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In this study, we present a technique to create a complex, high cholesterol-containing supported lipid bilayers (SLBs) using α-helical (AH) peptide-induced vesicle fusion. Vesicles consisting of POPC : POPE : POPS : SM : Chol (9.35 : 19.25 : 8.25 : 18.15 : 45.00) were used to form a SLB that models the native composition of the human immunodeficiency virus-1 (HIV-1) lipid envelope. In the absence of AH peptides, these biomimetic vesicles fail to form a complete SLB. We verified and characterized AH peptide-induced vesicle fusion by quartz crystal microbalance with dissipation monitoring, neutron reflectivity, and atomic force microscopy. Successful SLB formation entailed a characteristic frequency shift of -35.4 ± 2.0 Hz and a change in dissipation energy of 1.91 ± 0.52 × 10(-6). Neutron reflectivity measurements determined the SLB thickness to be 49.9 (+1.9) -1.5 Å, and showed the SLB to be 100 (+0.0) -0.1% complete and void of residual AH peptide after washing. Atomic force microscopy imaging confirmed complete SLB formation and revealed three distinct domains with no visible defects. This vesicle fusion technique gives researchers access to a complex SLB composition with high cholesterol content and thus the ability to better recapitulate the native HIV-1 lipid membrane.

Self-assembled microsphere monolayers (SMMs) hold significant promise for micro- and nanopatterning. Here we exploit, for the first time, SMMs as stamps for microcontact printing (μCP) and demonstrate this to fabricate patterned initiator templates that can subsequently be amplified into polymer brushes by surface initiated atom transfer radical polymerization (SI-ATRP). SMM stamps avoid the need for expensive and sophisticated instrumentation in pattern generation, and provide a broad range of accessible surface chemistries and pitch size control.

Chondrocytes in articular cartilage normally exhibit high expression of collagen II and aggrecan but rapidly dedifferentiate to a fibroblastic phenotype if passaged in culture. Previous studies have suggested that the loss of chondrocyte phenotype is associated with changes in the structure of the F-actin cytoskeleton, which also controls cell mechanical properties. In this study, we examined how dedifferentiation in monolayer influences the mechanical properties of chondrocytes isolated from different zones of articular cartilage. Atomic force microscopy was used to measure the mechanical properties of superficial and middle/deep zone chondrocytes as they underwent serial passaging and subsequent growth on fibronectin-coated, micropatterned self-assembled monolayers (MSAMs) that restored a rounded cell shape in 2D culture. Chondrocytes exhibited significant increases in elastic and viscoelastic moduli with dedifferentiation in culture. These changes were only partially ameliorated by the restoration of a rounded shape on micropatterned surfaces. Furthermore, intrinsic zonal differences in cell mechanical properties were rapidly lost with passage. These findings indicate that cell mechanical properties may provide additional measures of phenotypic expression of chondrocytes as they undergo dedifferentiation and possibly redifferentiation in culture.

