Molding micropatterns of elasticity on PEG-based hydrogels to control cell adhesion and migration

We present an innovative and simple, soft UV lithographic method “FIll-Molding In Capillaries” (FIMIC) that combines soft lithography with capillary force driven filling of micro-channels to create smooth hydrogel substrates with a 2D micro-pattern on the surface. The lithographic procedure involves the molding of a polymer; in our case a bulk PEG-based hydrogel, via UV-curing from a microfabricated silicon master. The grooves of the created regular line pattern are consequently filled with a second hydrogel by capillary action. As a result, a smooth surface is obtained with a well-defined pattern design of the two different polymers on its surface. The FIMIC method is very versatile; the only prerequisite is that the second material is liquid before curing in order to enable the filling process. In this specific case we present the proof of principle of this method by applying two hydrogels which differ in their crosslinking density and therefore in their elasticity. Preliminary cell culture studies on the fabricated elasticity patterned hydrogels indicate the preferred adhesion of the cells to the stiffer regions of the substrates, which implies that the novel substrates are a very useful platform for systematic cell migration studies, e.g. more fundamental investigation of the concept of “durotaxis”

Microfluidic device and interfacial transport: application to biomolecules and nanostructures

The aim of my dissertation is to provide new knowledge and applications of microfluidics in a variety of problems, from materials science, devices, and biomedicine, where the control on the fluid dynamics and the local concentration of the solutions containing the relevant molecules (either materials, precursors, or biomolecules) is crucial. The control of interfacial phenomena occurring in solutions at dierent length scales is compelling in nanotechnology for devising new sensors, molecular electronics devices, memories. Microfluidic devices were fabricated and integrated with organic electronics devices. The transduction involves the species in the solution which infills the transistor channel and confined by the microfluidic device. This device measures what happens on the surface, at few nanometers from the semiconductor channel. Soft-lithography was adopted to fabricate platinum electrodes, starting from platinum carbonyl precursor. I proposed a simple method to assemble these nanostructures in periodic arrays of microstripes, and form conductive electrodes with characteristic dimension of 600 nm. The conductivity of these sub-microwires is compared with the values reported in literature and bulk platinum. The process is suitable for fabricating thin conductive patterns for electronic devices or electrochemical cells, where the periodicity of the conductive pattern is comparable with the diusion length of the molecules in solution. The ordering induced among artificial nanostructures is of particular interest in science. I show that large building blocks, like carbon nanotubes or core-shell nanoparticles, can be ordered and self-organised on a surface in patterns due to capillary forces. The eective probability of inducing order with microfluidic flow is modeled with finite element calculation on the real geometry of the microcapillaries, in soft-lithographic process. The oligomerization of A40 peptide in microconfined environment represents a new investigation of the extensively studied peptide aggregation. The added value of the approach I devised is the precise control on the local concentration of peptides together with the possibility to mimick cellular crowding. Four populations of oligomers where distinguished, with diameters ranging from 15 to 200 nm. These aggregates could not be addresses separately in fluorescence. The statistical analysis on the atomic force microscopy images together with a model of growth reveal new insights on the kinetics of amyloidogenesis as well as allows me to identify the minimum stable nucleus size. This is an important result owing to its implications in the understanding and early diagnosis and therapy of the Alzheimer’s disease

