonitrile, and chloroform

	Coupling agent	C.A. T * A *	C *	T *	A *	C *
	a1: Si-C 10 -U-C 8 -N 3	63	60	64	0.98	0.40	1.4
	a2: Si-C10-U-C3-N3	65	66	62	1.08	0.56	2.63
	a3: Si-C10-U-C2-N3	65	55	58	0.89	0.43	2.36
	b1: Si-C3-U-C8-N3	60	54	59	0.69	0.30	4.85
	b2: Si-C3-U-C3-N3	55	53	48	0.64	0.52	1.81

* (±1°) AFM * /Roughness (nm)

Table 3 .

 3 3. Summary of the intensities of νaCH2 for all ureido SAMs prepared by the immersion method in acetonitrile compared to the simulated intensities of νaCH2

Table 3 .

 3 4. Summary of the intensities of νaCH2 for all ureido SAMs prepared by the immersion method in chloroform compared to the simulated intensities of νaCH2

Table 3 .

 3 5. Summary of the intensities of νaCH2 for all ureido SAMs prepared by the immersion method in toluene compared to the simulated intensities of νaCH2

Table 3 .

 3 6. Summary of the data of obtained from the PM-IRRAS spectra for the intensity of amideΙ and amideⅡ for all ureido SAMs prepared by the immersion method in toluene, acetonitrile, and chloroform

		Acetonitrile	Chloroform	Toluene
	Coupling agent
		IAΙ *	IAⅡ *	IAΙ *	IAⅡ *	IA Ι*	IAⅡ *
		(cm -1 )	(cm -1 )	(cm -1 )	(cm -1 )	(cm -1 )	(cm -1 )
	a1: Si-C 10 -U-C 8 -N 3	0.00049	0.00087	0.00297	0.00332	0.00162	0.00177
	a2: Si-C10-U-C3-N3	0 00063	0.00089	0.00219	0.00251	0.00182	0.00169
	a3: Si-C10-U-C2-N3	0.00071	0.00045	0.00129	0.00124	0.00118	0.00147
	b1: Si-C3-U-C8-N3	0.00018	0.00068	0.00427	0.00406	0.00300	0.00327
	b2: Si-C3-U-C3-N3	0.00092	0.00121	0.00438	0.00393	0.00153	0.00145

* IAΙ

is the intensity of amide Ι band and IAⅡ is the intensity of amide Ⅱ band

Table 3 .

 3 7. Summary of data obtained by the PM-IRRAS, AFM, and contact angle measurements for all azide-terminated ureido SAMs prepared by the immersion method in toluene

Table 3 .

 3 8. Summary of the ∆ν values for all azide-terminated SAMs and the ∆ν of all azide terminated ureide compounds (molecular silanes)

Table 3 .

 3 9. Summary of data obtained by PM-IRRAS spectra, AFM, and contact angle measurements of all ureido SAMs prepared by spin coatingStarting by PM-IRRAS data, the region in 3200-2600 cm -1 displays the characteristic bands of

							Spin coating
		0.004
								a1 5h/ Chloroform
								a2 24h/Toluene
								a3 24h/Toluene
								b1 24h/Chloroform
		0.003						b2 19h/Toluene
	1-[Rp(d)/Rp(0)]	0.002
		0.001
		0.000
			3000	2800	2600	2400	2200	2000	1800	1600	1400
						Wavenumbers, cm	-1
	Coupling agent	n	C.A.	Roughness	νaCH2
	Solvent/drying time		(±1°)	(nm)		(cm-

the methylene stretching vibrations. The methylene stretching region provides specific information about the conformation and Van der Waals interactions of the alkyl chains. For alkyl chain conformation, all ureido SAMs show disordered alkyl chain, as the positions of the

1 ) νsCH2 (cm-1 ) νN3 (cm-1 ) nRexp Amide I (cm-1 ) Amide II (cm-1 ) Δν (cm-1 ) a1: Si-C10-U-C8-N3