In last decades, the study of the interaction between inorganic nanoparticles and phospholipid mem-branes gained more attention as it may enable new attractive application possibilities in nanomedicine. In this context vesicles composed of phospholipid membranes serve as a model for biological mem-branes. Furthermore, the interaction with more or less soft nanoparticles has a great relevance, as the parameter softness will modulate the adsorption affinity towards membranes. Therefore, we studied the interaction of soft and hard nanoparticles with lipid membranes. The former was synthesized by means of surface-initiated atom transfer radical polymerization (ATRP). The soft properties arise from having a surface grafted polymer brush. Polyelectrolytes such as cationic poly-dimethylaminoethyl-methacry-late (PDMAEMA) and poly-methacrylicacid (PMAA) were used as they enhance the colloidal stability in solution due to electrostatic repulsion. As they are polyelectrolytes the charge density can be tuned by the solution pH. Furthermore, we can control the amount of grafted polymer via the synthetic proce-dure. A detailed analysis of such core-shell structures was done, where structural details (size, shape, shell thickness, molecular weight) were obtained by a combination of small angle scattering experiments (light, X-rays and neutrons). In the second part of the work previously synthesized cationic nanoparticles were mixed with anionic or zwitterionic lipid membranes on curved (vesicles) and planar (supported lipid bilayers) surfaces. As a result of attractive Van der Waals and electrostatic interactions nanoparticles adsorb onto the lipid membrane but until up to a certain amount of grafted polymer. Then the interaction becomes mainly repulsive, even if an electrostatic attraction is present. Static measurements were done via quartz crystal microbalance with dissipation (QCM-D), where the resonance frequency change records the spontane-ous supported lipid bilayer formation with subsequent nanoparticle adsorption. Essential parameters such as charge density of the polymer brush and the planar surface, amount of grafted polymer, particle size and material of the planar surface were studied. These experiments showed, that until a given amount of grafted polymer or charge density the nanoparticles do not adsorb at the surface. Beside static experiments the kinetics of the nanoparticle adsorption on vesicles of the same bilayers was investigated. Samples were used with nanoparticles containing a moderate amount of grafted polymer. The kinetic measurements were done by the stopped-flow technique, which allows rapid mixing of two small vol-umes composed of a vesicle and a nanoparticle solution into a measurement cell. The vesicle-nanopar-ticle aggregate formation was followed by time resolved turbidity and small angle X-ray scattering (SAXS) measurements. With increasing nanoparticle to vesicle ratio an increase of the kinetic time was observed. The net charge of the vesicles was inverted by adsorbing NPs and the net charge of decorated surfaces repels further approaching NPs away from the surface., In den letzten Jahren hat die Bedeutung der Untersuchung der Wechselwirkung zwischen anorganischen Nanopartikeln und Phospholipidmembranen zugenommen, denn diese ermöglichen neue attraktive Anwendungsmöglichkeiten von Nanopartikeln in der Medizin. In diesem Kontext fungieren Vesikel aus Phospholipidmembranen als Modellsysteme für biologische Zellmembranen. Neben harten anorganischen Nanopartikel kann die Interaktion auch mit weichen Nanopartikel von großer Bedeutung sein, denn der Parameter Weichheit wird das Adsorptions- und Desorptionsverhalten verändern. Die vorliegende Arbeit beschäftigt sich mit der Interaktion von weichen Silikananopartikel mit Phospholipidmembranen. Erstere wurden über eine oberflächeninitialisierte Atomtransfer Radikalpolymerisation (ATRP) synthetisiert, wobei die Weichheit der Nanopartikel durch aufgepfropfte Polymerbürsten auf den Oberflächen entsteht. Dabei wurden Polyelektrolyte verwendet, die die Oberflächenladung der Nanopartikel erhöht, um eine kolloidale stabile wässrige Lösung zu erhalten. Es handelte sich dabei um die Polyelektrolyte Poly-Dimethylaminoethyl-Methacrylat (PDMAEMA, kationisch) und Poly-Methacrylsäure (PMAA, anionisch), deren Ladungsdichte mittels des pH-Wertes gesteuert werden kann, da es sich um schwache Polyelektrolyte handelt. Des Weiteren wurde die aufgepfropfte Polymermenge variiert. Eine detaillierte Analyse der Kern-Schale