Kontaktmechanik und Strukturierung von festkörperunterstützen Lipidmembranen

Die vorliegende Arbeit beschreibt unter anderem die Realisierung eines Assays aus mikrostrukturierten und selektiv funktionalisierten künstlichen Membransegmenten auf einem Chip. Die Strukturierungsmethode kombiniert die softlithographische Technik des Mikroformens in Kapillaren mit der Vesikelspreittechnik und bietet ein elegantes Verfahren, einzeln adressierbare Lipidsegmente im Mikrometer Regime zu erzeugen. Unter Berücksichtigung des hydrodynamischen Fließverhaltens und der Stabilitätskriterien für PDMS-Elastomere wurden außerdem neue Strukturen entwi-ckelt, die für den kombinierten Einsatz von Rasterkraftmikroskopie und Fluoreszenz-mikroskopie optimiert sind. Die Anwendbarkeit des Lab-On-A-Chip-Devices als Bio-sensor wurde durch zwei prominente Protein-Rezeptor-Bindungsstudien fluores-zenzmikroskopisch und rasterkraftmikroskopisch belegt. Im zweiten Teil der Arbeit sind die mechanischen und adhäsiven Eigenschaften aus-gewählter Lipidsysteme mit einer neuen Charakterisierungstechnik untersucht wor-den, die die Kontaktmechanik von Rastersonden und Lipidmembranen auf Basis der Digitalisierung von Hochgeschwindigkeitskraftkurven und einer automatisierten Multi-parameteranalyse quantitativ erfasst. Dabei konnte die Korrelation zwischen der Ad-häsion und den materialspezifischen Durchbruchlängen und Durchbruchkräften, die charakteristische Stabilitätsparameter der Lipidmembran darstellen, auf Systemen mit variierenden Kopfgruppen und Kettenlängen analysiert werden. Das Verfahren erlaubte zudem die simultane Quantifizierung der elastischen Eigenschaften der Li-piddoppelschichten. Zu den Kraftkurven wurden Simulationen der Systemantwort durchgeführt, die ein tieferes Verständnis der Kontrastentstehung ermöglichen.

Untersuchung des Reorientierungsverhaltens und Strukturierung dünner amphiphiler Blockcopolymerfilme zum Aufbau von Kompositen

Grundlage für die hier gezeigte Arbeit stellt die Eigenschaft von amphiphilen Blockcopolymeren dar immer den Block mit der niedrigsten Grenzflächenenergie zum angrenzenden Medium an die Oberfläche zu bringen. Durch einen Austausch des Mediums an der Grenzfläche zum Blockcopolymer kann eine Reorientierung erzwungen werden, wenn die Grenzflächenenergie des anderen Blocks nun die niedrigere Grenzflächenenergie besitzt. Dieses Verhalten von dünnen amphiphilen Blockcopolymerfilmen wurde zur Strukturierung von Oberflächen ausgenutzt und in nachfolgenden Synthesen weiter verstärkt. Um dies zu erreichen wurde das zur Strukturierung erforderliche Poly(4-Octylstyrol)block(4-hydroxystyrol) durch kontrollierte radikalische Polymerisationsmethode mit dem Tempo Unimer (2,2,6,6-Tetramethyl-1-1(1-phenyl-ethoxy)-piperidin) synthetisiert. Für die geplanten Reorientierungen und Modifizierungen von Oberflächen wurden dünne Filme durch Schleuderbeschichtung auf verschiedenen Substraten (Siliziumwafern, Glassubstraten und Goldoberflächen) hergestellt. Das Verhalten der Oberflächen von diesen Filmen wurde durch Kontaktwinkelmessungen untersucht. Auf diese Weise konnte gezeigt werden, dass die Oberfläche von Polymerfilmen nach der Präparation aus dem hydrophoben Block des Polymers gebildet wird. Durch Kontakt des Polymerfilms mit Wasser kann dieser zur Reorientierung gebracht werden, so dass der hydrophile Block des Polymers an der Oberfläche erscheint. Dieses Verhalten wurde zur Strukturierung mit softlithographischen Techniken genutzt. Dazu wurden hydrophil/hydrophob strukturierte Oberflächen durch Aufsetzen von hydrophoben PDMS-Stempeln, die Teile der Oberfläche selektiv abdeckten, und Einbringen von Wasser in die dabei entstehenden Kapillaren hergestellt. Dies ermöglichte es die Oberfläche selektiv im Größenbereich von 500nm bis zu 50µm zu strukturieren und an den reaktiven Bereichen Materialien, wie z.B. Kupfer, Titandioxid, Polyelektrolyte, photonische Kristalle und angegraftete Polymere, mit verschiedenen Methoden selektiv auf die Oberfläche aufzubringen. Um den Reorganisationsprozess der Oberfläche genauer zu studieren, wurde ein für diese Aufgabe besser geeignetes Polymer (Poly(Styrol)-block-poly(essigsäure-2-(2-(4-vinyl-phenoxy)-ethoxy)ethylester)) synthetisiert. Aus diesem Blockcopolymer wurden wieder dünne Filme durch Spincoaten hergestellt. Die Reorientierung dieses Polymers in 70°C warmen Wasser konnte durch Kontaktwinkelmessungen und NEXAFS Spektroskopie nachgewiesen werden. Mit Hilfe der NEXAFS Spektroskopie konnte festgestellt werden, dass die Geschwindigkeit der Reorientierung durch eine exponentielle Funktion beschrieben werden kann. Eine Auswertung der Geschwindigkeitskonstante für die Reorientierung einer hydrophilen zu einer hydrophoben Oberfläche des Polymers bei 60°C führt zu =75min. Aufgrund des exponentiellen Charakters der Reorientierung macht es den Anschein, dass die Reorientierung bei verschiedenen Reorientierungstemperaturen bis zu einem gewissen Grad erfolgt und dann stoppt. Eine weitere Reorientierung scheint erst wieder bei einer Temperaturerhöhung zu beginnen. Aus AFM Messungen ist ein Beginnen der Reorientierung durch Bildung kleiner Löcher in der Polymeroberfläche zu erkennen, die sich zu runden Erhöhungen und Vertiefungen vergrößern, um letztendlich in ein spinodales Entmischungsmuster über zu gehen. Dieses heilt dann im Laufe der Zeit langsam durch Verschwinden der hydrophilen Bereiche langsam aus. Der Beginn des zuvor beschriebenen Reorientierungsprozesses einer hydrophilen Oberfläche in eine hydrophobe konnte sowohl in den AFM, als auch in den NEXAFS-Messungen zu ca. 50°C bestimmt werden.

