Detektion und Synthese eines liganden-gesteuerten Ionenkanals im mikrofluidischen Analysesystem

Membranproteine spielen eine wichtige Rolle bei physiologischen Prozessen wie Signalweiterleitung oder Immunreaktion. Deshalb stehen sie im Fokus der pharmakologischen Wirkstoffentwicklung und es besteht großes Interesse, Membranproteinbasierte Biosensoren zu entwickeln, die sich z.B. als Screening-Plattformen eignen. Allerdings stellt die Handhabung von Membranproteinen wegen ihrer amphiphilen Struktur eine große Herausforderung dar. Membranproteine werden meist in Zellkultur oder in bakteriellen Expressionssystemen synthetisiert. Diese Verfahren liefern aber oft nur eine geringe Ausbeute und erlauben wenig Kontrolle über die Expressionsbedingungen. Als alternativer Ansatz bietet sich stattdessen die in vitro Synthese von Proteinen an, die in einer zellfreien Umgebung stattfindet. Ziel der vorliegenden Arbeit war die Etablierung eines miniaturisierten Analysesystems, das Aktivitätsmessungen an in vitro synthetisierten Ionenkanälen erlaubt. Dafür wurde ein Labon- Chip entwickelt, der elektrochemische und optische Nachweismethoden in parallelen Anätzen ermöglicht. Als amphiphile Umgebung für die Inkorporation von Membranproteinen wurden vier verschieden biomimetische Membranaufbauten hinsichtlich ihrer Dichtigkeit und ihrer Reproduzierbarkeit untersucht. Als Methode fanden insbesondere die Impedanzspektroskpie und die Oberflächenplasmonen-Resonanzspektroskopie Anwendung. Die peptide cushioned Bilyer Lipid Membranes (pcBLM) eignete sich dabei am besten für Untersuchungen an Membranproteinen. Zur Detektion der Ionenkanalaktivität wurde eine neue Messmethode etabliert, die auf der Messung der Impedanz bei fester Frequenz basiert und u.a. eine Aussage über die Änderung des Membranwiderstandes bei Aktivierung erlaubt. Am Beispiel des nicotinischen Acetylcholinrezeptors (nAchR) konnte gezeigt werden, dass sich die Aktivität von Ionenkanälen mit dem entwickelten Chip-System nachweisen ließ. Die Spezifität der Methode konnte durch verschiedene Kontrollen wie die Zugabe eines nicht-aktivierenden Liganden oder Inhibition des Rezeptors nachgewiesen werden. Weiterhin konnte die in vitro Synthese des Ionenkanals a7 nAchR durch Radioaktivmarkierung nachgewiesen werden. Die Inkorporation des Rezeptors in die biomimetischen Membranen wurde mit Immunodetektion und elektrochemischen Methoden untersucht. Es zeigte sich, dass die funktionelle Inkorporation des a7 nAchR davon abhing, welcher biomimetische Membranaufbau verwendet wurde.

Spatio-temporal co-ordination of RhoA, Rac1 and Cdc42 activation during prototypical edge protrusion and retraction dynamics

The three canonical Rho GTPases RhoA, Rac1 and Cdc42 co-ordinate cytoskeletal dynamics. Recent studies indicate that all three Rho GTPases are activated at the leading edge of motile fibroblasts, where their activity fluctuates at subminute time and micrometer length scales. Here, we use a microfluidic chip to acutely manipulate fibroblast edge dynamics by applying pulses of platelet-derived growth factor (PDGF) or the Rho kinase inhibitor Y-27632 (which lowers contractility). This induces acute and robust membrane protrusion and retraction events, that exhibit stereotyped cytoskeletal dynamics, allowing us to fairly compare specific morphodynamic states across experiments. Using a novel Cdc42, as well as previously described, second generation RhoA and Rac1 biosensors, we observe distinct spatio-temporal signaling programs that involve all three Rho GTPases, during protrusion/retraction edge dynamics. Our results suggest that Rac1, Cdc42 and RhoA regulate different cytoskeletal and adhesion processes to fine tune the highly plastic edge protrusion/retraction dynamics that power cell motility.