Nanopartikel wurde mittels mehrerer Analysemethoden durchgeführt, wobei exakte strukturelle Details (Größe, Schalendicke, Molekulargewicht) über verschiedene Kleinwinkelstreumethoden (Licht, Röntgenstrahlung, Neutronen) untersucht wurden. Im zweiten Teil der Arbeit wurden die zuvor hergestellten Nanopartikel mit anionischen sowie zwitterionischen Phospholipidmembranen auf gekrümmten (Vesikel) sowie planaren Oberflächen (unterstüt-zende Lipidschichten) vermischt. Aufgrund anziehender Van-der-Waals und elektrostatischer Wechselwirkung adsorbieren die Nanopartikel auf die Lipidmembran, jedoch nur bis zu einer bestimmten Menge an aufgepfropftem Polymer. Danach ist die Wechselwirkung hauptsächlich repulsiv, obwohl eine elektrostatische Anziehung vorliegt. Statische Untersuchungen wurden mittels Quarz-Kristall-Mikrowaage mit Energieverlust auf planaren Oberflächen untersucht, wobei über die Resonanzfrequenzveränderung die Ausbildung der „supported lipid bilayer“ und die anschließende Nanopartikeladsorption beobachtet wurden. Wesentliche Parameter, wie die Ladungsdichte der Partikel und Lipide, aufgepfropfte Polymermenge, Partikelgröße und Material der planaren Oberfläche, wurden untersucht. Ergebnisse zeigen, dass eine zu große Polymermenge sowie eine zu hohe Ladungsdichte der Nanopartikel die Repulsion fördern. Neben den statischen Experimenten wurde auch die Dynamik der Nanopartikeladsorption auf Vesikeln untersucht. Es wurden ausschließlich Nanopartikel verwendet, welche eine moderate Menge an aufgepfropften Polymer erhalten. Dabei wurden mittels der Stopped-Flow-Technik Vesikel- und Nanopartikellösung schnell gemischt und dann über zeitaufgelöste Trübungs- und Kleinwinkelröntgenstreumessungen die Strukturbildung der Vesikel-Nanopartikel-Aggregate beobachtet. Es stellte sich heraus, dass sich mit zunehmendem Nanopartikel zu Vesikel Verhältnis das Gleichgewicht langsamer einstellte. Zuerst adsorbieren kationische Nanopartikel auf die anionischen Vesikel. Nach einer bestimmten Anzahl an adsorbierten Partikeln wird die Oberflächenladung umgepolt, sodass das Annähern weiterer Nanopartikel erschwert bzw. verhindert.

2D materials, e.g., graphene, and heterostructures have extraordinary properties compared to their 3D counterparts, and have great potential for a broad range of applications, including flexible electronic devices, nanocomposites, and transistors. However, in most of these applications the 2D materials need to interface with other materials such as substrates or other 2D heteroststructures for not only device functionality but also mechanical stability. The interfacial properties of 2D materials and heterostructures greatly affect the performance of these 2D materials-based devices and thus call for further investigation. In this dissertation, advanced scanning probe microscopy (SPM) techniques, including contact resonance atomic force microscopy (CR-AFM) and piezoresponse force microscopy (PFM), are applied to study the interfacial mechanical and piezoelectrical properties of graphene and 2D materials heterostructures. For the first time, CR-AFM is demonstrated with the sensitivity to local stiffness changes that arise from a single atomic layer of a van-der-Waals-adhered material. To this end, a new approach, combining CR-AFM with first-principles calculations and continuum mechanics modeling, is introduced, which can yield a quantitative subsurface atomic structure fingerprint for 2D materials and heterostructures, as demonstrated on an ideal model system – epitaxial graphene on SiC (0001). This model system is further investigated with PFM, which revealed a new source of piezoelectricity in graphene layers that arises from the presence of interfacial dipole moments induced by the polarization in the substrate. The last part of the dissertation discusses the interfacial mechanical properties of graphene deposited onto self-assembled-monolayers (SAMs). CR-AFM experiments and molecular dynamics (MD) simulations show that the surface energy of the SAM strongly affects the amount of water molecules present at the graphene-SAM interface, which in turn influences the elastic modulus of these graphene-SAM heterostructures. The SPM methods used in this dissertation can provide rich structure-property information about interfaces and surfaces, and can be used to understand other interfacial problems of fundamental and practical interest in 2D materials and heterostructures, such as nanoconfined water and 2D layered hybrid organic-inorganic perovskites.