Design and fabrication of biocompatible scaffolds for the regeneration of tissues

Regenerative medicine and tissue engineering attempt to repair or improve the biological functions of tissues that have been damaged or have ceased to perform their role through three main components: a biocompatible scaffold, cellular component and bioactive molecules. Nanotechnology provide a toolbox of innovative scaffold fabrication procedures in regenerative medicine. In fact, nanotechnology, using manufacturing techniques such as conventional and unconventional lithography, allows fabricating supports with different geometries and sizes as well as displaying physical chemical properties tunable over different length scales. Soft lithography techniques allow to functionalize the support by specific molecules that promote adhesion and control the growth of cells. Understanding cell response to scaffold, and viceversa, is a key issue; here we show our investigation of the essential features required for improving the cell-surface interaction over different scale lengths. The main goal of this thesis has been to devise a nanotechnology-based strategy for the fabrication of scaffolds for tissue regeneration. We made four types of scaffolds, which are able to accurately control cell adhesion and proliferation. For each scaffold, we chose properly designed materials, fabrication and characterization techniques.

Lung on Chip: In vitro HGF effects on injured alveolar A549 epithelial cells in microfluidic system

Microfluidic systems have become competitive tools in the invitro modelling of diseases and promising alternatives to animal studies. They allow obtaining more invivo like conditions for cellular assays. Research in idiopathic pulmonary fibrosis could benefit from this novel methodological approach to understand the pathophysiology of the disease & develop efficient therapies. The use of hepatocyte growth factor (HGF) for alveolar reepithelisation is a promising approach. In this study, we show a new microfluidic system to analyse the effects of HGF on injured alveolar epithelial cells. Microfluidic systems in polydimethylsiloxane were fabricated by soft lithography. The alveolar A549 epithelial cells (10,000 cells) were seeded and studied in these microfluidic systems with media perfusion (1μl/30min). Injury tests were made on the cells by the perfusion with media containing H2O2 or bleomycin. The degree of injury was then assessed by a metabolic and an apoptotic assays. Wound assays were also performed with a central laminar flow of trypsin. Monitoring of wound closure with HGF vs control media was assessed. The alveolar A549 epithelial cells grew and proliferated in the microfluidic system. In the wound closure assay, the degree of wound closure after 5 hours was (53.3±1.3%) with HGF compared to (9.8±2.4%) without HGF (P <0.001). We present a novel microfluidic model that allows culture, injury and wounding of A549 epithelial cells and represents the first step towards the development of an invitro reconstitution of the alveolar-capillary interface. We were also able to confirm that HGF increased alveolar epithelial repair in this system.

