Whole-cell sensors represent an emerging branch in biosensor development since they obviate the need for enzyme/antibody purification and provide the unique opportunity to assess global parameters such as genotoxicity and bioavailability. Yeast species such as Saccharomyces cerevisiae are ideal hosts for whole-cell sensor applications. However, current approaches almost exclusively rely on analyte-induced expression of fluorescent proteins or luciferases that imply issues with light scattering and/or require the supply of additional substrates. In this study, the yeast α-factor mating pheromone, a peptide pheromone involved in cell-cell communication in Saccharomyces cerevisiae, was utilized to create the whole-cell sensor read-out signal, in particular by employing engineered sensor cells that couple the response to a user-defined environmental signal to α-factor secretion. Two novel immunoassays - relying on hydrophobin-based surface engineering - were developed to quantify the α-factor. Hydrophobins are amphiphilic fungal proteins that self-assemble into robust monolayers at hydrophobic surfaces. Two recombinant hydrophobins, either lacking (EAS) or exposing the α-factor pheromone (EAS-α) upon self-assembly, were used to functionalize polystyrene supports. In a first approach (competitive immunoassay), pheromone-specific antibodies initially bound to the functionalized surface (due to the α-factor exposed by the hydrophobin layer) were competitively detached by soluble α-factor. In a second approach, the antibodies were first premixed with pheromone-containing samples and subsequently applied to functionalized surfaces, allowing for the attachment of antibodies that still carried available binding sites (inverse immunoassay). Both immunoassays enabled quantitative assessment of the yeast pheromone in a unique but partially overlapping dynamic range and allowed for facile tuning of the assay sensitivity by adjustment of the EAS-α content of the hydrophobin layer. With a limit of detection of 0.1 nM α-factor, the inverse immunoassay proved to be the most sensitive pheromone quantification assay currently available. Due to the high stability of hydrophobin monolayers, functionalized surfaces could be reused for multiple consecutive measurements. Favorably, both immunoassays proved to be largely robust against the changes in the sample matrix composition, allowing for pheromone quantification in complex sample matrices such as yeast culture supernatants. Hence, these immunoassays could also be applied to study the pheromone secretion of wild-type and engineered Saccharomyces cerevisiae strains. Additionally, a proof-of-concept whole-cell sensor for thiamine was developed by combining the hydrophobin-based immunoassays with engineered sensor cells of Schizosaccharomyces pombe modulating the secretion of the α-factor pheromone in response to thiamine. Since this read-out strategy encompasses intrinsic signal amplification and enables flexible choice of the transducer element, it could contribute to the development of miniaturized, portable whole-cell sensors for on-site application.

Smart surfaces are a source of innovation in the 21^st Century. Potential applications can be found in a wide range of fields where improved optical, mechanical or biological properties can enhance the functions of products. In the last years, a method called Direct LaserInterference Patterning (DLIP) has demonstrated to be capable of fabricating a wide range of periodic surface patterns even with resolution at the nanometer and sub-micrometer scales. This article describes recent advances of the DLIP method to process 2D and 3D parts. Firstly, the possibility to fabricate periodic arrays on metallic substrates with sub-micrometer resolution is shown. After that, different concepts to process three dimensional parts are shown, including the use of Cartesian translational stages as well as an industrial robot arm. Finally, some application examples aredescribed.

Starting from a simple concept, transferring the shape of an interference pattern directly to the surface of a material, the method of Direct Laser Interference Patterning (DLIP) has been continuously developed in the last 20 years. From lamppumped to high power diode-pumped lasers, DLIP permits today for the achievement of impressive processing speeds even close to 1 m²/min. The objective: to improve the erformance of surfaces by the use of periodically ordered microand nanostructures. This study describes 20 years of evolution of the DLIP method in Germany. From the structuring of thin metallic films to bulk materials using nano- and picosecond laser systems, going through different optical setups and industrial systems which have been recently developed. Several technological applications are discussed and summarized in this article including: surface micro-metallurgy, tribology, electrical connectors, biological interfaces, thin film organic solar cells and electrodes as well as decorative elements and safety features. In all cases, DLIP has not only shown to provide outstanding surface properties but also outstanding economic advantages compared to traditional methods.

