Imaging amyloid fibers at the nanoscale: Method development and applications for hybrid materials and biomedicine

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Física de la Materia Condensada. Fecha de lectura: 13-12-2019Esta tesis tiene embargado el acceso al texto completo hasta el 13-06-2021In the last decades, advanced imaging techniques have improved our ability to analyze biological systems at the nanoscale, enabling the observation of structural and molecular components. Different imaging tools are specialized in the characterization of a specific aspect of the sample and, when they are combined, complementary information is obtained providing a more comprehensive understanding of the system. This thesis focuses on the application of (super-resolution) fluorescence microscopy in combination with atomic force microscopy (AFM) for revealing specific chemical information in a high-resolution topography map. Particularly, correlative microscopy is applied to the characterization of amyloid fibers, which are misfolded protein aggregates with interest in nanomaterials research and biomedicine. This manuscript is organized in seven chapters. Chapter 1 introduces the imaging techniques used in the thesis. It also gives a general overview on amyloid fibers, their application as hybrid materials, their importance in biomedicine for being involved in different diseases, and the phototherapeutic approaches available to treat them. In Chapter 2, the general materials and methods used during the thesis are explained. Chapter 3 provides a detailed discussion about technical aspects of correlative super-resolution fluorescence microscopy and AFM such as sample preparation, data analysis and image alignment. Furthermore, the advantage of using AFM as a “ground truth” to evaluate different aspects of super-resolution techniques, such as labeling or image reconstruction, is highlighted. In Chapter 4, the methodology developed in Chapter 3 is applied to evaluate the functionalization of amyloid fibers with quantum dots or organic fluorophores. Thus, correlative microscopy is presented as a useful technique for characterizing luminescent hybrid materials at the nanoscale. In the context of biomedicine, amyloid aggregates are important for being involved in different diseases (e.g. Alzheimer or Parkinson). Photochemical strategies to degrade amyloid structures are becoming an interesting alternative. In this thesis, a thioflavin T (ThT) derivative (ROS-ThT), which is able to target pathogenic aggregates in the presence of functional proteins, is used to study photodamage effects on amyloid fibers. In addition to fluorescence, this photocatalyst or photosensitizer produces singlet oxygen Abstract upon blue light exposure, affecting amyloid structures through oxidation. The purpose of Chapter 5 is to select a useful amyloid model to evaluate photodamage at the nanoscale, and therefore different fibers were produced, fibrillated and characterized. In Chapter 6, the selected amyloid model is used to study photodamage induced by ROS-ThT at the single-fiber level through imaging techniques, and complemented by classical biochemical assays. These experiments highlight that the combination of fluorescence microscopy and AFM is useful to probe the heterogeneity of amyloid material and to disentangle the complex dependence between photocatalyst binding/activity and fiber morphology and/or composition. The aim of Chapter 7 is to provide coherence and perspective to the main results of the thesis, as well as an outlook on how advanced microscopy methods may impact the study of amyloids in different fields of researchQuiero agradecer también al Ministerio de Economía y Competitividad por financiar mi trabajo con la beca FPI BES-2016-076293 dentro del proyecto MAT2015-66605-P y al Ministerio de Ciencia, Innovación y Universidades por financiar el proyecto PCI2018‐093064

Fabrication and Characterization of Intricate Nanostructures

Encapsulation of nanoparticles within hexaniobate nanoscrolls presents interesting advances in the formation of nanocomposites exhibiting unique multi-dimensional properties. Building upon previous successes, facile yet versatile wet-chemical and microwave-irradiation synthetic protocols for the fabrication of a series of hexaniobate composites are presented herein. Solvothermal and, more recently, microwave-assisted methods have been developed that allow for the fabrication of peapod-like structures. During solvothermal treatment, exfoliated hexaniobate nanosheets scroll around highly ordered chains of preformed nanoparticles (NPs) to produce nanopeapods (NPPs). This approach offers versatility and high yields, in addition to the potential for advanced functional device fabrication. For the characterization of these materials, advanced techniques in atomic force microscopy (AFM) were used for investigating the surface of materials at the nanometer scale. Extensive physical, dynamic, and force modulation studies were performed on novel oxide nanocomposites by implementing particular scanning techniques to determine information such as topology, stress-induced behavior at the nanoscale, magnetic behavior, and frictional forces of the nanoscale materials. These composites were then analyzed by topological intermittent contact studies in tapping and contact mode, as well as with derivative techniques of these commonly used scanning probe approaches. In addition to studying surfaces using conventional modes of AFM, the mechanical properties of these nanocomposites were measured via dynamic lateral force modulation (DLFM) and magnetic properties of functionalized magnetic nanosheets were mapped via magnetic sampling modulation (MSM). By utilizing the capabilities of the DLFM imaging mode, elastic properties such as Young’s Modulus were measured from force-distance curves. In addition to this modulation mode, MSM was used to selectively map the vibrating magnetic nanomaterials from a modulated electromagnetic field. The information obtained from these AFM techniques can be helpful in determining the relative structural behavior of these nanocomposites and gauge their use in various applications such as structural engineering of nanoarchitectures as well as studying magnetic characteristics of metal oxide nanocomposites that exhibit characteristics different from their bulk counterparts