Thermal Tracking and Estimation for Microprocessors

Due to increasing integration density and operating frequency of today's high performance processors, the temperature of a typical chip can easily exceed 100 degrees Celsius. However, the runtime thermal state of a chip is very hard to predict and manage due to the random nature in computing workloads, as well as the process, voltage and ambient temperature variability (together called PVT variability). The uneven nature (both in time and space) of the heat dissipation of the chip could lead to severe reliability issues and error-prone chip behavior (e.g. timing errors). Many dynamic power/thermal management techniques have been proposed to address this issue such as dynamic voltage and frequency scaling (DVFS), clock gating and etc. However, most of such techniques require accurate knowledge of the runtime thermal state of the chip to make efficient and effective control decisions. In this work we address the problem of tracking and managing the temperature of microprocessors which include the following sub-problems: (1) how to design an efficient sensor-based thermal tracking system on a given design that could provide accurate real-time temperature feedback; (2) what statistical techniques could be used to estimate the full-chip thermal profile based on very limited (and possibly noise-corrupted) sensor observations; (3) how do we adapt to changes in the underlying system's behavior, since such changes could impact the accuracy of our thermal estimation. The thermal tracking methodology proposed in this work is enabled by on-chip sensors which are already implemented in many modern processors. We first investigate the underlying relationship between heat distribution and power consumption, then we introduce an accurate thermal model for the chip system. Based on this model, we characterize the temperature correlation that exists among different chip modules and explore statistical approaches (such as those based on Kalman filter) that could utilize such correlation to estimate the accurate chip-level thermal profiles in real time. Such estimation is performed based on limited sensor information because sensors are usually resource constrained and noise-corrupted. We also took a further step to extend the standard Kalman filter approach to account for (1) nonlinear effects such as leakage-temperature interdependency and (2) varying statistical characteristics in the underlying system model. The proposed thermal tracking infrastructure and estimation algorithms could consistently generate accurate thermal estimates even when the system is switching among workloads that have very distinct characteristics. Through experiments, our approaches have demonstrated promising results with much higher accuracy compared to existing approaches. Such results can be used to ensure thermal reliability and improve the effectiveness of dynamic thermal management 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.

PDMS encasing system for integrated lab-on-chip Ag/AgCl reference electrodes

Poly(dimethylsiloxane) (PDMS) is an organosilicon polymer widely used in the fabrication of microfluidic systems to integrate biochips. In this study, we propose the use of an adapted PDMS mould for the creation of a miniaturized, reusable, reference electrode for in-chip electrochemical measurements. Through its integrated microfluidic system it is possible to replenish internal buffer solutions, unclog critical junctions and treat the electrode’s surface, assuring a long term reuse of the same device. Planar Ag/AgCl reference electrodes were microfabricated over a passivated p-type Silicon Wafer. The PDMS mould, containing an integrated microfluidic system, was fabricated based on patterned SU-8 mould, which includes a lateral horizontal inlet access point. Surface oxidation was used for irreversible permanent bondage between flat surfaces. The final result was planar Ag/AgCl reference electrode with integrated microfluidic that allows for electrochemical analysis in biochips

System-on-chip field-programmable gate array design for onboard real-time hyperspectral unmixing

Hyperspectral instruments have been incorporated in satellite missions, providing large amounts of data of high spectral resolution of the Earth surface. This data can be used in remote sensing applications that often require a real-time or near-real-time response. To avoid delays between hyperspectral image acquisition and its interpretation, the last usually done on a ground station, onboard systems have emerged to process data, reducing the volume of information to transfer from the satellite to the ground station. For this purpose, compact reconfigurable hardware modules, such as field-programmable gate arrays (FPGAs), are widely used. This paper proposes an FPGA-based architecture for hyperspectral unmixing. This method based on the vertex component analysis (VCA) and it works without a dimensionality reduction preprocessing step. The architecture has been designed for a low-cost Xilinx Zynq board with a Zynq-7020 system-on-chip FPGA-based on the Artix-7 FPGA programmable logic and tested using real hyperspectral data. Experimental results indicate that the proposed implementation can achieve real-time processing, while maintaining the methods accuracy, which indicate the potential of the proposed platform to implement high-performance, low-cost embedded systems, opening perspectives for onboard hyperspectral image processing.

Microfluidic based on aqueous two-phase system for plasmidic DNA purification

Dissertação de mestrado em Bioquímica Aplicada (área de especialização em Biotecnologia)

Monitoratge remot d'esdeveniments per al control i supervisió utilitzant "System on chip" i "w-USB"

Aquesta memòria descriu el procés de desenvolupament d'un projecte que consisteix en un conjunt de hardware, “PSoC” (Programmable System on Chip), i un software, C#, mitjançant els quals s'automatitza la gestió de comandes a les taules d'un restaurant. A cada taula trobem un aparell anomenat “WaiterClient”, a través del qual els clients sol·liciten l'atenció d'un cambrer. Aquest hardware té una pantalla on es mostrarà informació i un conjunt de polsadors per demanar. Per una altra banda, trobem un altre aparell, “WaiterServidor”, encarregat de rebre els senyals enviats per wireless des dels “WaiterClients” que hi ha a cada taula. Un cop rebudes, les transmet a un ordinador central per cable sèrie RS-232.