	Chloroform/5hours	18	68	1.21	2929	2856	2098	1.65	1640	1572	68
	a2: Si-C10-U-C3-N3 Toluene/24hours	13	64	1.4	2928	2857	2098	3.76	1635	1573	62
	a3: Si-C10-U-C2-N3 Toluene/24hours	12	67	1.61	2928	2857	2103	4.54	1644	1573	71
	b1: Si-C3-U-C8-N3 Chloroform/24 hours	11	67	0.93	2931	2858	2098	8.25	1634	1575	59
	b2: Si-C3-U-C3-N3 Toluene/19hours	6	63	1.52	2928	2856	2098	6.54	16 35	1575	60

4.1. Introduction ................................................................................................................................

Acknowledgements

The realisation of this work was only possible due to the several people's collaboration, to

Chapter 2

Azide-terminated SAMs with alkyl spacer

Preparation of alkyl azide-terminated SAMs

In the first part of this section, azide-terminated SAMs have been prepared by the postfunctionalisation strategy by using both the immersion and the spin coating methods. Secondly, direct grafting strategy has been investigated by also using both methods (the immersion and the spin coating). Then, all resulted SAMs have been analysed in PM-IRRAS, AFM, XPS, and contact angle to produce a deep study related to the properties of SAMs.

Azide-terminated SAMs prepared by the post-functionalisation strategy

In this part azide-terminated SAMs have been prepared by the commonly used postfunctionalisation method. In this method (as shown in figure 2.6), first 11bromoundecyltrimethoxysilane should be grafted onto the surface then the produced SAM-Br is substituted by NaN3 to afford SAM-N3. Two different processes were investigated to elaborate SAM-Br: the immersion method and the spin coating method.

Using the immersion method:

Two kinds of substrates were functionalised. The first type is silica-coated gold mirrors (SiO2/Au), this type has a gold layer under the silica layer (20 nm) for reflecting the polarized light in the PM-IRRAS analysis. The second type is silicon wafers for AFM analysis. At the beginning, slides have been cleaned by MilliQ water, sonicated in chloroform, and activated under UV-O3 for 30 minutes to provide highly hydrophilic surfaces devoid of organic

Chapter 3

A new family of azide-terminated ureido SAMs reaction. Then the acyl azide is heated resulting in its rearrangement (figure 3.8). At this step, the leaving group is nitrogen gas (N2), followed by the formation of the isocyanate group [28].

Synthesis of compound (a1):

This compound is the first compound of this family. Its synthesis has previously been optimised and described in detail in our laboratory [19]. This molecule is prepared by the coupling of two parts. The first part is the isocyanate part of ten-carbon chain with an alkene terminal group which is already described in the previous section. The second part is an amino part of eight-carbon chain with an azide terminal side which is prepared by multistep reactions. As shown in figure 3.9, to prepare the amino part; first, starting by 8aminooctan-1-ol reagent, the amine group was protected by Boc2O reagent to be masked from reacting with the mesyl chloride in the next step and this step produced compound 1 with a very good yield. The second step is the mesylation of the hydroxyl group by reacting the protected compound 1 with mesyl chloride in the presence of triethyl amine to produce compound 2 (98% yield). Then, in the third step, compound 2 was reacted with sodium azide, in which azide substituted the methylsulfate group (nucleophilic substitution) to produce compound 3 with a satisfactory yield of 84% over the two steps. The amine group is still protected in compound 3 and it should be deprotected just before the condensation reaction.