Ühedimensionaalsete nanostruktuuride tribomehaanilised omadused: lõplike elementide meetodi simulatsioonidega toetatud eksperimentaalmõõtmised

Väitekirja elektrooniline versioon ei sisalda publikatsiooneVäitekirja raames uuriti mitmeid olulisi küsimusi, mis käsitlevad 1D nanostruktuuride mehaanilisi ja triboloogilisi omadusi ja käitumist. Põhitegevused ja uudsed aspektid on esitatud allpool. 1DNS elemente manipuleeriti tasasel pinnal ja analüüsiti vastavaid triboloogilisi protsesse. Alljärgnevalt tuvastati: • Tasasel pinnal asetsev elastselt painutatud nanotraat on väga oluline nanotriboloogilistes mõõtmistes, kuna võimaldab nanotraadi profiilist lähtuvalt leida alusega seotud triboloogilised väärtused ilma välist jõusensorit kasutamata. Meetodi täpsuse parandamiseks töötati välja uudne analüütiline meetod, mis võtab arvesse staatilise hõõrde jaotuse tasasel pinnal asetseva elastselt painutatud nanotraadil. Erinevalt varasematest meetoditest pakub uus mudel realistlikuma jõuspektri ja arvesteb ääretingimusi. Meetodit rakendati edukalt staatilise hõõrdumise arvutamiseks ZnO nanotraatide korral, mis olid ränialusel AFM-i teravikuga manipuleerimisega suvalisse kujusse painutatud. • Uus FEM mudel töötati välja sellise konfiguratsiooni jaoks, kus osa nanotraadist toetub lamedale substraadile, samal ajal kui teine osa on vabalt üle serva. Üleulatuva vaba otsa painutatakse alusele fikseeritud osa nihkumiseni. Registreerides paindprofiili vahetult enne fikseeritud osa nihkumist, saame sisendi fikseeritud osa mõjutatava jõu arvutamiseks. Vanemate mudelite puhul võeti eelduseks staatilise hõõrdejõu ühtlast jaotust fikseeritud osale. Uue mudeli puhul näidati staatilise hõõrdumise ületamist väga lokaliseeritud protsessina, mis sarnaneb pragude tekkimisega. Näidati, et olemasolevad mudelid on staatilisest hõõrdumise rolli tunduvalt alahinnatud, samas kui uus mudel pakub reaalsusega paremat kooskõla. • Töötati välja dünaamiline FEM-mudel lamedal aluspinnal asetsevast mõlemast otsast sulanud Ag nanotraadi kirjeldamiseks. Näidati, et nanotraadis tekitatud mehaanilised pinged on tingitud asjaolust, et sulanud otsad moodustavad ümarad elemendid, mille tulemusena on võimalik ületada nanotraadi ja ränialuse vahelist adhesiooni. Selle tulemusena saavutatakse konfiguratsioon, kus ainult saadud nanoosakeste otsaelmemendid puutuvad kokku pinnaga, samas kui keskosa on pinna kohal. Selline “hantlisarnane” struktuur ja konfiguratsioon on tribologiliste mõõtmiste jaoks äärmiselt atraktiivne, kuna seda saab hõlpsasti manipuleerida väikese kontaktiala tõttu ja samal ajal säilivad kõik 1D geomeetria eelised. Lisaks uuriti alljärgnevid 1DNS mehaanilisi omadusi: • Karakteriseeriti paksude seintega torukujuliste 1DNS elastseid omadusi kasutades nii eksperimentaalseid kui ka teoreetili meetodeid. o SiO2 nanotorude elastset moodulit mõõdeti kolme erineva meetodi abil, kasutades konsooltala painutamist, nanoindetatsiooni ja kolme punkti paindekatseid. Tuvastati, et kolme punkti paindekatse on kõige täpsem meetod paksuseinaliste torukujuliste 1DNS elastusmooduli mõõtmiseksA number of important issues concerning mechanical and tribological properties and behavior of 1D nanostructures were studied within the framework of the thesis. Main activity and the novelty aspects are summarized below. First, tribological aspects of 1DNS manipulated on a flat substrate were considered. In particular: • Nanowire elastically bent of a flat substrate is highly attractive for nanotribological studies as profile of nanowire can be used for extracting frictional data without using external force sensors. In