Periodic surfaces structures with micrometer or submicrometer resolution produced on the surface of components can be used to improve their mechanical, biological or optical properties. In particular, these surfaces can control the tribological performance of parts, for instance in the automotive industry. In the recent years, substantial efforts have been made to develop new technologies capable to produce functionalized surfaces. One of these technologies is Direct Laser Interference Patterning (DLIP), which permits to combine high fabrication speed with high resolution even in the sub-micrometer range. In DLIP, a laser beam is split into two or more coherent beams which are guided to interfere on the work piece surface. This causes modulated laser intensities over the component’s surface, enabling the direct fabrication of a periodic pattern based on selective laser ablation or melting. Depending on the angle between the laser beams and the wavelength of the laser, the pattern’s spatial period can be perfectly controlled. In this study, we introduce new modular DLIP processing heads, developed at the Fraunhofer IWS and the Technische Universität Dresden for high speed surface laser patterning of polymers and metals. For the first time it is shown that effective patterning speeds of up to 0.90 m2/min and 0.36 m²/min are possible on polymer and metals, respectively. Line- and dot-like surface architectures with spatial periods between 7 μm and 22 μm are shown.

L’ARN polymérase dépendante d’ADN (RNAP) est une enzyme responsable de la polymérisation de ribonucleotides dans une séquence d'ARN complémentaire de l'ADN de matrice. La famille de RNAP a plusieurs membres, comme de protéines sous-unité unique (par exemple du bactériophage T7) ou multiple sous-unité (bactériennes et eucaryotes). Transcription de l'ARN - un événement crucial dans l'expression des gènes - varie en fonction de l'origine de RNAP. Bien que le processus de transcription est relativement bien caractérisée, de nombreux éléments restent mal compris, surtout par rapport à la dynamique de la reconnaissance de promoteur, d'évasion et de l'allongement dans une contexte de cellule où la densité moléculaire, les concentrations et les effets plus proches environs sont importants. L'objectif de cette thèse était la développement d’une méthode qui permettrait suivre la réaction RNAP in vitro en temps réel dans des conditions très contrôlées. Un axe majeur a été mis pour développer un biocapteur basé surface qui permettrait à la caractérisation des principales étapes de la réaction de transcription. Par conséquent, les interactions entre des molécules d'ADN immobilisés sur une surface du capteur et RNAP libre délivré par un système microfluidique de la surface ont été examinées. Changements de l'indice de réfraction, corrélés avec les changements de masse sur la surface ont été suivis en utilisant l'imagerie par résonance de plasmon de surface (SPRi). SPRi est une technique sensible dédiée à l'analyse des interactions entre deux ligands en temps réel. Les bases du mécanisme sont la détection de légères différences dans la réflexion de la lumière polarisée à un angle fixe qui est associé avec une variation de masse à l'interface. Les données obtenues à partir SPRi sont utilisées pour déterminer la cinétique des interactions. Géométrie d’ADN puces permet de suivre plusieurs échantillons simultanément, qui raccourcit considérablement le temps que de manipulation et améliore la qualité et la reproductibilité des résultats obtenus. Autres biocapteurs optofluidique: résonateur de microring et microscopie de fluorescence par réflexion totale interne (TIRF) ont été développés en parallèle. Nous avons biofunctionalisé et caractérisé des surfaces de capteur (de verre couvert de polymère pour un résonateur de microring et la microscopie TIRF et 50 nm couche mince d’or sur des prismes de SPRi) afin d'immobiliser ADN d'une manière contrôlée, par création d’une monocouche auto-assemblée (SAM). Fonctionnalisation de polymères SU-8 concernées deux méthodes: covalent immobilisation de (bio) molécules et la conjugaison non covalente sur la base de couplage hydrophobe. Pour la fonctionnalisation de surface d’or, quatre stratégies différentes d'immobilisation des molécules ont été comparés: formation de la liaison de thiol - or, la formation des liaisons amide, interactions extrAvidin - biotine et le couplage hydrophobe. Les études de la conjugaison de l'ADN à la surface d'or fonctionnalisé ont été effectuées en ce qui concerne la spécificité et la densité d'ADN immobilisées de longueurs différentes: 50, 500 et 1000 pb. Enfin, les surfaces biofunctionalized ont été utilisées pour suivre en temps réel des réactions de transcription de deux RNAP: bactériophage T7 RNAP monomère et l'holoenzyme d'Escherichia coli RNAP. Les analyses cinétiques de la formation d’un complexe nucléoprotéine et la transcription d'ARN ont été fait par report de la densité et la longueur de l'ADN immobilisé, la position de la séquence du promoteur spécifique. Transcription de l'ARN dans l'appareil SPRi a été confirmée par la collection, la détection et l'analyse des produits ARN.L'objectif final comprends une synthèse de l'ARN contrôlée qui serait une étape intermédiaire d'enquêter en temps réel la production de protéines in vitro. / DNA-dependent RNA polymerase (RNAP) is an enzyme responsible for the polymerization of ribonucleotides into an RNA sequence complementary to the template DNA. RNAP family has several members being single subunit (e.g. T7 bacteriophage) or multi subunit (bacterial and eukaryote) proteins. RNA transcription – a crucial event in gene expression – differs depending on the RNAP origin. Although the transcription process is relatively well characterized, many elements remain poorly understood, especially with respect to the dynamics of promoter recognition, escape and elongation in a cell like context where molecular density, concentrations and nearest neighbour effects are prevalent.The goal of this thesis was to develop a robust method that would allow real time monitoring of RNAP reaction in vitro in thoroughly controlled conditions. A major axis was to develop a surface-based biosensor that would allow the characterization of the main steps of the transcription reaction. Consequently, interactions between DNA molecules immobilized on a sensor surface and free RNAP delivered through a microfluidic flow system to the surface were examined. Changes in refractive index, correlated with changes in mass at a surface were followed using surface plasmon resonance imaging (SPRi). SPRi is a sensitive technique dedicated to analysis of interactions between two ligands in real time. The mechanism bases on the detection of slight differences in the reflectivity of polarized light at a fixed angle that are associated with a mass variation at the interface. Data obtained from SPRi are used to determine the kinetics of the interactions. Microarray geometry of SPRi allows monitoring several samples simultaneously that significantly shortens manipulation time and improves a quality and reproducibility of obtained results. Other label-free optofluidic biosensors: microring resonator and total internal reflection fluorescence (TIRF) microscopy were developed in parallel.We firstly biofunctionalized and characterized sensor surfaces (polymer coated glass for microring resonator and TIRF microscopy and 50-nm thin layer gold coatings on glass prisms for SPRi) in order to immobilize DNA strands in a controlled manner, using a self-assembled monolayer (SAM). Functionalization of photoresist polymer SU-8 concerned two methods: covalent (bio)molecule grafting and non-covalent conjugation based on hydrophobic coupling. Regarding gold surface functionalization, four different strategies of antifouling (bio)molecule immobilization were compared: thiol – gold bond formation, amide bond formation, extrAvidin – biotin interactions and hydrophobic coupling. Studies of DNA conjugation to the functionalized gold surface were performed with respect to specificity and density of immobilized DNA molecules of different lengths: 50, 500 and 1000 bp.Finally, biofunctionalized surfaces were used for real time monitoring of transcription reactions using two RNAPs: monomeric bacteriophage T7 RNAP and the holoenzyme of Escherichia coli RNAP. Kinetic analyses of nucleoprotein complex formation and RNA transcription were performed as a function of immobilized DNA density, the length of the immobilized DNA, the position of the specific promoter sequence with respect to the point of immobilization and the direction of subsequent transcription. RNA transcription in the SPRi apparatus was confirmed by collection, detection and analysis of relevant products.The future development of biosensors dedicated to in vitro gene expression will include the adaptation of the methods presented above to other optofluidic systems and further development of the technique. The final goal comprises a controlled RNA synthesis that would be an intermediate step to investigate real time in vitro protein production.

