1,044 research outputs found

    Lithograph regulation of cellular mechanical properties c by Tsu-Te Judith Su.

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    Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2004.Includes bibliographical references (leaves 34-36).A magnetic trap in combination with two-photon fluorescence microscopy was used to determine the cytoskeletal stiffness and three-dimensional (3D) cytoskeletal structure of NIH 3T3 fibroblast cells plated on micropatterned substrates. Microcontact printing of self-assembled monolayers (SAMs) of alkanethiolates on gold was used to create a planar substrate of islands surrounded by non-adhesive regions. The cells were physically constrained within nanometer high adhesive cylindrical posts of defined size on the surface of a titanium and gold coated coverslip. The islands were coated with the extracellular matrix protein fibronectin (FN) and a protein inhibiter was used to restrict cellular extension. After plating, the cells were fixed and stained with phalloidin. A high-speed, two-photon scanning microscope was used to resolve actin architecture in three dimensions and a fractal dimension measurement was performed to quantify the distribution of actin within the cell as a function of adhesion area. The experiments intend to test the hypothesis that cytoskeletal mechanical properties are a function of cellular adhesion area. We further try to understand these mechanical changes by seeking a con-elation between these mechanical parameters and actin stress fiber distribution. It was discovered that the fractal dimension is a weak inverse function of cell adhesion area but that there is a significant change in fractal dimension between patterned and control cells which can freely spread to their natural dimensions. Microrheological experiments using the magnetic trap show that the mechanical properties of patterned cells are similar within statistical error while significantly softer than the control cells.S.M

    Nanoscale Studies of Proteins and Thin Films Using Scanning Probe Microscopy

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    Nanostructures of organosilanes, thin metal films, and protein nanopatterns were prepared and analyzed with atomic force microscopy (AFM). Organosilanes with designed functional groups were used to selectively pattern green fluorescent protein at the nanoscale using protocols developed with particle lithography. Mesospheres are deposited onto a substrate to produce a surface mask. Organosilanes are deposited to form a matrix film surrounding nanopores for depositing proteins. The nanopatterns were characterized using AFM, after steps of particle lithography for directly visualizing surface changes. Studies with AFM also provide a compelling tool for teaching undergraduates to introduce concepts of nanoscience. An undergraduate laboratory was developed with particle lithography to introduce the concepts of nanoscience and surface chemistry. Nanopatterns of organosilane films are prepared using protocols of particle lithography. An organic thin film is applied to the substrate using steps of either heated vapor deposition or immersion in solution. At the molecular level, two types of sample morphology can be made depending on the step for depositing organosilanes. Experience with advanced AFM instrumentation is obtained for data acquisition, digital image processing and analysis. Skills with chemical analysis are gained with bench methods of sample preparation. Concepts such as the organization of molecules on surfaces and molecular self-assembly are demonstrated with the visualization of nanopatterns prepared by students. Experiments with particle lithography can be used as a laboratory module or for undergraduate research projects, and are suitable for students with a multidisciplinary science background. The kinetics and properties of thin gold films during dewetting were studied using AFM. Thin films of gold with varying initial thickness were first deposited onto fire polished glass slides and imaged with AFM. Next, the films were annealed for two hours, and then imaged after annealing. Gold islands with varying degrees of separation were formed. Surface plasmon spectroscopy was also used to analyze the gold films. To further this study, a kinetic study was done. Two gold thin films of 10 nm each were imaged after being annealed for 15, 30, 45, 60 and 120 minutes. It was found that after the first 15 minutes of annealing, gold islands were observed

    Development of experimental setups for the characterization of the mechanoelectrical coupling of cells in vitro

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    The field of mechanobiology emerged from the many evidences that mechanical forces acting on cells have a central role in their development and physiology. Cells, in fact, convert such forces into biochemical activities and gene expression in a process referred as mechanotransduction. In vitro models that mimic cell environment also from the mechanical point of view represent therefore a key tool for modelling cell behaviour and would find many applications, e.g. in drug development and tissue engineering. In this work I introduce novel tools for the study of mechanotransduction. In particular, I present a system for the evaluation of the complex response of electrically active cells, such as neurons and cardiomyocytes. This system integrates atomic force microscopy, extracellular electrophysiological recording, and optical microscopy in order to investigate cell activity in response to mechanical stimuli. I also present cell scaffolds for the in vitro study of cancer. Obtained results, although preliminary, show the potential of the proposed systems and methods to develop accurate in vitro models for mechanobiology studies

