High-voltage 10 ns delayed paired or bipolar pulses for in vitro bioelectric experiments

International audienc

Experimental and numerical characterization of a grounded coplanar waveguide for nanoelectroporation applied to liposomes

AbstractElectroporation has become a powerful technological platform for the electromanipulation of cells and tissues for various medical and biotechnological applications. Recently, nanoporation based on nanosecond pulsed electric fields (nsPEFs) has gained great attention due to its potential to permeabilize the membrane of small vesicles. Here, the authors propose and characterize, both experimentally and through multiphysics modeling, a grounded coplanar waveguide compliant with the wideband requirements for nanosecond pulses to be used for experiments of drug delivery with liposomes activated by nsPEFs

Nanomedicine applications mediated by electromagnetic fields

Recently, the introduction of nanotechnologies into medical applications has become more frequent due to the growing of several diseases originating from alteration of biological processes at molecular and nanoscale level (e.g. mutated genes, cell malfunction due to viruses or bacteria). The nanomedicine combines the innovation of the nanotechnology materials (shape and size of nm scale) to health care, providing new promising techniques for the diagnosis, the prevention, the tissue regeneration and therapeutic fields. Disorders like cancer, Alzheimer’s, Parkinson’s disease, cardiovascular problems or inflammatory diseases are serious challenges to be dealt with. For this reason researches are focusing their attention to the nanomaterials unique properties [Murty et al., 2013, Xia et al., 2009]. The progress in nanomedicine ranges from nanoparticles for molecular diagnostics, imaging and therapy to integrated medical nanosystems [Nune et al., 2009, Shi, 2009] to act at the cellular level inside the body. For a recent review on challenges, opportunities, and clinical applications in nanomedicine an interesting review is the one of Wicki et al. [Wicki et al., 2015]. Despite the concerns raised by the authors in their review, the expert opinion on clinical opportunities finds a generalized consensus on stimuli-responsive systems for targeting the compound (drug, gene, biomolecule) at the site of interest and on the use of lipid based nanosystems for the biocompatible platform to be used in clinical trials. In this scenario is placed the main activity of this Ph.D. thesis whose aim is to provide a multiscale and multidisciplinary approach to demonstrate the capability to activate lipid-based nanosystems by means of electromagnetic fields (EMFs). Specifically, the attention will be focused, on a first part, on the liposome-based systems mediated by EMF to provide a proof-of-concept of EMF stimuli-response systems for applications of drug delivery. This aspect will be approached both form a theoretic, technological and experimental point of view. Moreover, because proteins are considered a fundamental pattern as bio-sensors for signaling cell processes, a molecular dynamics simulation approach will be provided to study the interaction mechanisms between EMFs and proteins structures for potential protein activation

Defining cellular microenvironments using multiphoton lithography

textTo understand the chemistry of life processes in detail is largely a challenge of resolving them in their native, cellular environment. Cell culture, first developed a century ago, has proven to be an essential tool for reductionist studies of cellular biochemistry and development. However, for the technology of cell culture to move forward and address increasingly complex problems, in vitro environments must be refined to better reflect the cellular environment in vivo. This dissertation work has focused on the development of methods to define cellular microenvironments using the high resolution, 3D capabilities of multiphoton lithography. Here, site-specific photochemistry using multiphoton excitation is applied to the photocrosslinking of proteins, providing the means to organize bioactive species into well-defined 3D microenvironments. Further, conditions have been identified that enable microfabrication to be performed in the presence of cells -- allowing cell outgrowth and motility to be directed in real time. In addition to the intrinsic chemical functionality of microfabricated protein structures, 3D protein matrices are shown to respond mechanically to changes in the chemical environment, enabling new avenues for micro-scale actuation to be explored. Complex 2D and 3D protein photocrosslinking is further facilitated by integrating transparency and automated reflectance photomasks into the fabrication system. These advances could be transformative in efforts to fabricate precise cellular scaffolding that replicates the morphological (and potentially biochemical) features of in vivo tissue microenvironments. Finally, these methods are applied to the study of microorganism behavior with single-cell resolution. Microarchitectures are designed that allow the position and motion of motile bacterial to generate directional microfluidic flow -- providing a foundation to develop micro-scale devices powered by cells.Chemistry and BiochemistryBiochemistr