The use of DNA as a polymeric building material transcends its function in biology and is exciting in bionanotechnology for applications ranging from biosensing, to diagnostics, and to targeted drug delivery. These applications are enabled by DNA’s unique structural and chemical properties, embodied as a directional polyanion that exhibits molecular recognition capabilities. Hence, the efficient and precise synthesis of high molecular weight DNA materials has become key to advance DNA bionanotechnology. Current synthesis methods largely rely on either solid phase chemical synthesis or template-dependent polymerase amplification. The inherent step-by-step fashion of solid phase synthesis limits the length of the resulting DNA to typically less than 150 nucleotides. In contrast, polymerase based enzymatic synthesis methods (e.g., polymerase chain reaction) are not limited by product length, but require a DNA template to guide the synthesis. Furthermore, advanced DNA bionanotechnology requires tailorable structural and self-assembly properties. Current synthesis methods, however, often involve multiple conjugating reactions and extensive purification steps.The research described in this dissertation aims to develop a facile method to synthesize high molecular weight, single stranded DNA (or polynucleotide) with versatile functionalities. We exploit the ability of a template-independent DNA polymerase−terminal deoxynucleotidyl transferase (TdT) to catalyze the polymerization of 2’-deoxyribonucleoside 5’-triphosphates (dNTP, monomer) from the 3’-hydroxyl group of an oligodeoxyribonucleotide (initiator). We termed this enzymatic synthesis method: TdT catalyzed enzymatic polymerization, or TcEP.Specifically, this dissertation is structured to address three specific research aims. With the objective to generate high molecular weight polynucleotides, Specific Aim 1 studies the reaction kinetics of TcEP by investigating the polymerization of 2’-deoxythymidine 5’-triphosphates (monomer) from the 3’-hydroxyl group of oligodeoxyribothymidine (initiator) using in situ 1H NMR and fluorescent gel electrophoresis. We found that TcEP kinetics follows the “living” chain-growth polycondensation mechanism, and like in “living” polymerizations, the molecular weight of the final product is determined by the starting molar ratio of monomer to initiator. The distribution of the molecular weight is crucially influenced by the molar ratio of initiator to TdT. We developed a reaction kinetics model that allows us to quantitatively describe the reaction and predict the molecular weight of the reaction products.Specific Aim 2 further explores TcEP’s ability to transcend homo-polynucleotide synthesis by varying the choices of initiators and monomers. We investigated the effects of initiator length and sequence on TcEP, and found that the minimum length of an effective initiator should be 10 nucleotides and that the formation of secondary structures close to the 3’-hydroxyl group can impede the polymerization reaction. We also demonstrated TcEP’s capacity to incorporate a wide range of unnatural dNTPs into the growing chain, such as, hydrophobic fluorescent dNTP and fluoro modified dNTP. By harnessing the encoded nucleotide sequence of an initiator and the chemical diversity of monomers, TcEP enables us to introduce molecular recognition capabilities and chemical functionalities on the 5’-terminus and 3’-terminus, respectively.Building on TcEP’s synthesis capacities, in Specific Aim 3 we invented a two-step strategy to synthesize diblock amphiphilic polynucleotides, in which the first, hydrophilic block serves as a macro-initiator for the growth of the second block, comprised of natural and/or unnatural nucleotides. By tuning the hydrophilic length, we synthesized the amphiphilic diblock polynucleotides that can self-assemble into micellar structures ranging from star-like to crew-cut morphologies. The observed self-assembly behaviors agree with predictions from dissipative particle dynamics simulations as well as scaling law for polyelectrolyte block copolymers.In summary, we developed an enzymatic synthesis method (i.e., TcEP) that enables the facile synthesis of high molecular weight polynucleotides with low polydispersity. Although we can control the nucleotide sequence only to a limited extent, TcEP offers a method to integrate an oligodeoxyribonucleotide with specific sequence at the 5’-terminus and to incorporate functional groups along the growing chains simultaneously. Additionally, we used TcEP to synthesize amphiphilic polynucleotides that display self-assemble ability. We anticipate that our facile synthesis method will not only advance molecular biology, but also invigorate materials science and bionanotechnology.