Lung on a chip: Co-culture of alveolar epithelial cells and bone marrow derived stromal stem cells in a microfluidic system

Background: Microfluidics system are novel tools to study cell-cell interactions in vitro. This project focuses on the development of a new microfluidic device to co-culture alveolar epithelial cells and mesenchymal stem cells to study cellular interactions involved in healing the injured alveolar epithelium. Methods: Microfluidic systems in polydimethylsiloxane were fabricated by soft lithography. The alveolar A549 epithelial cells were seeded and injury tests were made on the cells by perfusion with media containing H2O2 or bleomycin during 6 or 18hrs. Rat Bone marrow derived stromal cells (BMSC) were then introduced into the system and cell-cell interaction was studied over 24 hrs. Results: A successful co-culture of A549 alveolar epithelial cells and BMS was achieved in the microfluidic system. The seeded alveolar epithelial cells and BMSC adhered to the bottom surface of the microfluidic device and proliferated under constant perfusion. Epithelial injury to mimic mechanisms seen in idiopathic pulmonary fibrosis was induced in the microchannels by perfusing with H2O2 or bleomycin. Migration of BMSC towards the injured epithelium was observed as well as cell-cell interaction between the two cell types was also seen. Conclusion: We demonstrate a novel microfluidic device aimed at showing interactions between different cell types on the basis of a changing microenvironment. Also we were able to confirm interaction between injured alvolar epithelium and BMSC, and showed that BMSC try to heal the injured epitelium.

Fabrication of a Large, Ordered, Three-Dimensional Nanocup Array

Metallic nanocups provide a unique method for redirecting scattered light by creating magnetic plasmon responses at optical frequencies. Despite considerable development of nanocup fabrication processes, simultaneously achieving accurate control over the placement, orientation, and geometry of nanocups has remained a significant challenge. Here we present a technique for fabricating large, periodically ordered arrays of uniformly oriented three-dimensional gold nanocups for manipulating light at subwavelength scales. Nanoimprint lithography, soft lithography, and shadow evaporation were used to fabricate nanocups onto the tips of polydimethylsiloxane nanopillars with precise control over the shapes and optical properties of asymmetric nanocups.

Improving the Manufacturability of Active Microfluidic Devices using Stereolithography (SL)

Thesis (Master's)--University of Washington, 2016-06

Absorption and fluorescence spectroscopy of rhodamine 6G in titanium dioxide nanocomposites

A comparison has been made between the spectroscopic properties of the laser dye rhodamine 6G (R6G) in mesostructured titanium dioxide (TiO2) and in ethanol. Steady-state excitation and emission techniques have been used to probe the dye-matrix interactions. We show that the TiO2-nanocomposite studied is a good host for R6G, as it allows high dye concentrations, while keeping dye molecules isolated, and preventing aggregation. Our findings have important implications in the context of solid state dye-lasers and microphotonic device applications. (C) 2003 Elsevier B.V. All rights reserved.

Optical fiber sensors embedded in flexible polymer foils

In traditional electrical sensing applications, multiplexing and interconnecting the different sensing elements is a major challenge. Recently, many optical alternatives have been investigated including optical fiber sensors of which the sensing elements consist of fiber Bragg gratings. Different sensing points can be integrated in one optical fiber solving the interconnection problem and avoiding any electromagnetical interference (EMI). Many new sensing applications also require flexible or stretchable sensing foils which can be attached to or wrapped around irregularly shaped objects such as robot fingers and car bumpers or which can even be applied in biomedical applications where a sensor is fixed on a human body. The use of these optical sensors however always implies the use of a light-source, detectors and electronic circuitry to be coupled and integrated with these sensors. The coupling of these fibers with these light sources and detectors is a critical packaging problem and as it is well-known the costs for packaging, especially with optoelectronic components and fiber alignment issues are huge. The end goal of this embedded sensor is to create a flexible optical sensor integrated with (opto)electronic modules and control circuitry. To obtain this flexibility, one can embed the optical sensors and the driving optoelectronics in a stretchable polymer host material. In this article different embedding techniques for optical fiber sensors are described and characterized. Initial tests based on standard manufacturing processes such as molding and laser structuring are reported as well as a more advanced embedding technique based on soft lithography processing.