    Functional surface micropatterns by dewetting of thin polymer films

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    Patterned polymer surfaces are of great importance with respect to an increasing number of technological and bio-medical applications, due to their great versatility in terms of chemical composition, properties and processing techniques. Surface micro-patterning by spontaneous dewetting of thin polymer films represents a versatile and robust process to fabricate surfaces with controlled topography and chemistry at the micro-scale. In this Thesis, we used polymer dewetting in combination with complementary approaches to engineer both surface chemistry and the ordering of the dewetting patterns. The dewetting of poly(D,L-glycolide-co-lactide) (PLGA) thin films on polystyrene (PS) was combined with the grafting of protein-repellent poly(ethylene glycol) (PEG), in order to form topographical and chemical surface micropatterns consisting in protein-adhesive PS domains surrounded by protein-repellent PEG-grafted PLGA films. The produced micropatterned surfaces were used for site-specific protein adsorption, and represent a promising platform for biological applications, such as proteomics, single-cell studies and tissue engineering. Spatially ordered surface micropatterns were obtained by combining polymer dewetting with microcontact printing and colloidal lithography, respectively. The dewetting of thin PS films was guided within specific regions of the substrate by prestamping of the silicon substrate with self-assembled monolayers of an alkylsilane by microcontact printing. Ordered micropatterns consisting in arrays of holes with tunable size were obtained by exploiting the spontaneous dewetting of poly(4-vinyl pyridine) (P4VP) thin films on PS from the holes produced by colloidal imprinting with two-dimensional colloidal crystals assembled on the polymer bilayer

    Functional surface micropatterns by dewetting of thin polymer films

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    Patterned polymer surfaces are of great importance with respect to an increasing number of technological and bio-medical applications, due to their great versatility in terms of chemical composition, properties and processing techniques. Surface micro-patterning by spontaneous dewetting of thin polymer films represents a versatile and robust process to fabricate surfaces with controlled topography and chemistry at the micro-scale. In this Thesis, we used polymer dewetting in combination with complementary approaches to engineer both surface chemistry and the ordering of the dewetting patterns. The dewetting of poly(D,L-glycolide-co-lactide) (PLGA) thin films on polystyrene (PS) was combined with the grafting of protein-repellent poly(ethylene glycol) (PEG), in order to form topographical and chemical surface micropatterns consisting in protein-adhesive PS domains surrounded by protein-repellent PEG-grafted PLGA films. The produced micropatterned surfaces were used for site-specific protein adsorption, and represent a promising platform for biological applications, such as proteomics, single-cell studies and tissue engineering. Spatially ordered surface micropatterns were obtained by combining polymer dewetting with microcontact printing and colloidal lithography, respectively. The dewetting of thin PS films was guided within specific regions of the substrate by prestamping of the silicon substrate with self-assembled monolayers of an alkylsilane by microcontact printing. Ordered micropatterns consisting in arrays of holes with tunable size were obtained by exploiting the spontaneous dewetting of poly(4-vinyl pyridine) (P4VP) thin films on PS from the holes produced by colloidal imprinting with two-dimensional colloidal crystals assembled on the polymer bilayer

    Selective placement of actin filaments on protein patterned surfaces

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    Motors proteins are used by living organisms to convert chemical energy into mechanical energy. The human body uses such motors proteins to transport materials through cells and, in the case of the biomolecular motor system of actin and myosin, to contract muscle. By understanding how these biological motors work, artificial motors with improved function may be possible and may be engineered to work in complex biological and non-biological environments. Recent research efforts have focused on understanding how to harness the power of, and manipulate the functioning of biological motors for integration into useful nanoscale systems. One important step towards this integration is the binding of motor proteins onto substrates and the full characterization of the system. The aim of this thesis was to study the feasibility of selective immobilization of actin filament motor protein based on the bioaffinity reaction between patterned streptavidin on a substrate and biotinylated actin filaments on an aminopropyltriethoxysilane (APTES)-functionalized glass surface. Gelsolin was used to cap the barbed/positive end of actin and to link actin to biotin molecules on the functionalized surface. Results demonstrate significant binding of actin filaments on streptavidin patterned surfaces via bioaffinity immobilization. Fluorescent microscopy and image processing software were used to characterize these results. Characterization of the APTES-functionalized surface was conducted using atomic force microscopy (AFM). The relationship between actin and gelsolin capping protein was examined as well as non-specific binding control of actin filaments

    Use of Self-Assembled Monolayers to Tailor Surface Properties: From Lubrication to Neuronal Development

