Various aspects of single bacterial cell: from encapsulating nanoparticle, cell patterning and studying gene expression

Behaviour of single bacterium have been mysterious for many years due to the technical difficulty and nature of bacteria forming communities. It has not been long since engineers and scientists have started to develop various techniques and devices to study and isolate single bacterium. In the present thesis, three subprojects with a common theme of applying single bacterium cells for engineering application or investigating regulation of gene expressions is introduced. Recently, metal structures with micrometer dimensions have been fabricated and successfully utilized to isolate single bacterial cells with dimensions similar to the designed pillars. First project further develops previously discovered feature of single bacterial cell isolation by metal hollow structures to capture polymeric nanoparticles inside the hollow structures. High achievement rates of filling the nanoparticles inside the structures and subsequently capping the top opening of hollow pillars have been attained. The study demonstrates that the shape of structures affects both the capturing rate and capping rate of nanoparticles. In the second project, a fabrication method and result of simple template to obtain well organized array of single bacterial cells is shown. The bacterial cells with size less than micrometer are patterned on a gold substrate in a uniform matter. The technique developed here requires no additional binding agent nor chemical modification of the substrate which provides an advantage in saving the processing time and cost in comparison to existing techniques developed to isolate single bacterial cells. The isolated bacterial cells are further engineered with magnetic nanoparticles to illustrate bacterial cell wall property remaining the constant after deposition. In the last project, promoter activity of single cells has been analyzed to study the population behaviour of the bacterial culture. In contrast to the assumption that the behaviour of a single bacterium would be identical as a group, the opposite behaviour was found. In the thesis, phenotypic heterogeneity within a promoter that may correlate with the external morphology is shown. During the experiment, two different morphology of cells were observed to exist in a single culture, a short cell and a long elongated cell and the promoter activity correlated with the morphology where the short body ones had higher activity in the promoter compared to the long ones. A fluorescence reporter assays are experimented and evaluated in attempt to reveal the reason behind such heterogeneity

Various aspects of single bacterial cell: from encapsulating nanoparticle, cell patterning and studying gene expression

Behaviour of single bacterium have been mysterious for many years due to the technical difficulty and nature of bacteria forming communities. It has not been long since engineers and scientists have started to develop various techniques and devices to study and isolate single bacterium. In the present thesis, three subprojects with a common theme of applying single bacterium cells for engineering application or investigating regulation of gene expressions is introduced. Recently, metal structures with micrometer dimensions have been fabricated and successfully utilized to isolate single bacterial cells with dimensions similar to the designed pillars. First project further develops previously discovered feature of single bacterial cell isolation by metal hollow structures to capture polymeric nanoparticles inside the hollow structures. High achievement rates of filling the nanoparticles inside the structures and subsequently capping the top opening of hollow pillars have been attained. The study demonstrates that the shape of structures affects both the capturing rate and capping rate of nanoparticles. In the second project, a fabrication method and result of simple template to obtain well organized array of single bacterial cells is shown. The bacterial cells with size less than micrometer are patterned on a gold substrate in a uniform matter. The technique developed here requires no additional binding agent nor chemical modification of the substrate which provides an advantage in saving the processing time and cost in comparison to existing techniques developed to isolate single bacterial cells. The isolated bacterial cells are further engineered with magnetic nanoparticles to illustrate bacterial cell wall property remaining the constant after deposition. In the last project, promoter activity of single cells has been analyzed to study the population behaviour of the bacterial culture. In contrast to the assumption that the behaviour of a single bacterium would be identical as a group, the opposite behaviour was found. In the thesis, phenotypic heterogeneity within a promoter that may correlate with the external morphology is shown. During the experiment, two different morphology of cells were observed to exist in a single culture, a short cell and a long elongated cell and the promoter activity correlated with the morphology where the short body ones had higher activity in the promoter compared to the long ones. A fluorescence reporter assays are experimented and evaluated in attempt to reveal the reason behind such heterogeneity