Cell-cell communication in three dimensional microenvironments

Cellular behavior is dependent on a variety of extracellular cues required for normal tissue function, wound healing, and activation of the immune system. Removed from their in vivo microenvironment and cultured in vitro, cells lose many environmental cues and that may result in abberant behavior, making it difficult to study cellular processes. In order to mimic native tissue environments, optical tweezer and microfluidic technologies were used to place cells within defined areas of the culture environment. To provide three dimensional supports found in natural tissues, hydrogel scaffolds of poly (ethylene glycol) diacrylate and the basement membrane matrix Matrigel were used. Optical tweezer technology allowed precision placement and formation of homotypic and heterotypic arrays of human U937, HEK 293, and porcine mesenchymal stem cells. Alternatively, two microfluidic devices were designed to pattern Matrigel scaffolds. The first microfluidic device utilized laminar flow to spatially pattern multiple cell types within the device. Gradients of soluble molecules were then be formed and manipulated across the Matrigel scaffolds. Patterning Matrigel using laminar flow techniques require microfluidic expertise and do not produce consistent patterning conditions, limiting their use difficult in most cell culture laboratories. Thus, a buried Matrigel polydimethylsiloxane (PDMS) device was developed for spatial patterning of biological scaffolds. Matrigel is injected into micron sized channels of PDMS fabricated by soft lithography and allowed to thermally cure. Following curing, a second PDMS device was placed on top of the buried Matrigel channels to support media flow. In order to validate these systems, a cell-cell communication model system was developed utilizing LPS and TNFα signaling with fluorescent reporter systems to monitor communication in real time. We demonstrated the utility of microfluidic devices to support the cell-cell communication model system by co culturing three cell types within Matrigel scaffolds and monitoring signaling activity via fluorescent reporters.