One promising HIV-1 vaccine target is the membrane-proximal external region (MPER) of viral gp41. MPER is poorly immunogenic, however, the two rare neutralizing antibodies (NAbs), 2F5 and 4E10, bind to MPER with great neutralizing ability. Although their neutralizing mechanism represents a promising framework for the design of new HIV-1 liposomal vaccine candidates, this mechanism remains poorly understood. It is known that 2F5 and 4E10 are required to first associate with HIV-1 lipids before binding to the target MPER antigen, however, little is known about how lipid membranes contribute to NAb-antigen binding. To this end we have developed model membrane systems to study NAb and antigen lipid interactions. We first created a surface plasmon resonance (SPR) spectroscopy based assay that monitors antibody binding to thiol monolayers, which mimic the surface chemical properties of lipid membranes. Next, we focused on mimicking the lipid phase organization (i.e., domain formation) of native membranes by using supported lipid bilayers (SLBs). We used simple SLB compositions to model the liquid-disordered (Ld) and gel phases. To model the HIV-1 envelope, we used a complex SLB composition that contains an Ld and liquid-ordered (Lo) phase. To reliably create model HIV-1 SLBs, we developed an SLB formation technique that uses amphipathic, α-helical peptides as a catalyst to generate complex SLBs that have a high cholesterol content and contain multiple lipid types. For all SLB surfaces we used atomic force microscopy (AFM) to visualize membrane domains, antigen presentation, and antibody-membrane interactions.Results from experiments using thiol surfaces showed that NAb binding to hydrophobic thiol surfaces was significantly greater than that of control monoclonal antibodies. This supports the hypothesis that these NAbs embed into the hydrophobic membrane core. Our results demonstrate that 2F5/4E10 do not interact with the highly ordered gel and Lo domains in the SLB but exclusively bind to the Ld phase. This suggests that 2F5/4E10 require low membrane order and weak lateral lipid-lipid interactions to insert into the hydrophobic membrane interior. Thus, vaccine liposomes that primarily contain an Ld phase are more likely to elicit the production of lipid reactive, 2F5- and 4E10-like antibodies, compared to liposomes that contain an Lo or gel phase. In the context of liposomal antigen presentation, our results show that the presence of the MPER656 antigen can severely limit the Ld area available for antibody interactions. Subsequently, this reduces the amount of MPER656 that is accessible for 2F5/4E10 binding, since MPER656 preferentially localizes to the Ld area. If Ld forming lipid components are used in vaccine liposomes, it is important to ensure that the presence of antigen does not inhibit large-scale Ld formation.

International audience; After a brief reminder of the basics of carbon nanotubes regarding their morphology, structure, texture, and nanotexture, this chapter attempts to summarize the knowledge gathered on every aspect of their properties as the result of the extensive investigation carried out in this field since the 1990s. The properties covered include electrical, thermal, optical, electronic, and adsorptive (chemical reactivity) completed by a summary of the behavior of carbon nanotubes in biological environment, both from the point of view of eco- and cytotoxicity and that of positive (e.g., therapeutic) interactions. An abundant literature is cited for enabling the reader to deepen any selected topic among those addressed here.

Engineering thin, organic materials with tailored properties requires both the understanding of the conformation of thin organic films and their conformational response to changes in the environment, and the accurate characterization the mechanical properties of the materials as a thin layer on surfaces. These issues have not yet been sufficiently addressed due to the paucity of appropriate tools and data interpretation approaches to reveal the nanometer scale conformation and mechanics of surface-grafted, thin, organic films. In this dissertation, I report on the characterization of conformational and mechanical properties of thin organic films, and the development of techniques that allow more detailed and reliable measurement of these material properties. First, I co-developed a novel approach to evaluate neutron reflectivity data and to simulate the conformational structure for thin stimulus-responsive polymer brushes. In this approach, we used a molecular-based lattice mean-field theory, augmented with experimentally obtained parameters to describe the polymer chains. The approach and fitting results required fewer fitting parameters, and captured the thermal response of the sample self-consistently. Second, I demonstrated the capability of force-modulation microscopy in imaging surface-grafted, organic thin films in aqueous environments, with high spatial resolution and sensitivity to conformational details that affect the contact mechanics. To this end, I developed a new parameter-selection approach. This approach allowed both highly sensitive mapping of subtle differences in the molecular packing of thiol molecules on the substrate surface, and generation of high-contrast contact-stiffness images of end-grafted protein patterns on a surface. Finally, I discussed model selection and error estimation in calculating the reduced Young's modulus of soft materials on surfaces. I found that the detailed characterization of probe apex profiles, using probe-reconstruction techniques, provide only marginal improvements in calculating the reduced Young's modulus of thin films, compared with analytical models of equivalent probe radii; however, I found that a hybrid worn-cone model is appropriate for large indentations on soft materials, and benefits from the characterization of the probe apex profile. Additionally, we rendered error maps of several common scenarios, referenced to indentation and probe radius values, in the determination of the reduced Young's modulus.