En vue de vectoriser de façon ciblée des microcapsules de parfum vers un substrat textile pour des applications lessivielles, ce travail de thèse s’est consacré à la modification chimique de la surface de ces microcapsules en milieu aqueux par des polysaccharides ayant des affinités particulières pour les substrats de cellulose (agent d’aide au dépôt). Pour ce faire, une approche mettant en jeu des fonctionnalités époxy a été développée en deux étapes : fonctionnalisation de la surface des microcapsules par des molécules relais, puis greffage covalent d’un polysaccharide via la fonctionnalité époxy. Après chaque étape de greffage covalent en surface des microcapsules, différentes stratégies de caractérisations ont été mises en place (spectroscopies infrarouge, RAMAN, RMN du solide, XPS, ATG, mesure du potentiel zêta, gravimétrie, microscopie optique et fluorescente). Des expériences témoins ont également été réalisées pour prouver la non-adsorption des greffons de surface sur les microcapsules. La synthèse et le greffage de polysaccharides marqués avec des sondes fluorescente, alcyne et méthacrylate nous ont également permis d’appuyer nos conclusions. Afin d’envisager des modifications chimiques en milieu aqueux, la stabilité des composés époxy dans l’eau a dû être étudiée de façon précise par spectroscopie RMN en solution et nous avons abouti avec succès à une meilleure compréhension des phénomènes réactionnels époxy-amine et époxy-hydroxyle en milieu aqueux.Enfin, une enzyme (la lipase) a également pu être greffée de façon covalente via la fonctionnalisation époxy tout en conservant son activité catalytique. / Colloidal suspensions are of paramount significance in industrial applications. They are employed in various domains like paintings, inks, pigments, pharmacology, cosmetics, food,textile, composite materials or waste water treatment. Properties of colloids strongly depend on parameters such as the chemical composition, dimensions or morphology. To confer additional features to the colloids, i.e. stability, compatibilization, targeting, stealth properties and so on, it is also crucial to tailor their surface functionalization. In this work, we intend to develop a methodology allowing for tuning the surface properties of highly cross-linked fragrance microcapsules to graft polysaccharides. To do so, the first objective of this work is to identify functionalities at the surface (of the colloids) amenable to post-modifications. Based on this crucial insight, suitable surface chemistries are further explored to impart new properties to the colloids. Thus the presence of amine functions is highlighted by ninhydrine tests and then exploited to incorporate new functionalities at the surface of colloids.Incorporation of fluorescent tags (such as  Rhodamine Isothiocyanate, RITC), intermediate polymer epoxy chains (α,ω-epoxy functionalized polyethylene glycol or PGMA) are performed. Depending on the nature of the moieties to be grafted, the resulting colloids are subsequently characterized by Confocal Laser Scanning Microscopy (CLSM), FTIR, XPS, and RAMAN Spectroscopy. After this first step of functionnalization, epoxy rings at the surface are used to postgraft polysaccharides.