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    The subsequent work describes advances in modifying the chemical properties of various substrates to tailor the surface properties for specific applications. This is achieved by making use of a molecular assembly known as self-assembled monolayers, or SAMs. SAMs are composed of tightly packed organic molecules that form a well-ordered structure on a substrate. Typically, the head group of the monomer is covalently anchored to the substrate, and monolayer order and self-assembly is achieved through van der Waals interactions between the long alkyl chains of the monomer\u27s tail group. Monolayers containing head groups consisting of thiols, siloxanes, and phosphonates have been demonstrated on gold, glass, and metal oxides, respectively. We have expanded upon existing monolayer technology and designed monolayers with either new head group or new tail group functionalities. The resulting surfaces have been characterized by a variety of techniques including infrared spectroscopy, contact angle analysis, quartz crystal microbalance analysis, surface plasmon resonance imaging, and atomic force microscopy. We have also explored applications for these functionalized surfaces in areas ranging from microelectromechanical systems: MEMS) lubrication to platforms for studying neuronal development in vitro. In the area of MEMS lubrication, the development of new surface coatings is critical for combating wear and increasing the device lifetime. We reported a class of arsonic acid SAMs that form readily on oxide substrates including silicon oxide, borosilicate glass, and titanium oxide. The monolayers are easily prepared using a straightforward soaking technique, which is amenable to large-scale commercial applications. We have characterized monolayer formation on borosilicate glass and titanium oxide using infrared spectroscopy. Monolayers on borosilicate glass, native silicon oxide and titanium oxide were also evaluated with contact angle measurements, and as wear measurements using nanoscratching experiments. On titanium oxide and borosilicate glass, monolayers prepared from hexadecylarsonic acid provide significantly greater surface protection than surfaces reacted under similar conditions with hexadecylphosphonic acid, a common modifying agent for oxide substrates. To develop a platform for in vitro studies of neuronal development, we have utilized mixed-monolayers incorporating low densities of cell-adhesive peptides. The monomers feature a tetraethylene glycol moiety in the tail group to prevent the non-specific adsorption of proteins, and a low density of monomers were terminated with an azide moiety to specifically attach a laminin-derived peptide: IKVAV) terminated with an alkyne group via the copper-mediated azide-alkyne cycloaddition: CuAAC) reaction. To achieve this, a pentynoic acid molecule was appended to the N-terminus of the peptide during solid phase synthesis. Surfaces containing 0.01% and 0.1% azide-coupled peptide were determined to be resistant to the non-specific adsorption of proteins. Hippocampal neurons dissected from embryonic mice were cultured on these surfaces and the effects of the peptides on neurite outgrowth were observed. Similar neurite numbers per cell were observed on both substrates, but longer neurites were measured on the 0.1% azide-coupled peptide substrate. Unfortunately, further studies revealed that aldehyde fixation methods for immunohistochemistry did not successfully attach neuronal cells to the surface due to limited attachment points on the surface. Many developmental cell biology experiments require downstream immunohistochemical analysis. As such, to overcome this limitation and to simplify the surface preparation, a protein-resistant intermolecular zwitterionic monolayer, which supports cell fixation, was utilized. We have shown that the intermolecular zwitterionic monolayer has well-defined, non-receptor mediated cellular attachment provided by cell-surface sugar interactions. Exploiting these properties, we have developed a monolayer stripe assay, where the interactions between neurons: cell bodies and neurites) and extracellular matrix: ECM) proteins or guidance cues can be observed and quantified. This system goes beyond current technologies and is capable of evaluating neuronal response to the extracellular matrix protein, laminin, which has previously been considered a control molecule in neuronal stripe assays. Taken together, this work highlights advancements in the field of self-assembled monolayer chemistry with practical applications. In particular, we have focused on the functionalization of glass and oxides surfaces for applications in device lubrication. As well, we have developed two alkanethiol self-assembled monolayer approaches for generating surfaces that are both protein resistant and cell permissive, advancing the tools available for studying neuronal development in vitro