To deprotect the amine group, compound 3 was reacted with trifluoroacetic acid and produced compound 4. At the end, in the last step of the synthesis, compound 4 was condensed with the 10-isocyanato-1-decene in the presence of triethylamine resulting in the formation compound a1 precursor. (the detailed protocol for each step is in the experimental part). Finally, the hydrosilylation reaction is applied to compound a1 precursor, as usually preferred, just before grafting. Here the hydrosilylation reaction follows the same procedure than the one used for the alkyl azide molecules; by reacting compound a1 precursor with trimethoxysilane in the presence of Karstedt catalyst (the source of [Pt] 0 ). Here, the reaction is preferable in toluene at 60°C under inert atmosphere for 3 h. The final product of the hydrosilylation reaction is 1-(8-

Deposition of azide-terminated ureido SAMs using the immersion method in chloroform

Chloroform (the solvent of intermediated polarity) was expected to give higher intensity of the organic layer than acetonitrile and less than toluene (the solvent of the least polarity).

Surprisingly, chloroform gives the highest quantity of matter on the surface (from PM-IRRAS data in figure 3.14). Also, the intensity of νaCH2 in the resulted SAMs is higher than the simulated values for all azide-terminated ureido SAMs except for compound a3 ( table 3.4), which could be due to having some aggregations or multilayers rather than an organic monolayer on the surface. This behaviour is not easy to explain. We can hypothesise that chloroform polarity is able to remove a quantity of water layer from the substrates, which can induce hydrolysis-condensation in solution resulting in the formation of aggregates. vertical to the surface. Meanwhile, changing the grafting method is also affecting the azide orientation and more vertical azide is produced by the spin coating method.

Deposition of SC-SAM-b1 using the spin coating

This compound shows a kind of different behaviour compared to the previously discussed compounds. Figure 3.39 shows all analytical data for SC-SAM-b1 in toluene at different drying times. First, we can observe that the spin coating process is efficient to form SC-SAM-b1, which is confirmed by the appearance of all distinguished bands related to compound b1 on the surface (CH2, azide and amides). Second, the intensity of aCH2 is 0.0022 for both 3 and 24 hours, which is also less than the immersion method (0.0037) indicating having less organic matter on the surface. Surprisingly, the intensity of the azide band for 3 hours of drying time is much less than the 24 hours despite having the same intensity of aCH2 band and amides bands.

It is obvious that the azide is more vertical to the surface for the 24 h than 3 hours, which could be due to reorganisation of the azide dipole by increasing the drying time. This behaviour has been previously observed for SC-SAM-a2 and SC-SAM-a3, which is another evidence to confirm the hypothesis of the azide reorganisation by increasing drying time in the spin coating method. However, the contact angle is 63° for 3 hours and more for 24 hours (68°) and those values are slightly more than the value of 60° obtained by the immersion method. AFM analysis is done with toluene as solvent for the 24 hours drying time as it has the higher azide intensity.

However, spin coating method is optimised for each ureido coupling agent and we found that the best solvent is chloroform for compounds a1 and b1, while for compounds a2, a3 and b2 toluene is preferred. The click reaction of some spin coated azide-terminated ureido SAMs was successfully tested and showed not complete consumption of azide. For compound a2 and a3, both methods (immersion and spin coating process) lead to the same reactivity of the terminal azide group around 80%.

Both methods allow to obtain azide-terminated SAMs with a H-bonding self-assembly.

However, for the same compound the quality of this self-assembly will depend on the deposition method. By immersion, the strength of the hydrogen bond increases with the number of carbon atoms while for the spin coating process it is the opposite. In general, for both methods the compounds with the lowest number of carbon (C6) seem to be more favourable to have a vertical azide dipole.

The variation of monolayers intensities, azide intensities, azide orientation, surface hydrophilicity and topography by changing conditions in both methods (immersion and spin coating) could be a tool for future applications to control SAMs properties. Understanding the result of each change leads to have a guide or a catalogue for preparing SAMs depending on the preferred characteristics as a kind of molecular engineering of surfaces.