order to improve accuracy of the method, a novel analytical method was developed for the calculation of distributed static friction in elastically bent nanowire resting on a flat substrate. Unlike previously available methods, new model provides more realistic force spectrum and comply with boundary conditions. The method was successfully applied for calculation of distributed static friction in ZnO nanowires bent into arbitrary shapes in AFM manipulations on a Si substrate. • A novel FEM model was developed for configuration in which part of the nanowire is resting on a flat substrate while other part is suspended over the trench. Measurements consist in bending the free end until fixed part is displaced. The bending profile prior the displacement of fixed part is used for calculation of force acting on a fixed part. In older models static friction was considered to be uniformly distributed in adhered part. The new model considered overcoming of static friction as a highly localized process similar to crack formation. It was shown, that existing models severely underestimated static friction, while novel model provides more realistic results. • Dynamic FEM model of Ag nanowire that is being melted from both ends while resting on a flat substrate was created. It was shown that mechanical stresses, generated in nanowire due to the fact that molted ends form rounded bulbs, are able to overcome the adhesion between nanowire and silicon substrate. As a result, a configuration is achieved where only the end-bulbs of the obtained nanodumbell are in contact with the surface while intact midpart is suspended above the substrate. Such structure and configuration is highly attractive for tribological measurements as it can be easily manipulated due to the small contact area and at the same time it preserves all benefits of 1D geometry. Further, mechanical properties of 1DNS were considered: • Elastic properties of tubular 1DNS with thick walls were treated both experimentally and theoretically. o Elastic modulus of SiO2 nanotubes was measured by three different methods including cantilever beam bending, nanoindentation and three-point bending tests. Three-point bending tests were found to be the most appropriate method for measuring the Young’s modulus of thick-walled tubular 1DNS. o FEM model was created to investigate the behavior of tubular 1DNS in nanoindentation test. It was shown there are both compression and indentation present. Thus, neither of existing models where walls of nanotube are considered either as a thin membrane or rigid wall cannot be used for given system as they underestimate the Young modulus. • FEM model of composite core-shell nanowire consisting of elastic core and viscous shell was created to simulate the behavior of Ag/SiO2 core-shell nanowire in bending test under electron beam irradiation. By fitting the experimental result with FEM model it was found that even at moderate current and voltage e-beam is capable of inducing glass transition in amorphous oxide shell. Finally, two variations of three-point bending test of Au nanowires were compared: freely sliding ends and rigidly fixed ends. The effect of different boundary conditions on experimental results was determined and the adhesion forces acting between Au and substrate were estimated using the FEM modeling. In total, it was demonstrated that FEM is a powerful method for studying mechanical and tribological properties of nanoscale systems when used in combination with experimental result

Controlled surface manipulation at the nanometer scale based on the atomic force microscope