Solid-liquid interactions in microscale structures and devices

Liquid-solid interactions become important as dimensions approach mciro/nano-scale. This dissertation focuses on liquid-solid interactions in two distinct applications: capillary driven self-assembly of thin foils into 3D structures, and droplet wetting of hydrophobic micropatterned surfaces. The phenomenon of self-assembly of complex structures is common in biological systems. Examples include self-assembly of proteins into macromolecular structures and self-assembly of lipid bilayer membranes. The principles governing this phenomenon have been applied to induce self-assembly of millimeter scale Si thin films into spherical and other 3D structures, which are then integrated into light-trapping photovoltaic (PV) devices. Motivated by this application, we present a generalized analytical study of the self-folding of thin plates into deterministic 3D shapes, through fluid-solid interactions, to be used as PV devices. This study consists of developing a model using beam theory, which incorporates the two competing components — a capillary force that promotes folding and the bending rigidity of the foil that resists folding into a 3D structure. Through an equivalence argument of thin foils of different geometry, an effective folding parameter, which uniquely characterizes the driving force for folding, has been identified. A criterion for spontaneous folding of an arbitrarily shaped 2D foil, based on the effective folding parameter, is thus established. Measurements from experiments using different materials and predictions from the model match well, validating the assumptions used in the analysis. As an alternative to the mechanics model approach, the minimization of the total free energy is employed to investigate the interactions between a fluid droplet and a flexible thin film. A 2D energy functional is proposed, comprising the surface energy of the fluid, bending energy of the thin film and gravitational energy of the fluid. Through simulations with Surface Evolver, the shapes of the droplet and the thin film at equilibrium are obtained. A critical thin film length necessary for complete enclosure of the fluid droplet, and hence successful self-assembly into a PV device, is determined and compared with the experimental results and mechanics model predictions. The results from the modeling and energy approaches and the experiments are all consistent. Superhydrophobic surfaces, which have unique properties including self-cleaning and water repelling are desired in many applications. One excellent example in nature is the lotus leaf. To fabricate these surfaces, well designed micro/nano- surface structures are often employed. In this research, we fabricate superhydrophobic micropatterned Polydimethylsiloxane (PDMS) surfaces composed of micropillars of various sizes and arrangements by means of soft lithography. Both anisotropic surfaces, consisting of parallel grooves and cylindrical pillars in rectangular lattices, and isotropic surfaces, consisting of cylindrical pillars in square and hexagonal lattices, are considered. A novel technique is proposed to image the contact line (CL) of the droplet on the hydrophobic surface. This technique provides a new approach to distinguish between partial and complete wetting. The contact area between droplet and microtextured surface is then measured for a droplet in the Cassie state, which is a state of partial wetting. The results show that although the droplet is in the Cassie state, the contact area does not necessarily follow Cassie model predictions. Moreover, the CL is not circular, and is affected by the micropatterns, in both isotropic and anisotropic cases. Thus, it is suggested that along with the contact angle — the typical parameter reported in literature quantifying wetting, the size and shape of the contact area should also be presented. This technique is employed to investigate the evolution of the CL on a hydrophobic micropatterned surface in the cases of: a single droplet impacting the micropatterned surface, two droplets coalescing on micropillars, and a receding droplet resting on the micropatterned surface. Another parameter which quantifies hydrophobicity is the contact angle hysteresis (CAH), which indicates the resistance of the surface to the sliding of a droplet with a given volume. The conventional methods of using advancing and receding angles or tilting stage to measure the resistance of the micropatterned surface are indirect, without mentioning the inaccuracy due to the discrete and stepwise motion of the CL on micropillars. A micronewton force sensor is utilized to directly measure the resisting force by dragging a droplet on a microtextured surface. Together with the proposed imaging technique, the evolution of the CL during sliding is also explored. It is found that, at the onset of sliding, the CL behaves as a linear elastic solid with a constant stiffness. Afterwards, the force first increases and then decreases and reaches a steady state, accompanied with periodic oscillations due to regular pinning and depinning of the CL. Both the maximum and steady state forces are primarily dependent on area fractions of the micropatterned surfaces in our experiment. The resisting force is found to be proportional to the number of pillars which pin the CL at the trailing edge, validating the assumption that the resistance mainly arises from the CL pinning at the trailing edge. In each pinning-and-depinning cycle during the steady state, the CL also shows linear elastic behavior but with a lower stiffness. The force variation and energy dissipation involved can also be determined. This novel method of measuring the resistance of the micropatterned surface elucidates the dependence on CL pinning and provides more insight into the mechanisms of CAH.

Low dimensional fabrication of giant dielectric CaCu3Ti4O12 through soft e-beam lithography

We report low-dimensional fabrication of technologically important giant dielectric material CaCu3Ti4O12 (CCTO) using soft electron beam lithographic technique. Sol-gel precursor solution of CCTO was prepared using inorganic metal nitrates and Ti-isopropoxide. Employing the prepared precursor solution and e-beam lithographically fabricated resist mask CCTO dots with similar to 200 nm characteristic dimension were fabricated on platinized Si (111) substrate. Phase formation, chemical purity and crystalline nature of fabricated low dimensional structures were investigated with X-ray diffraction (XRD), energy dispersive X-ray spectroscopy (EDS) and selected area electron diffraction (SAED), respectively. Morphological investigations were carried out with the help of scanning electron microscopy (SEM) and transmission electron microscopy (TEM). This kind of solution based fabrication of patterned low-dimensional high dielectric architectures might get potential significance for cost-effective technological applications. (C) 2012 Elsevier B.V. All rights reserved.

Plasmonic Sensing Using Nanodome Arrays Fabricated by Soft Nanoimprint Lithography

Arrays of gold-coated nanodomes were fabricated on glass substrates using a soft
nanoimprint lithography technique. Optical transmission measurements revealed complex
plasmonic resonances that proved highly sensitive to the array dimensions, the thickness of
the gold layer, and the refractive index of the surrounding medium. As one promising
application for these structures, the refractive index sensing capabilities of the nanodome
arrays were assessed.