Glioma on Chips Analysis of glioma cell guidance and interaction in microfluidic-controlled microenvironment enabled by machine learning

In biosystems, chemical and physical fields established by gradients guide cell migration, which is a fundamental phenomenon underlying physiological and pathophysiological processes such as development, morphogenesis, wound healing, and cancer metastasis. Cells in the supportive tissue of the brain, glia, are electrically stimulated by the local field potentials from neuronal activities. How the electric field may influence glial cells is yet fully understood. Furthermore, the cancer of glia, glioma, is not only the most common type of brain cancer, but the high-grade form of it (glioblastoma) is particularly aggressive with cells migrating into the surrounding tissues (infiltration) and contribute to poor prognosis. In this thesis, I investigate how electric fields in the microenvironment can affect the migration of glioblastoma cells using a versatile microsystem I have developed. I employ a hybrid microfluidic design to combine poly(methylmethacrylate) (PMMA) and poly(dimethylsiloxane) (PDMS), two of the most common materials for microfluidic fabrication. The advantages of the two materials can be complemented while disadvantages can be mitigated. The hybrid microfluidics have advantages such as versatile 3D layouts in PMMA, high dimensional accuracy in PDMS, and rapid prototype turnaround by facile bonding between PMMA and PDMS using a dual-energy double sided tape. To accurately analyze label-free cell migration, a machine learning software, Usiigaci, is developed to automatically segment, track, and analyze single cell movement and morphological changes under phase contrast microscopy. The hybrid microfluidic chip is then used to study the migration of glioblastoma cell models, T98G and U-251MG, in electric field (electrotaxis). The influence of extracellular matrix and chemical ligands on glioblastoma electrotaxis are investigated. I further test if voltage-gated calcium channels are involved in glioblastoma electrotaxis. The electrotaxes of glioblastoma cells are found to require optimal laminin extracellular matrices and depend on different types of voltage-gated calcium channels, voltage-gated potassium channels, and sodium transporters. A reversiblysealed hybrid microfluidic chip is developed to study how electric field and laminar shear can condition confluent endothelial cells and if the biomimetic conditions affect glioma cell adhesion to them. It is found that glioma/endothelial adhesion is mediated by the Ang1/Tie2 signaling axis and adhesion of glioma is slightly increased to endothelial cells conditioned with shear flow and moderate electric field. In conclusion, robust and versatile hybrid microsystems are employed for studying glioma biology with emphasis on cell migration. The hybrid microfluidic tools can enable us to elucidate fundamental mechanisms in the field of the tumor biology and regenerative medicine.Okinawa Institute of Science and Technology Graduate Universit

Functional carbon nanotubes for photonic applications

Carbon nanomaterials are an active frontier of research in current nanotechnology. Single wall Carbon Nanotube (SWNT) is a unique material which has already found several applications in photonics, electronics, sensors and drug delivery. This thesis presents a summary of the author’s research on functionalisation of SWNTs, a study of their optical properties, and potential for an application in laser physics. The first significant result is a breakthrough in controlling the size of SWNT bundles by varying the salt concentrations in N-methyl 2-pyrrolidone (NMP) through a salting out effect. The addition of Sodium iodide leads to self-assembly of CNTs into recognizable bundles. Furthermore, a stable dispersion can be made via addition polyvinylpyrrolidone (PVP) polymer to SWNTs-NMP dispersion, which indicates a promising direction for SWNT bundle engineering in organic solvents. The second set of experiments are concerned with enhancement of photoluminescence (PL), through the formation of novel macromolecular complexes of SWNTs with polymethine dyes with emission from enhanced nanotubes in the range of dye excitation. The effect appears to originate from exciton energy transfer within the solution. Thirdly, SWNT base-saturable absorbers (SA) were developed and applied to mode locking of fibre lasers. SWNT-based SAs were applied in both composite and liquid dispersion forms and achieved stable ultrashort generation at 1000nm, 1550nm, and 1800 nm for Ytterbium, Erbium and Thulium-doped fibre laser respectively. The work presented here demonstrates several innovative approaches for development of rapid functionalised SWNT-based dispersions and composites with potential for application in various photonic devices at low cost