    Engineering biocompatible surfaces from the nano to the micro scale

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    One of the important challenges in surface bioengineering is the fabrication of robust and regular supramolecular structures, tuning the chemical and topographical properties of the surface, in order to include functional biomolecules, which preserve their activity and/ or induce the desired biological process.Laccases, are redox enzyme, that catalyse the oxidation of a broad range of polyphenols and aromatic substrates. Wide variety of application in the industry has been reported, and lately their use for biosensors development. Bacterial S-layers are very interesting systems since they self-assemble forming 2-D crystals (S-layers) on many type of surfaces, and they can be fused with other biomolecules maintaining their functionality.HegG2 cell line is an hepatoma cell line that has been used for cancer research. These cells maintain part of the normal metabolic capacity of hepatocytes, what make them a useful tool for high-throughput in vitro toxicity assays, as well as in the development of bioartificial livers. The objective of this work was to generate biocompatible surfaces to immobilise lacase, S-layers and HepG2 cells, on which they mantain they active. Different surfaces with defined functionalities have been constructed with synthetic polyelectrolytes, using the layer-by-layer technique and soft lithography. At the nanoscale, an enzyme (laccase) was covalently immobilised on a gold/polyethylenimineI/glutaraldhyde layer, preserving its activity. The immobilisation was studied with a quartz crystal microbalance with dissipation monitoring (QCM-D) and its activity assayed with a spectrophotometer.Besides, bacterial surface proteins (SbpA, SbpA-EGFP and SbpA-STV) were adsorbed on polyelectrolytes multilayers. The combination of soft-lithography with a protein resistant polyelectrolyte (PLL-g-PEG) led to the construction of micro-structured surfaces of functional bacterial proteins. Surface wetability, fluorescence and atomic force microscopy (AFM) were used to characterise those interfaces.Increasing the complexity, attachment of HepG2 cells on polyelectrolytes was studied. Cell adopted different morphologies depending on the hosting underlying polyelectrolyte as observed by transmission, scanning electron and atomic force microscopes. The adhesion and spreading of the cells that were monitored with QCM-D and transmission microscopy, and assayed with crystal violet, showed a higher affinity of the cells toward the adlayer formed on PEI, PAH and PLL in comparison with PSS and PLL-g-PEG. Force spectroscopy studies with AFM showed higher repulsion between PSS surfaces and the cell surface, and different local cell mechanical properties between cells attached to PEI and PSS.La ingeniería de biomateriales busca obtener materiales biológicamente activos, ajustando las propiedades químico-físicas (y topográficas) de la superficie de interés. Las lacasas, son enzimas que catalizan la oxidación de un gran número de polifenoles con aplicación en la industria, así como en el desarrollo de biosensores. Las proteínas bacterianas S, son sistemas muy interesantes ya que se auto-ensamblan formando cristales en 2-D y se pueden fusionar con otras biomoléculas. La línea celular hepática cancerosaHepG2, se ha empleado en estudios relacionados con el cáncer, citotoxicidad , se ha incorporado en dispositivos extracorporales para suplir funciones hepáticas, ya que a pesar de su transformación mantienen ciertas funciones de los hepatocitos normales. El objetivo de este trabajo fue generar superficies biocompatibles en las que se inmovilizó lacasa y se adsorbieron proteínas (de fusión) bacterianas y células, comprobando su funcionalidad.El ajuste de la química superficial se realizó por adsorción capa tras capa de polielectrolitos y para su estructuración se utilizó litografía blanda ("micro-contact printing"). La lacasa se inmobilizó covalentemente sobre oro cubierto con PEI (polietilenimina) través de gluraldehído, para posteriormente recubrirla con otros polielectrolitos. Dicho proceso se monitoreó con QCM (microblanza de cuarzo) y espectrofotometría (actividad enzimática). Las proteínas bacterianas se adsobieron selectivamente sobre muliticapas de polielectrolitos previamente estructuradas. Su funcionalidad se comprobó usando AFM (microscopía de fuerza atómica) y microscopía de fluorescencia. Las células HepG2 se inmobilizaron sobre superficies homogéneas y estructuradas de con distintos polielectrolitos. Se comprobó su viabilidad por ensayos con MTT y se observaron con SEM (microscopía de escaneo de electrones). El proceso de adhesion de las células sobre las multicapas de polielectrolos se estudió en función del tiempo con QCM y microscopía de transmisión, y fue testado con cristal violeta. Se utilizó AFM para estudiar la interacción entre las células con los polielectrolitos.El protocolo de inmovilización usado es aplicable para la lacasa, la cual conserva su actividad catalítica en la presencia de ABTS. La enzima se recubrió con multicapas de polielectrolitos pero la determinación de su actividad se ve dificultada por la interferencia con el ABTS.Por primera vez se utilizó "micro-contact printing" para construir superficies estructuradas, en las que se adsorbieron proteínas (de fusión) S, generando superficies funcionales en escala nanométrica dentro de microestructuras. Cabe destacar que la posibilidad de manipular la distribución de la proteína en escala micro es un requerimiento básico para el desarrollo de biosensores. En lo que se refiere a la interacción célula/superficie, se encontró que las células HepG2 adoptan diferente morfología dependiendo del substrato al que se adhieren. Mientras polielectrolitos positivos (PEI, PAH) adsorben moléculas que inducen la expansión celular, PLL-g-PEG (interfase neutra e hidrofílica) y PSS (polielectrolito negativo) no favorecieron la expansión celular. El QCM-D revelan que las células no son detectadas cuando se depositan sobre PSS (lo opuesto sucede cuando se adhieren sobre PEI) aunque se mantiene adheridas. Las propiedades mecánicas de las células varían de acuerdo al sustrato al que se adhieren, más rígidas sobre PEI. La máxima adhesión de las puntas (funcionarizadas con PEI y PSS) del AFM a la superficie celular es independiente de la carga aplicada y de su recubrimiento, para un tiempo nulo de residencia de la punta sobre la superficie celular. La máxima adhesión observada fue de 750 pN para un tiempo de residencia de residencia de 3 s, cuando el tip fue funcionalizado con PEI
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