The object of this thesis is the development of theoretical and experimental methods for the controlled manipulation of surfaces at the nanometer scale, including the design, construction and experimental demonstration of an atomic force microscope (AFM) based manipulator. The transfer function description of an AFM system not only offers a theoretical dynamic characterization but, additionally, it is appropriate for the analysis of stability and controllability of different system configurations, i.e. different inputs and outputs. In this thesis, transfer functions are derived that correspond to a realistic model of the AFM sensor, including all its resonance modes and the tip-sample interaction. This theoretical description is then validated using the frequency response along an AFM cantilever. Different experimental and control techniques have been combined in the NanoManipulator system to optimize AFM lithography. Optical video microscopy allows a fast recognition of the sample and exact positioning of the AFM tip in the particular region of interest, while UV-laser ablation offers the possibility of noncontact manipulation of a wide range of materials, including biological specimens. Two different control approaches have been implemented in the NanoManipulator system: (i) automated control using a vector-scan module, and (ii) interactive control based on the use of a haptic interface. Using the NanoManipulator, the two different standard AFM lithography techniques based on dynamic methods (namely dynamic and modulated plowing) are compared by performing nanopatterning on thin resist films. The results reflect that modulated plowing, where the AFM tip is in permanent contact with the resist surface while the force is being modulated, offers the highest reliability, minimizing undesired side effects. The isolation and extraction of localized regions of human metaphase chromosomes represents a promising alternative to standard methods for the analysis of genetic material. The NanoManipulator is an excellent tool for such application, as it is here illustrated by comparing AFM based mechanical dissection and noncontact ablation on side by side chromosomes. The results are analyzed in situ using AFM imaging, revealing the high precision of mechanical dissection. Acoustical force nanolithography is a novel method for AFM based lithography where the cantilever is actuated using an acoustic wave coupled through the sample surface. The influence of acoustic wave frequency and magnitude, along with the preloading force of the cantilever are studied in detail. Acoustical force nanolithography can be used as a stand alone method or as a complement for the fine adjustment of manipulation forces

Conformations of single polymer chains on surfaces

In dieser Arbeit wurden auf Substratoberflächen adsorbierte Polymermoleküle mit Rasterkraftmikroskopie (RKM) untersucht. Dabei war die Form der Moleküle (Konformation) von besonderem Interesse. Sie ist von zentralerer Bedeutung in der Polymerphysik und wird üblicherweise in Lösung und mit Streumethoden untersucht. Polymerkonformationen auf Oberflächen sind heutzutage noch wenig untersucht. Üblicherweise wird das Verhalten nach dem so genannten Wormlike-Chain Modell angenommen. Es basiert auf der Annahme, dass die Kettenbiegung nur aus thermischen Fluktuationen resultiert, so dass sich die Kettenform durch statistische Mechanik beschreiben lässt. Es wurden für verschiedene Modellsystem einzelne Moleküle hochaufgelöst abgebildet und die Konformation aus den Bildern bestimmt. Es hat sich gezeigt, dass die idealisierte Vorstellung des Wormlike-Chain Modells tatsächlich nur für wenige der untersuchten Systeme erfüllt ist. Abweichende Konformationen sind oft auffallend regelmäßig: entweder sinusartig mäandrierte oder spiralförmig gedrehte. Beide Charakteristika lassen sich aus dem Prozess der Adsorption erklären, was zeigt, dass die Moleküle auf dem Substrat immobil sind, so dass eine thermische Relaxation der Konformation verhindert ist. Konformtionen lassen sich mit RKM nicht nur beobachten, sondern auch gezielt verändern (Nanomanipulation). Für dendronisierte Polymere konnte so gezeigt werden, dass es einen glasartigen Zustand für das einzelne Molekül gibt. In diesem Zustand verhält sich das Molekül nicht mehr wie eine bewegliche Kette, sondern formstabil, ähnlich einem makroskopischen festen Körper.In this work single polymer molecules adsorbed onto substrate surfaces were investigated by scanning force microscopy (SFM). The focus was on the shape (conformation) of the molecules, which is of central importance in polymer physics. It is commonly investigated in solutions and with scattering methods. Conformations on surfaces are only little investigated thus far. Often a behavior according to the so-called worm-like chain model is assumed. It is based on the assumption that chain bending results entirely from thermal fluctuations so that the overall chain shape can be described by statistical mechanics. For several model systems single molecules were imaged and the conformation was determined from the images. It was found that the idealistic wormlike chain behavior is only valid for a few systems. Deviations are often remarkable regular: either sine-like undulated or spiral wound. Both characteristics can be explained from the process of adsorption, indicating that molecules are immobile on the substrate so that thermal relaxation is inhibited. Conformations can not only be imaged using the SFM, but also changed in a defined way (nanomanipulation). Manipulation experiments with dendronized polymers the existence of a glassy state for the single polymer. In this state the molecule no longer behaves as a flexible chain but remains its shape, similar to a macroscopic solid body