Chapter 4

A new concept of mixed SAMs kind of SAMs, such as the mixed nitrobenzyl ester-/methyl-terminated SAMs [6], aiming to prepare hydrogen bonding mixed SAMs and control the concentration of the reactive terminal groups in the monolayers, those mixed SAMs have been prepared using the one step deposition method. This study showed that the monolayer composition for a small mole fraction of the ester is identical to the molar ratio of the two organosilanes in the deposition solution revealing a non-preferential surface adsorption of one compound, this resulted from the presence of polar urea groups in the molecular structure of both organosilanes, allowing them to have the same ability to self-assemble by hydrogen-bonding and to have also the same affinity to the hydrophilic surfaces.

The role of the presence of polar urea groups in the molecular structure of both organosilanes could be the leading force of the distribution of organosilanes in the SAMs, this point motivated us to create a new concept of mixed SAMs (figure 4.2). We propose a new and original approach, using only one silylated compound which will simplify the process. This organosilane will be responsible for the anchorage on the surface and the urea group will ensure the self-assembly. The terminal group of interest will be carried by a non-silylated compound which will facilitate the synthesis and will also allow to integrate other functions to obtain multifunctional surfaces. Indeed, this concept has been previously investigated in our research group [7]. In this way, the effect of the length of the silylated compound will be investigated at the beginning, then the effect of changing the length of the non-silylated compound d will be studied depending on the first part results. To summarise, in this chapter we will apply the new concept of mixed SAMs by using two models of different lengths between the urea function and the silicon atom, in order to study the effect of this change on the flexibility of the silylated molecule to incorporate the non-silylated one in the mixed SAMs.

Moreover, both methods: the immersion and the spin coating will be investigated to study the impact of the deposition method. The PM-IRRAS technique will be the main analysis of this chapter.

triethylamine resulting in the formation of compound d1, compound d2 and compound d3 in good yields. (the detailed protocol for each step is in the experimental part).

Formation of the mixed SAMs by the immersion method

The classical immersion protocols described for SAMs in chapter 2 and 3 are used here, and the conditions for both compounds c1 and c2 are previously optimised in the lab [10]. The pretreated surfaces have been immersed in silanes solution of 2.5*10 -4 M for 100% SAMs, while for other mixed SAMs the two compounds are mixed by the preferred percentages prior to the hydrosilylation process to have a concentration of 2.5*10 -4 M for the final immersion solution.

The immersion time and temperature were fixed to the reference procedure of each compound.

At the end of the process, SAMs were washed, dried and analysed in the PM-IRRAS. We started by 100% of compound c1 to form I-SAM-c1-100%, as a reference to notice changes in properties after mixing. For the mixed SAMs (figure 4.6a), we started by a ratio of 75%:25%

of compound c1 to compound d2 and the produced SAMs have been analysed in the PM-IRRAS. Figure 4.6b shows the spectra of I-SAM-c1-100% and I-SAM-c1-75% prepared by the immersion method in toluene. We can observe all distinguished bands: in the 3100-2700 cm -1 region characteristic of the methylene vibrations and in the 1800-1500 cm -1 region characteristic for urea groups vibrations as the amide I and amide II bands for both SAMs. The presence of those bands confirms the successful deposition of compound c1 on the surface.

The proof of successful formation of the mixed SAMs (I-SAM-c1-75%) is observing the azide band, in the 2100 cm -1 region characteristic of N3 vibrations, but it is clear that there is no azide

In this section, we will start by changing two parameters: the spacer length and the mixing percentage. We will try the silylated compound with a longer spacer to enable more flexibility of the molecules to introduce the non-silylated compounds. Compound c2 with a ten-carbon chain between the urea unit and the silicon atom will be used to form a mixed SAMs with compound d2 as shown in figure 4.7a. We started by the immersion method following the same protocol as the one applied with compound c1. For this trial, we used first a solution of 100% compound c2, and then a solution mixture of 50 % Compound c2 and 50% Compound d2 to produce I-SAM-c2-100% and I-SAM-c2-50%, respectively. Both SAMs have been analysed

in PM-IRRAS as shown in figure 4.7b. We can observe all the characteristic bands of compound c1: the methylene vibrations in the 3100-2700 cm -1 region and the amide I and amide II bands in the 3100-2700 cm -1 region. No azide band at 2100 cm -1 is visible, indicating there is no formation of the mixed SAMs in this trial also. However, the intensity of amide bands for I-SAM-c2-100% are compatible with the monolayer in contrast with the I-SAM-c1-100%, which shows that a long alkyl chain is more favourable to obtain a monolayer.