No description available.

Applying a thin film coating is a vital strategy to enhance long term and interface stability of Ni-rich layered oxide cathode materials (NRLOs), especially when they are matched with sulfidic solid electrolytes (SSEs) in solid-state batteries (SSBs). The coating prevents direct contact between the cathode active material (CAM) and the SSE, shielding against parasitic side reactions at the cathode electrolyte interface (CEI). Conventional coatings are based on wet-chemical methods and therefore harmful to the environment and require long-lasting processing and high costs. In this study, we present a versatile, facile and highly-scalable dry-coating method (with suitable equipment up to 500 kg per batch) successfully employed for both multiand single-crystalline LiNi₇.₀Co₀.₁₅Mn₀.₁₅O₂ (NCM70) particles by fumed Li₂ZrO₃ nanostructured particles (LZONPs) via high intensity mixing process. The resulting porous coating layer stays firmly attached at the CAM particle surface without a need of post-calcination step at elevated temperatures. The electrochemical testing results signify enhanced rate capability up to 1.5 mAcm⁻² for both particle types and cyclic stability up to 650 cycles with a capacity retention of 86.1% for singlecrystalline NCM70. We attribute the enhanced performance to the reduced CEI reactions as cathodic charge transfer resistance depressed significantly after dry-coating by LZONPs, being an important step towards sulfidic solid-state batteries.

Resolving the mismatch between the practical potential window (PPW) and the available capacitive potential window of supercapacitor electrodes provides a feasible way to expand the operating voltage of supercapacitors, which further boosts energy density. Here, our research unveils a unique approach to manually control the PPW of the corresponding carbon-based supercapacitors (CSCs) by rational functionalization with amino groups. The extra pair of electrons from amino N atoms naturally adsorbs cations in the electrolyte, which rationalizes the surface charge of the carbon electrode and adjusts the PPW. A remarkable voltage expansion is achieved for CSCs, from 1.4 V to its maximum limit, 1.8 V, correspondently resulting in an approximately 1-fold increase in the energy density. Importantly, such a simple strategy endows our CSCs with an outstanding maximum energy density of 7.7 mWh cm⁻³, which is not only among the best values reported for thin-film CSCs but also comparable to those reported for Li thin-film batteries. These encouraging results are believed to bring fundamental insights into the nature of potential control in energy storage devices.