MEMS-based Lab-on-chip platform with integrated 3D and planar microelectrodes for organotypic and cell cultures

La presente tesis doctoral se centra en el desarrollo y la validación de plataformas lab on chip (LOC) para su aplicación en el campo de la Biología, la Medicina y la Biomedicina, particularmente relacionados con el cultivo de células y tejidos, así como su tratamiento mediante electroestimulación y su actividad eléctrica. Actualmente, las investigaciones centradas en el desarrollo de LOCs han experimentado un crecimiento considerable, gracias, en gran medida, a la versatilidad que ofrecen. Dicha versatilidad se traduce en numerosas aplicaciones, de las cuales, aquellas relacionadas con la Biología y la Medicina, están alcanzando especial relevancia. La integración de sensores, actuadores, circuitos microfluídicos y circuitos electrónicos en la misma plataforma, permite fabricar sistemas con múltiples aplicaciones. Esta tesis se centra fundamentalmente en el desarrollo de plataformas para el cultivo in vitro de tejidos y células, así como para la monitorización y la interacción con dicho cultivo. Los cultivos in vitro resultan de vital importancia para realizar estudios en células o tejidos, experimentar con medicamentos o estudiar su proliferación y morfología. De esta manera, se cubriría la creciente necesidad de encontrar una alternativa para replicar modelos humanos de enfermedades in vitro para poder desarrollar nuevos fármacos y avanzar en la medicina personalizada. Por tanto, la posibilidad de realizar cultivos de media o larga duración en plataformas que no precisen de un equipamiento costoso como las incubadoras de CO2 y que puedan ser monitorizadas mediante aplicaciones ópticas, supone un salto cualitativo en el desarrollo de los cultivos in vitro. En este contexto, se presenta el trabajo relacionado con esta tesis que ha sido desarrollada dentro del grupo de Microsistemas de la Escuela Superior de Ingeniería de la Universidad de Sevilla. La tesis está estructurada de manera que a lo largo de este escrito se da respuesta a los distintos aspectos anteriormente descritos. En primer lugar, se hace una breve introducción a la tecnología MEMS y a los principios básicos de la microfluídica. Dado que este trabajo se ha desarrollado en un ambiente multidisciplinar, esta sección resulta necesaria para dar una visión general a aquellos no familiarizados con esta disciplina. Tras esa introducción se realiza una descripción del estado del arte en el que se encuadra este trabajo, incluyendo los sistemas LoCs, y sus principales aplicaciones en el campo de la Biología, Medicina y Biomedicina, prestando especial atención a las aplicaciones de los LoCs relacionadas con cultivos organotípicos y de células. Tras la introducción y el estado del arte en el que se enmarca la tesis, se explican los resultados obtenidos durante este trabajo. Durante la primera parte, se describe el desarrollo, fabricación y caracterización de un sistema autónomo para el cultivo y electroestimulación de tejidos que integra un lab on PCB (LOP) formado por un array de microelectrodos en 3D (MEA) formado por hilos de oro de 25 µm en sustrato transparente, sensores y actuadores, junto con una plataforma microfluídica fabricada en metacrilato (PMMA) y polidimetilsiloxano (PDMS). El LOP permite mantener las condiciones de temperatura idóneas para llevar a cabo cultivos de media-larga duración sin necesidad de usar incubadoras deCO2 , así como su seguimiento de forma continua a través de un microscopio, gracias al uso de materiales transparentes. Este sistema también incluye una electrónica suplementaria y un programa que permite la monitorización del sistema y la electrostimulación de la muestra biológica. Tras explicar detalladamente el diseño y el novedoso proceso de fabricación del LOP, se presentan los resultados experimentales. En primer lugar, se ha demostrado que es posible desarrollar cultivos organotípicos de retinas de ratón durante más de 7 días, obteniendo resultados muy similares a los conseguidos para las mismas muestras biológicas, pero mediante métodos de cultivo tradicionales. Además, se ha logrado la neuro-protección mediante la electroestimulación de retinas de ratón con la enfermedad de la retinosis pigmentaria, logrando de esta manera ralentizar la degeneración de la muestra debido a la enfermedad. Otra de las aplicaciones que se quiere conseguir con el desarrollo del LOP anteriormente descrito se centra en la adquisición de señales eléctricas procedentes de las muestras biológicas cultivadas en el dispositivo, así como extrapolar su uso a cultivos celulares. Para la adquisición de señales procedentes del cultivo, la impedancia de los electrodos fabricados con hilos de oro de 25 µm ha resultado ser demasiado alta como para discernir entre el ruido base y la actividad eléctrica del cultivo. Por ello, la segunda parte de esta tesis doctoral se centra en la mejora de la MEA para la adquisición de actividad eléctrica. Dado el objetivo marcado en esta segunda parte, durante esta tesis se ha realizado una estancia en la Universidad de Bath. En dicha estancia, se ha caracterizado la actividad eléctrica de células del cáncer de próstata (PC-3), que fueron cultivadas en chips de silicio con electrodos de oro. La experiencia obtenida durante la estancia ha permitido avanzar en el desarrollo y la fabricación de nuevas MEAs para la adquisción de señales eléctricas de cultivos celulares. La primera aproximación para mejorar la MEA se ha realizado sobre PCB. Se trata de un dispositivo compuesto por pilares de oro en 3D