NANOPILLAR BASED ELECTROCHEMICAL BIOSENSOR FOR MONITORING MICROFLUIDIC BASED CELL CULTURE

In-vitro assays using cultured cells have been widely performed for studying many aspects of cell biology and cell physiology. These assays also form the basis of cell based sensing. Presently, analysis procedures on cell cultures are done using techniques that are not integrated with the cell culture system. This approach makes continuous and real-time in-vitro measurements difficult. It is well known that the availability of continuous online measurements for extended periods of time will help provide a better understanding and will give better insight into cell physiological events. With this motivation we developed a highly sensitive, selective and stable microfluidic electrochemical glucose biosensor to make continuous glucose measurements in cell culture media. The performance of the microfluidic biosensor was enhanced by adding 3D nanopillars to the electrode surfaces. The microfluidic glucose biosensor consisted of three electrodes - Enzyme electrode, Working electrode, and Counter electrode. All these electrodes were enhanced with nanopillars and were optimized in their respective own ways to obtain an effective and stable biosensing device in cell culture media. For example, the `Enzyme electrode\u27 was optimized for enzyme immobilization via either a polypyrrole-based or a self-assembled-monolayer-based immobilization method, and the `Working electrode\u27 was modified with Prussian Blue or electropolymerized Neutral Red to reduce the working potential and also the interference from other interacting electro-active species. The complete microfluidic biosensor was tested for its ability to monitor glucose concentration changes in cell culture media. The significance of this work is multifold. First, the developed device may find applications in continuous and real-time measurements of glucose concentrations in in-vitro cell cultures. Second, the development of a microfluidic biosensor will bring technical know-how toward constructing continuous glucose monitoring devices. Third, the methods used to develop 3D electrodes incorporated with nanopillars can be used for other applications such as neural probes, fuel cells, solar cells etc., and finally, the knowledge obtained from the immobilization of enzymes onto nanostructures sheds some new insight into nanomaterial/biomolecule interactions

Patterned magnetic nanostructures for biomedical applications

155 p.Las nanoestructuras magnéticas en forma de disco presentan características distintivas para aplicaciones nanomédicas. Este tipo de aplicaciones demandan un control preciso de la geometría y las dimensiones de las nanoestructuras para evaluar tanto su eficiencia como funcionalidad. En esta tesis se presenta una estrategia de fabricación sin máscara, versátil y económica para el diseño de elementos con momentos magnéticos altos a escala submicrométrica. Se evaluaron dos rutas de fabricación mediante litografía de interferencia utilizando resinas positivas y negativas en cada caso, incluyendo las ventajas y desventajas de cada ruta. Los pasos más críticos del proceso de fabricación, como es la liberación de los discos del sustrato, se optimizaron para preservar completamente las propiedades magnéticas del material. Una vez elegida la ruta más óptima y dada la posibilidad de la técnica de depositar multicapas de distintos materiales magnéticos y no magnéticos, se fabricaron y caracterizaron tres tipos de nanoestructuras con remanencia nula para aplicaciones biomédicas: nanodiscos de Permalloy y nanodiscos de hierro en estado vórtice e imanes antiferromagnéticos sintéticos. Dada su mayor facilidad de fabricación y mejores propiedades magnéticas para las aplicaciones biomédicas se eligieron las nanoestructuras en estado vórtice para los ensayos posteriores. Se estudió la dinámica de los discos sujetos a campos magnéticos alternos con distintas condiciones, y se evaluó la citotoxicidad en células de melanoma primario. No se observaron efectos de citotoxicidad, validando el uso de estas nanoestructuras en aplicaciones biomédicas