Σήμερα η μελέτη των βιοϋλικών προσανατολίζεται σε δύο κατευθύνσεις, την ανάπτυξη συστημάτων μεταφοράς φαρμάκων και συστημάτων κατάλληλων να διεγείρουν κυτταρικές λειτουργίες. Η έρευνά μας έχει σχέση με την συγκριτική μελέτη συστημάτων μεταφοράς φαρμάκων κατάλληλων για εφαρμογή σε οστικούς καρκίνους. Τέτοιου είδους συστήματα θα πρέπει, αρχικά να είναι ικανά να μεταφέρουν τα φαρμακευτικά μόρια και στη συνέχεια να μπορούν να επάγουν οστεογένεση. Η δεύτερη λειτουργικότητα είναι ιδιαιτέρως σημαντική καθώς έχει σαν αποτέλεσμα την πλήρωση του οστικού ελλείμματος που προκαλείται από την δράση των καρκινικών κυττάρων. Για τον σκοπό αυτό, διερευνήθηκε η ικανότητα του υαλώδους δικτύου να μεταφέρει φαρμακευτικά μόρια μέσω παραδοσιακών συστημάτων μεταφοράς. Συνεπώς, ακολούθησε η ex vitro μελέτη pH-ευαίσθητων τροποποιημένων πυριτικών ξηρών πηκτών στα οποία είχε συνδεθεί το αντικαρκινικό φάρμακο δοξορουπμυσίνη. Συγκεκριμένα, πυριτικά ξηρά πηκτώματα συντέθηκαν με την μέθοδο sol-gel και τροποποιήθηκαν περαιτέρω με χημεία καρβοδιϊμιδίου. Η τροποποίηση είχε σαν αποτέλεσμα την επιφανειακή σύνδεση υδροπηκτών δεξτράνης που παρουσιάζουν ευαισθησία στο pH. Στη συνέχεια, ακολούθησε η σύνθεση των ανόργανων βιοενεργών νανοσφαιρών. Για την σύνθεση των υαλωδών νανοσφαιρών με εσωτερική κοιλότητα ακολουθήθηκε η διαδικασία επικάλυψης sol-gel, κατά την οποία έγινε η ηλεκτροστατική επικάλυψη νανοσωματιδίων πολυστυρενίου με αποτέλεσμα την σύνθεση ανόργανων πυριτικών και φωσφοπυριτικών νανοσφαιρών. Επιπλέον, μελετήθηκε η εφαρμογή του συμπολυμερούς του πολυμεθακρυλικού μεθυλεστέρα – υδρομεθακρυλικού προπυλεστέρα ως υποστρώματος καθώς το PMMA αποτελεί βασικό συστατικό των οστικών τσιμέντων και παρουσιάζει βελτιωμένες μηχανικές ιδιότητες. Προκειμένου να ολοκληρωθεί η συγκριτική μελέτη μας, ακολούθησε η ex vitro ανάλυση των παραπάνω υβριδικών φωσφοπυριτικών νανοσφαιρών καθώς επίσης των πυριτικών νανοσφαιρών PS και PMMA-co-HPMA. Η επώαση σε διάλυμα SBF οδήγησε στον σχηματισμό ανθρακικού πυριτικού υδροξυαπατίτη με το μέγεθος των κρυσταλλιτών να μην υπερβαίνει τα 45 nm και έντονη παρουσία συσσωματωμάτων. Βέλτιστες ιδιότητες βιοενεργότητας παρουσιάζουν οι τροποποιημένες με αμίνες υβριδικές νανοσφαίρες PMMA-co-HPMA, οι οποίες έχουν επίσης την δυνατότητα να χρησιμοποιηθούν ως μεταφορείς φαρμακευτικών μορίων σε όξινο καθώς επίσης και σε φυσιολογικό pH με παρατεταμένη δυνατότητα αποδέσμευσης. / Recently the study of biomaterials has moved in two directions, the evolution of drug delivery systems and of systems that can stimulate specific cellular responses. Our investigation aims to the study of drug delivery systems for bone cancer therapy. These systems must fulfill two important functionalities. At first, they should be able to deliver drug molecules to bone cancer environment through loading or surface conjugation and subsequently to cause osteogenesis. Their second functionality is especially important since it leads to substitution of bone defects caused from the action of cancer cells. For this purpose, the ability of the glassy network to deliver drug molecules was studied. For this purpose, expanded ex vitro research was followed in DOX conjugated pH-sensitive functionalized silica xerogels. Specifically, silica xerogels were synthesized through a sol-gel process and further functionalized with carbodiimide chemistry. The functionalization process resulted in pH-sensitive dextran hydrogels. The study of the enhanced properties of glassy substrates was followed by the synthesis of amorphous bioactive nanospheres. Moreover, the change of the organic core and the use of PMMA-co-HPMA were advantageous due to the enhanced mechanical properties of PMMA-co-HPMA and its use in bone cements. In order to accomplish our comparative investigation, we followed the ex vitro study of the above hybrid binary silicate, ternary and quaternary phosphosilicate nanospheres as well as the silicate PS and PMMA-co-HPMA nanospheres. The incubation in SBF solution resulted in the formation of a silica-substituted carbonate hydroxyapatite (Si-HCA) a with crystallite size of around 45 nm and extended surface aggregates. The amino-modified PMMA-co-HPMA hybrid nanospheres present enhanced biocompatible properties, with prolonged release ability as drug delivery systems, in acidic as well as physiological pH.