fabricados mediante la técnica de Resumen XXV electroplating. Estos electrodos tienen 100 µm de diámetro y una altura de 25 µm que aseguran el contacto en el caso de cultivos de tejidos. Se ha demostrado una mejora significativa, traducida tanto en una impedancia más baja, como en una línea base de ruido menor con respecto a la MEA con hilos de oro. Asimismo, se han obtenido patrones de actividad eléctrica en las células PC-3 muy similares a los obtenidos con el chip de silicio y oro empleado en la estancia. Como mejora de la MEA 3D se ha cambiado el sustrato por otro transparente, como vidrio o PMMA, para permitir su uso en aplicaciones ópticas. Dichas MEAs integran electrodos planares fabricados mediante la técnica de sputtering de oro sobre su superficie. Estas MEAs están en una fase preliminar de desarrollo, y se está probando en primer lugar su biocompatibilidad y viabilidad para el desarrollo de cultivos celulares. Para finalizar, se exponen las conclusiones de esta tesis doctoral, entre las que destacan: el proceso de fabricación del LOP con electrodos de oro y la aplicación del sistema completo para desarrollar cultivos organotípicos, monitorizarlos y aplicar electroestimulación, logrando la neuro-protección de retinas de ratón con la retinosis pigmentaria; la transición hacia el desarrollo de una plataforma para cultivos celulares mejorando la MEA y su fabricación usando diferentes sustratos; la caracterización de la actividad eléctrica de las células PC-3. También se incluyen las líneas de investigación abiertas para continuar lo que se ha empezado en esta tesis doctoral. Para facilitar la comprensión del lector, se adjuntan los apéndices complementarios a esta tesis doctoral.The presented thesis is focused on the development and validation of lab on chip (LOC) platforms for their application on Biology, Medicine and Biomedicine, particularly those related with cells and tissues cultures, as well as their treatment through electrostimulation and their electrical behavior. Nowadays, research works focused on the development of LOCs have significantly increased, mostly thanks to its high versatility, which involves countless applications. Among all this applications, those related with Biology and Medicine are becoming more and more important. The integration of sensors, actuators, microfluidic circuits and electronic circuits in the same platform allows the fabrication of systems with lots of applications. This thesis is focused on the development of platforms for in vitro cultures of cells and tissues, to monitor their behavior and interact with the biological samples. The importance of in vitro cultures lies on the study of cells and tissues proliferation and morphology or performing drug delivery experiments. In this respect, through LOC technologies, it would be possible to model human diseases in vitro, in order to improve the development of new drugs and advance personalized medicine. Thus, the possibility of carrying out medium-long term cultures on platforms without the need of any expensive equipment, such as CO2 incubators, with software and monitoring, implies a qualitative step forward in the development of in vitro cultures. Within this framework, the work related to this thesis is presented. This PhD has been undertaken in the Microsystem group of the High School Engineering of the University of Seville. The structure of this thesis is organized in such a way that, all along the text, the different aspects previously described are explained in detail. Firstly, a brief introduction about MEMS technology and the basic principles of Microfluidics is presented. Due to this work has been developed in a multidisciplinary environment, this section becomes necessary in order to give a wide view to those non XXVII XXVIII Abstract directly familiarized with these fields. Subsequently, a description of the state of the art is presented, including LOC systems and their applications in Biology, Medicine and Biomedicine, taking special attention to those applications related to organotypic and cell cultures. After the introduction and the state of the art of the framework of this thesis, the results obtained are presented. In the first part of this PhD, the development, fabrication and characterization of the autonomous system for culture and electrostimulation of tissues is described. This system includes a lab on PCB (LOP) composed of a 3D microelectrode array (MEA), with gold wires of 25 µm on transparent substrate, sensors and actuators, together with a microfluidic platform made of PMMA and PDMS. This LOP allows to maintain the appropriate temperature conditions to carry out medium-long term cultures without using a CO2 incubator, as well as its continuous monitoring through an inverted microscope, thanks to the transparent materials used for its fabrication. This system is connected to an external electronic circuit and a software to control the whole system, including the electrostimulation of the biological sample. After explaining the design and the innovative fabrication process of the LOP, the experimental results are presented. Firstly, it has been demonstrated the suitability of this system to perform organotypic cultures of mice retinas for longer than 7 days, obtaining similar results to the same samples, but cultured through traditional methods. In addition, it has been provided neuroprotection to mice retinal explants with the retinitis pigmentosa (RP) disease through the electrostimulation of the samples, being able to slowdown the degeneration of the retinas caused by RP