Moreover, the contact angle is 71° for both SAMs confirming that there is no change on the surface hydrophilicity, due to the absence of any new functional group to the surface. The general intensity of I-SAM-c2-50% spectrum is clearly lower (divided by two) than I-SAM-c2-100% showing that compound d2 prefers to remain in the solvent phase because it has little affinity for the surface due to its less polar molecular structure. We can conclude that even using longer silylated molecules is not producing mixed SAMs by the immersion method for this new concept. Thus, the spin coating method should be tested for this concept. GNLs are of particular interest for biomedical applications due to their intrinsic properties, in particular the non-covalent nature of the molecular interactions network (π-π stacking between the nucleobases and/or the triazole moieties, intermolecular hydrogen bond, etc). Obviously, their biological interest is closely related to their gelling properties [11]. Hydrogels are particularly attractive for drug delivery or tissue engineering applications [12] [13], whereas organogels are promising systems for designing and constructing, among others, sensors, During cells growth, the extracellular environment plays a role in regulating cell behaviour, and the overarching requirement of cell culture is to recreate the cellular micro-environment, bringing physiologic conditions to an in vitro setting that supports healthy cell growth and proliferation as well as attentive maintenance to propagate healthy cultures free of contamination [24]. At the beginning, surfaces should be sterilised prior to cell culture, and it is important to ensure the efficiency of the sterilisation process with no contamination. For the sterilisation process, all surfaced have been introduced to wells and cleaned by ethanol 20 minutes, then washed 4 times with sterile H2O and air dried for 2 hours. After one week, no contamination was observed, indicating that the sterilisation process is efficient. Then, all surfaces in wells have been coated with 0.2% gelatine for mESC or 1µg/mL Matrigel (for hiPSC) and seeded with 25 000 cells (figure 5.8). Cells were grown for a week in their respective cell growth medium: cell medium containing leukaemia inhibitory factor (LIF) for mESC and complete medium specific for hiPSC, with medium change every other day.

General conclusion

General conclusion

The combination of both SAMs and biomolecules immobilisation opens the possibility to create functional structured substrates, which might be used in bioapplications such as tissue engineering, cell-based assay or biosensors, etc. The control of both orientation and distribution of the biomolecules on the artificial surface is a great challenge in this field. Moreover, factors influencing biomolecules immobilisation on a solid surface are numerous and among them, the chemistry and the topographical properties of the surface are of paramount importance. One class of molecules, which attracted significant attention during the last decades, are silane-based self-assembled monolayers on hydroxyl terminated substrates, e.g. silicon and glass. These systems are physically and chemically robust and can be applied in various fields of technology which relies on further modification.

In this thesis, we designed self-assembled monolayers (SAMs) with azide terminal groups on silica surfaces and evaluate their structural characteristics, as well as their performance for biomolecules immobilisation using the Cu(I)-catalysed 1,3-dipolar azide-alkyne cycloaddition (CuAAC). Two types of azide-terminated SAMs have been prepared: SAMs bearing an alkyl spacer and SAMs bearing a urea spacer.