Ferroelectric thin films (FETFs) offer a promising new platform for advancing liquid-phase interfacial sensing devices. FETFs are capable of expressing surface charge densities that are an order of magnitude higher than those of traditional charged surfaces in liquid environments (e.g., common oxides, self-assembled monolayers, or electrets). Furthermore, the switchable polarization state of FETFs enables patterning of charge-heterogeneous surfaces whose charge patterns persist over a range of environmental conditions. Integration of FETFs into liquid-phase interfacial sensing devices, however, requires the fabrication of films with nanometer-scale surface roughness, high remnant polarization values, and interfacial stability during prolonged exposure. The objectives of my research were to i) fabricate ferroelectric ultra-smooth lead zirconium titanate (US-PZT) thin films with nanometer-scale surface roughness, ii) establish the interfacial stability of these films after prolonged exposure to aqueous environments, iii) measure the interfacial forces as a function of film polarization and ionic strength, iv) calculate the surface potential of the US-PZT surface using electric double layer (EDL) theory, and v) demonstrate the guided deposition of charged colloidal particles onto locally polarized US-PZT thin films from solution. I demonstrate the use of ferroelectric US-PZT thin films to manipulate EDL interaction forces in aqueous environments. My work conclusively shows that the polarization state of US-PZT controls EDL formation and can be used to induce the guided deposition of charged colloidal particles in solution. I present a robust fabrication scheme for making ferroelectric US-PZT thin films from a sol-gel precursor. By optimizing critical thermal processing steps I am able to minimize the in-plane stress of the film and reliably produce US-PZT thin films on the wafer-scale with mean surface roughness values of only 2.4 nm over a 25 μm2 area. I then establish US-PZT film stability in water by measuring changes in film topography, crystallinity, surface chemistry, and electrical properties as a function of exposure duration. My results show that fabrication of crack-free US-PZT thin film is critical for long-term film fidelity in aqueous environments. Furthermore, I found no change in film topography or bulk composition with increasing exposure duration. Prolonged exposure to aqueous environments, however, gradually oxidizes the surface of the US-PZT wich results in a decrease in film resistivity and polarization saturation. Next, I used colloidal probe force microscopy (CPFM) to measure the EDL interaction force as a function of separation distance between polarized US-PZT thin films and a clean borosilicate probe. CPFM measurements were performed on oppositely polarized US-PZT thin films, which expressed either a positive or negative surface charge, and over a range of ionic strengths. The inner-Helmholtz plane (IHP) potential of the US-PZT was determined by fitting the CPFM force-separation data to number of EDL models, including; an analytical EDL model using a constant potential boundary condition with a Stern layer, a charge regulation EDL model, and a numerical EDL model using the non-linear Poisson-Boltzmann equation. Each model provides good agreement with the experimentally measured and predict high IHP surface potential for the polarized US-PZT thin films in solution. Finally, I demonstrate the use of polarized US-PZT to induce the guided deposition of positively or negatively charged colloidal particles from aqueous environments. I explore the effects of ionic strength, particle size, surface roughness, and pH on particle deposition. Overall, this work demonstrates, for the first time, that FETFs can be used as a platform to manipulate colloidal particles in aqueous environments. The experimental results demonstrate that the surface charge of the FETF is reduced by charge shielding and perform similarly to traditional, charged surfaces in aqueous environments.