Optimization of Continuous Flow Polymerase Chain Reaction with Microfluidic Reactors

The polymerase chain reaction (PCR) is an enzyme catalyzed technique, used to amplify the number of copies of a specific ~gion ofDNA. This technique can be used to identify, with high-probability, disease-causing viruses and/or bacteria, the identity of a deceased person, or a criminal suspect. Even though PCR has had a tremendous impact in clinical diagnostics, medical sciences and forensics, the technique presents several drawbacks. For example, the costs associated with each reaction are high and the reaction is prone to cont,amination due its inherent efficiency and high sensitivity. By employing microfluidic' systems to perform PCR these advantages can be circumvented. This thesis addresses implementation issues that adversely affect PCR . in microdevices and aims to improve the efficiency of the reaction by introducing novel materials and methods to existing protocols. Molecule-surface-interactions and ,' temperature control/determination are the main focus within this work. Microchannels and microreactors are char:acterized by extremely high surface-tovolume ratios. This dictates that surfaces play a dominant role in defining the efficiency ofPCR (and other synthetic processes) through increased molecule-surface interactions. In a multicomponent reaction system where the concentration of several components needs to be maintained the situation is particularly complicated. For example, inhibition of PCR is commonly observed due to polymerase adsorption on channel walls. Within??????? this work a number of different surface treatments have been investigated with a view to minimizing adsorption effects on microfluidic channels. In addition, novel studies introducing the use of superhydrophobic coatings on microfluidic channels are presented. Specifically superhydrophobic surfaces exhibiting contact angles in excess of 1500 have been created by growing Copper oxide and Zinc oxide' nanoneedles and silica-sol gel micropores on microfluidic channels. Such surfaces utilize additional surface roughness to promote hydrophobicity. Aqueous solutions in contact with superhydrophobic surfaces are suspended by bridging-type wetting, and therefore the fraction of the surface in contact with the aqueous layer is significantly lower than for a flat surface. An additional difficulty associated with PCR on microscale is the detennination and control of temperature. When perfonning PCR, the ability to accurately control system temperatures is especially important since both primer annealing to singlestranded DNA and the catalytic extension of this primer to fonn the complementary strand will only proceed in an efficient manner within relatively narrow temperature ranges. It is therefore imperative to be able to accurately monitor the temperature distributions in such microfluidic channels. In this thesis, fluorescence lifetime imaging (FLIM) is used as a novel method to directly quantify temperature within microchannel environments. The approach, which includes the use of multiphoton e'xcitation to achieve optical sectioning, allows for high spatial and temporal resolution, operates over a wide temperature range and can be used to rapidly quantify local temperatures with high precision.Imperial Users onl