Azide-terminated SAMs bearing an alkyl spacer have been deposited successfully by using three different deposition methods (post functionalisation and direct grafting by immersion as well as spin coating) in three different solvents (acetonitrile, toluene and chloroform). Chemical modifications of the surfaces have been investigated using Polarisation Modulation Infrared Reflection Adsorption Spectroscopy (PM-IRRAS) and X-ray Photoelectron Spectroscopy (XPS). Atomic Force Microscopy (AFM) was also used to image the surface topography. We found that changing the grafting solvent could affect the quantity of organic matter within the monolayer and the azide orientation as well as changing the deposition method. Moreover, we concluded that the reactivity of the azido group on the surface with the alkyne in solution is not trivial and seems to be closely related to the orientation of the azide. Indeed, more the azide is vertically oriented more it is accessible and reactive. The orientation of azido dipoles at the surface depends strongly on the method used to prepare the monolayer. The postfunctionalisation method allows to have azide groups on the surface with a better vertical orientation than that obtained using direct grafting by immersion or spin coating processes.

Whatever the type of azide-terminated SAMs, the reactivity of the accessible vertical azido Vellutini, 2012).The PM-IRRAS spectra were recorded at a resolution of 4 cm -1 during 4 h acquisition time. The PM-IRRAS spectra were calibrated in order to be presented in IRRAS units [3] (Z. Li, C. N. Weeraman, J. M. Gibbs-Davis, 2014). All spectra were collected in a dryair atmosphere after 30 min of incubation in the chamber.

X-ray Photoelectron Spectroscopy (XPS) experiments:

XPS analyses were performed using an Omicron Argus X-ray photoelectron spectrometer, equipped with a monochromated AlKα radiation source (hν = 1486.6 eV) and a 280 W electron beam power. The emission of photoelectrons from the sample was analyzed at a take-off angle of 45° under ultra-high vacuum conditions (≤ 10 -10 Torr). For the molecular compounds (powders embedded in an indium foil) a flood gun was used in order to compensate the charging effect of the insulating powder.

Spectra were recorded with a 100 eV pass energy for the survey scan and 20 eV pass energy for the C1s, O1s, N1s, Si2p regions. Binding energies were calibrated against the C1s binding energy at 285.0 eV and element peak intensities were corrected by Scofield factors. The peak areas were determined after subtraction of a Shirley background. The spectra were fitted using Casa XPS v.2.3.15 software (Casa Software Ltd., U.K.) and applying a Gaussian/Lorentzian ratio G/L equal to 70/30.

Synthesis of Reagents Synthesis of 11-azidoundecene

Sodium azide (1.34 g, 1.3 eq., 22.29 mmol) was added to a solution of 11-bromoundecene (4 g, 17.15 mmol) in DMF (40 mL). After vigorous stirring for 6 hours at ambient temperature, the precipitate was removed by filtration and the organic phase was then concentrated under reduced pressure. Water (30 mL) was added to the residue and then extracted with pentane ( 4x 30 mL). The combined organic phases were dried over MgSO4, filtered and concentrated under reduced pressure to afford a colourless oil (3.5 g, 80 % yield).

Synthesis of tert-butyl N-(8-hydroxyoctyl)carbamate (compound 1)

This compound was synthesised following the protocol in reference [6].

A solution of Boc2O (1.8 g, 8.2 mmol, 1.2 eq in 10 mL dichloromethane) was added dropwise to a solution of 8-aminooctan-1-ol (1.0 g, 6.8 mmol in 10 mL dichloromethane) at 0°C, and stirred for 20 hours at ambient temperature. Then the mixture was concentrated under reduced pressure and the residue was purified by chromatography over silica gel (cyclohexane / ethyl acetate 50/50) to afford a white solid (1.5 g, 93% yield). Rf=0.