Bio-oriented Micro- and Nano- Structures Based on Stimuli-responsive Polymers

Nowadays, the ability to pattern surfaces on the micro- and nano- scale is the basis for a wide range of research fields. Over last few decades, a lot of processing technologies offer the possibility to fabricate complex 2D and 3D polymeric designs which are mostly static in nature since they cannot be physically and chemically modified once fabricated. The aim of the present thesis is to overcome such a limitation, exploiting stimuli-responsive materials (Chapter I). We allow to engineer polymeric architectures adding interesting functionalities, by providing an active manipulation of pre-structured systems, which could be helpfully in a wide variety of applications, such as biosensing and cell conditioning. In the first part of the present dissertation (Chapter II), a thermos-sensitive material is employed. We investigate the thermo-responsive behavior of Poly(N-isopropylacrylamide) (pNIPAAm)-based crosslinkable hydrogel as active binding matrix in optical biosensors. In this study, we propose an extension of surface plasmon resonance (SPR) and optical waveguide mode (OWS) spectroscopy, for in situ observation of nano-patterned hydrogel film that are allowed to swell and collapse by varying the external temperature of the aqueous environment. Weak refractive index contrast of hydrogel structures arranged in periodic pattern, is generally associated with intrinsically low diffraction efficiency. In order to enhance the intensity of diffracted light, the surface is probed by resonantly excited optical waveguide modes, taking advantage of the fact that the hydrogel can serve as optical waveguide (HOW) enabling the excitation of additional modes besides surface plasmons. Thus, we provide a hydrogel optical waveguide-enhanced diffraction measurements, taking advantage of strong electromagnetic field intensity enhancements that amplifies the weak diffracted light intensity. The main part of the thesis is focused in the study of azopolymer-containing materials, a specific class of light-responsive materials. Upon photon absorption, azobenzene undergo reversible trans-cis photoisomerization, which induces a substantial geometrical change of its molecular structure, that can be translated into larger-scale movements of the material below the glass transition temperature (Tg) of the polymer. In Chapter III, by exploiting the light-induced mass migration phenomenon, we demonstrate that an azopolymeric film patterned by soft imprinting technique, can be anisotropically deformed and consequent restored in its initial shape via single irradiation just by controlling the polarization state of the incident laser beam. We also propose that the light-driven morphological manipulation can induce anisotropic wettability changes. Lastly, a polarization driven birefringence effect on flat and structured surfaces is discussed. Chapter IV focuses in the design of novel azopolymeric systems, where the optical response is provided by azobenzene molecules, which doped two different host materials. The photo-responsive behavior and potential applications of azo compounds incorporated into either a soft elastomeric and in rigid matrix is discussed. Azo-embedded poly(dimethylsiloxane) (PDMS) is studied as tunable optical lens and an azo-doped photocurable commercial polymeric resin is developed to study the photo-mechanical transduction of a 3D suspended membrane fabricated by two photon lithography technique. In Chapter V, we propose a light-deformable azopolymeric micro-pillars patterned substrate as a biocompatible and “smart” platform for dynamic material-cell observation in 2D environment, modified by a holographic optical conditioning. The aim is to observe by time-lapse acquisitions, how an in situ deformation of a pre-patterned structure can influence cell functions and fate. Finally, in Chapter VI, general remarks of the present work are discussed, and directions for future perspective are summarized

Fabricación y caracterización de electrodos nanoestructurados para interfaces neurales no invasivas de eficiencia mejorada

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Físicas, leída el 07-04-2022Neurological disorders produce serious cognitive and motor disabilities, and they account for 7% of total global disease burden, measured in disability‐adjusted life years. In addition, the life expectancy has been continuously growing during the last decades and, as a consequence, neurodegenerative disorders are becoming more prevalent representing a larger part of the healthcare efforts and expenses. Every year, treating brain conditions accounts for 35% of Europe’s disease burden with a yearly cost of €798.000 million. Despite the efforts performed in the medical and science fields, the cure for most of the neurological disorders is far from being achieved. One of the most efficient strategies to treat neurological disorders is the use of implanted electrodes to produce neural electrical stimulation. Furthermore, electrodes are one of the main neural interfaces used in diagnostics techniques, and for the study of the neural activity in basic investigation, in vitro and in vivo. Despite their enormous potential, these electrodes face nowadays limitations. Generally, they are too big, producing unspecific stimulation that can lead to secondary effects. Their size reduction is limited by the associated impedance increase, which restricts their charge‐injection to the tissue...Los trastornos neurológicos producen graves discapacidades cognitivas y motoras y suponen actualmente el 7% de la carga global de morbilidad, medida en años de vida ajustados por discapacidad. Además, el aumento progresivo en la esperanza de vida de las últimas décadas ha incrementado la prevalencia de las enfermedades neurodegenerativas, produciéndose una mayor demanda de recursos médicos y un incremento del coste económico asociado. Cada año, el tratamiento de enfermedades cerebrales supone el 35% del gasto total médico europeo, con un coste de 798.000millones de euros. A pesar de los esfuerzos realizados en medicina y otras ciencias, actualmente no existe una cura para la mayoría de las enfermedades neurológicas. Una de las terapias más utilizadas en clínica es la estimulación eléctrica neuronal con electrodos implantados. Los electrodos se usan extensamente en técnicas de diagnóstico y en el estudio de la actividad neuronal, in vitro e in vivo. Sin embargo, a pesar de su enorme potencial, su uso lleva asociadas algunas limitaciones. En general son grandes, por lo que la estimulación neuronal no es localizada, pudiendo producir efectos secundarios, y la reducción de su tamaño lleva asociada un incremento de su impedancia, reduciéndose su capacidad de inyección de carga...Fac. de Ciencias FísicasTRUEunpu