Synthesis of 8-((tert-butoxycarbonyl) amino)octyl methanesulfonate ( compound 2)

This compound was synthesised following the protocol in reference (Y. Mousli, L; Rouvière, I. Traboulsi, J. Hunel, T. Buffeteau, K. Heuzé, L. Vellutini, E. Genin, 2018)

Compound 1 (0.7 g, 2.85 mmol) was dissolved in 20 mL of dichloromethane, then triethylamine (0.59 mL, 4.28 mmol, 1.5 eq) and mesyl chloride (0.24 mL, 3.14mmol, 1.1 eq) were added respectively at 0°C and stirred for 20 hours at ambient temperature. At the end of reaction, the mixture was diluted by 10 mL dichloromethane, washed with brine (4x 10 mL), dried over MgSO4, filtered, and concentrated under reduced pressure to afford yellow oil (0.9 g, 98% yield). 1

Synthesis of N-tert-butyl (8-azidooctyl)carbamate (compound 3)

This compound was synthesised following the protocol in reference [6].

Sodium azide (0.55 g, 8.45 mmol, 3 eq.) was added to a solution of compound 2 (0.9 mg, 2.83 mmol in 25 mL of anhydrous DMF) under inert atmosphere, and stirred for 36 hours at ambient temperature. At the end, brine (50 mL) was added. Then the mixture was extracted with dichloromethane (3x 50 mL). the combined organic extracts were washed with brine (2x 50 mL), dried over MgSO4, filtered and concentrated under reduced pressure. DMF traces were removed by co-evaporation under reduced pressure with toluene (2x 15 mL) to obtain the product as a slightly yellow oil (0.64 g, 84% yield). 3-azidopropan-1-amine (compound 6) (0.33 g, 3.3 mmol, 1eq.) was dissolved in 60 mL of CH2Cl2, followed by dropwise addition of 3-isocyanatoprop-1-ene (0.34 mg, 3.9 mmol, 1.2 eq).

The mixture was stirred for 1 night at ambient temperature under inert atmosphere. Then solvent was removed under reduced pressure and the residue was purified by chromatography over silica gel (cyclohexane / ethyl acetate 20/80) to afford pale yellow oil (0.53 g, 88 % yield). A solution of 11-azidoundecene (0.16 g, 1 eq., 0.82 mmol) and 1-heptyne (0.16 g, 2 eq., 1.64 mmol) in THF was added to a solution of CuSO4.5H2O (0.019 g, 0.15 eq., 0.12 mmol) and sodium ascorbate (0.071 g, 0.45 eq., 0.37 mmol) in water and the reaction mixture was stirred at ambient temperature for 24 hours. After completion of the reaction, ammonium chloride (10 mL) was added to the whole reaction mixture and the product was extracted with ethyl acetate (4 x 30 mL). The combined organic phases were dried over MgSO4, filtered and concentrated under reduced pressure. Then, the product was purified by column chromatography on silica gel (ethyl acetate/cyclohexane, 10/90) to afford a white solid (0.21 g, 56 % yield) [9]. 1

Ⅱ. Formation of the Self-Assembled Monolayers (SAMs) by the spin coating method

Freshly prepared hydrosilylated self-assembling molecule was dissolved in dry solvent under inert atmosphere to prepare a solution at 4 x 10 -3 M. 40 µL of this freshly prepared solution was used to spin-coat the substrate (rotated at 6000 rpm for 40 seconds). The sample was dried at ambient temperature for 30 min in a laminar flow hood, then washed by sonicating in solvent (1 x 5 min), chloroform (1 x 5 min) and dried under nitrogen flow.

Ⅲ. Post-functionalisation of SAM-Br to SAM-N3

The bromine terminated monolayers were immersed in a saturated solution of sodium azide in dry DMF for 48 h at room temperature under argon atmosphere. The substrates were subsequently sonicated for several minutes in DMF, water and chloroform, respectively, and dried under nitrogen flow.

Click reaction of azide-terminated SAMs and alkynes

1,3-dipolar cycloaddition reactions were carried out in a steriplan soda-lime petri dish (diameter: 60 mm; height: 15 mm) under inert atmosphere by immersing the azide-terminated substrate in a mixture of degassed Milli-Q water (6.36 mL) and degassed DMSO (1.