Magnetic vortex nanodiscs for cancer cell destruction

145 p.Los nanodiscos de Permalloy (Ni80Fe20), pueden presentar el estado magnético vórtice, es decir, una configuración de espines curling. Gracias a esta configuración, las nanopartículas con forma de disco presentan remanencia nula, por lo que no se aglomeran en disolución, y responden rápidamente a un campo magnético de baja amplitud y frecuencia alineando el plano del disco con la dirección del campo. Esta oscilación, genera una fuerza capaz de dañar la membrana de células de cáncer o de liberar fármacos. En la presente Tesis, se han fabricado nanodiscos mediante una novedosa técnica de litografía, en estado magnético vórtice que se ha estudiado tanto experimentalmente como teóricamente. Por último, se ha evaluado la capacidad magneto-mecánica de los nanodiscos fabricados para destruir células de cáncer de pulmón, obteniendo resultados prometedores

RESISTIVE SWITCHING CHARACTERISTICS OF NANOSTRUCTURED AND SOLUTION-PROCESSED COMPLEX OXIDE ASSEMBLIES

Miniaturization of conventional nonvolatile (NVM) memory devices is rapidly approaching the physical limitations of the constituent materials. An emerging random access memory (RAM), nanoscale resistive RAM (RRAM), has the potential to replace conventional nonvolatile memory and could foster novel type of computing due to its fast switching speed, high scalability, and low power consumption. RRAM, or memristors, represent a class of two terminal devices comprising an insulating layer, such as a metal oxide, sandwiched between two terminal electrodes that exhibits two or more distinct resistance states that depend on the history of the applied bias. While the sudden resistance reduction into a conductive state in metal oxide insulators has been known for almost 50 years, the fundamental resistive switching mechanism is a complex phenomenon that is still long-debated, complex process. Further improvements to existing memristor performance require a complete understanding of memristive properties under various operation conditions. Additional technical issues also remain, such as the development of facile, low-cost fabrication methods as an alternative to expensive, ultra-high vacuum (UHV) deposition methods. This collection of work explores resistive switching within metal oxide-based memristive material assemblies by analyzing the fundamental physical insulating material properties. Chapter 3 aims to translate the utility and simplicity of the highly ordered anodic aluminum oxide (AAO) template structure to complex, yet more functional (memristive) materials. Functional oxides possessing ordered, scalable nanoporous arrays and nanocapacitor arrays over a large area is of interest to both the fields of next-generation electronics and energy storing/harvesting devices. Here their switching performance will be evaluated using conductive atomic force microscopy (C-AFM). Chapter 4 demonstrates a convective self-assembly fabrication method that effectively enables the synthesis of a low-cost solution processed memristor comprising binary oxide and perovskite ABO3 nanocrystals of varying diameter. Chapter 5 systematically compares the influence of inter-nanoparticle distance on the threshold switching SET voltage of hafnium oxide (HfO2) memristors. Utilizing shorter phosphonic acid ligands with higher binding affinity on the nanocrystal surface enabled a record-low SET voltage to be achieved. Chapter 6 extends the scope to the fine tuning of solution processed memristors with two types of perovskites nanocrystals. The primary advantage of nanocrystal memristors is the ability to draw from additional degrees of freedom by tuning the constituent nanocrystal material properties. Recent advancement of solution phase techniques enables a high degree of controllability over the nanocrystal size and structure. Thus, this work found in this dissertation aims to understand and decouple the effects of the geometric size and substitutional nanocrystal parameters on resistive switching

Present and future of surface-enhanced Raman scattering

The discovery of the enhancement of Raman scattering by molecules adsorbed on nanostructured metal surfaces is a landmark in the history of spectroscopic and analytical techniques. Significant experimental and theoretical effort has been directed toward understanding the surface-enhanced Raman scattering (SERS) effect and demonstrating its potential in various types of ultrasensitive sensing applications in a wide variety of fields. In the 45 years since its discovery, SERS has blossomed into a rich area of research and technology, but additional efforts are still needed before it can be routinely used analytically and in commercial products. In this Review, prominent authors from around the world joined together to summarize the state of the art in understanding and using SERS and to predict what can be expected in the near future in terms of research, applications, and technological development. This Review is dedicated to SERS pioneer and our coauthor, the late Prof. Richard Van Duyne, whom we lost during the preparation of this article