Sistema de monitorització vital portable amb System on Chip i interfície SD Card

Aquesta memòria descriu el procés de desenvolupament del projecte de fi de carrera “Sistema de monitorització vital portable amb System on Chip i interfície SD Card”. Aquest es tracta d’un dispositiu de dimensions reduïdes, baix consum i portable amb capacitat d’enregistrar els biopotencials cardíacs dins d’una targeta de memòria flash SD Card. En temps real es mostra una representació d’aquests biopotencials mitjançant una pantalla LCD gràfica. El projecte, a més, inclou el desenvolupament d’un software de visualització per PC que permet l’anàlisi posterior més detallada dels registres emmagatzemats a la targeta SD Card.

Combined dielectrophoretic and impedance system for on-chip controlled bacteria concentration: Application to Escherichia coli

The present paper reports a bacteria autonomous controlled concentrator prototype with a user-friendly interface for bench-top applications. It is based on a micro-fluidic lab-on-a-chip and its associated custom instrumentation, which consists in a dielectrophoretic actuator, to pre-concentrate the sample, and an impedance analyser, to measure concentrated bacteria levels. The system is composed by a single micro-fluidic chamber with interdigitated electrodes and a instrumentation with custom electronics. The prototype is supported by a real-time platform connected to a remote computer, which automatically controls the system and displays impedance data used to monitor the status of bacteria accumulation on-chip. The system automates the whole concentrating operation. Performance has been studied for controlled volumes of Escherichia coli (E. coli) samples injected into the micro-fluidic chip at constant flow rate of 10 μL/min. A media conductivity correcting protocol has been developed, as the preliminary results showed distortion of the impedance analyser measurement produced by bacterial media conductivity variations through time. With the correcting protocol, the measured impedance values were related to the quantity of bacteria concentrated with a correlation of 0.988 and a coefficient of variation of 3.1%. Feasibility of E. coli on-chip automated concentration, using the miniaturized system, has been demonstrated. Furthermore, the impedance monitoring protocol had been adjusted and optimized, to handle changes in the electrical properties of the bacteria media over time.

One chip solution for low-cost active magnetic bearing system

In this work the implementation of the active magnetic bearing control system in a single FPGA is studied. Requirements for the full magnetic bearing control system are reviewed. Different control methods for active magnetic bearings are described shortly. Flux and the current base controllers are implemented in a FPGA. Suitability of the con-trollers for a low-cost magnetic bearing application is studied. Floating-point arithmetic’s are used in the controllers to ease designing burden and improve calculation precision. Per-formance of the flux controller is verified with simulations.

A Model-Based Development and Verification Framework for Distributed System-on-Chip Architecture

The capabilities and thus, design complexity of VLSI-based embedded systems have increased tremendously in recent years, riding the wave of Moore’s law. The time-to-market requirements are also shrinking, imposing challenges to the designers, which in turn, seek to adopt new design methods to increase their productivity. As an answer to these new pressures, modern day systems have moved towards on-chip multiprocessing technologies. New architectures have emerged in on-chip multiprocessing in order to utilize the tremendous advances of fabrication technology. Platform-based design is a possible solution in addressing these challenges. The principle behind the approach is to separate the functionality of an application from the organization and communication architecture of hardware platform at several levels of abstraction. The existing design methodologies pertaining to platform-based design approach don’t provide full automation at every level of the design processes, and sometimes, the co-design of platform-based systems lead to sub-optimal systems. In addition, the design productivity gap in multiprocessor systems remain a key challenge due to existing design methodologies. This thesis addresses the aforementioned challenges and discusses the creation of a development framework for a platform-based system design, in the context of the SegBus platform - a distributed communication architecture. This research aims to provide automated procedures for platform design and application mapping. Structural verification support is also featured thus ensuring correct-by-design platforms. The solution is based on a model-based process. Both the platform and the application are modeled using the Unified Modeling Language. This thesis develops a Domain Specific Language to support platform modeling based on a corresponding UML profile. Object Constraint Language constraints are used to support structurally correct platform construction. An emulator is thus introduced to allow as much as possible accurate performance estimation of the solution, at high abstraction levels. VHDL code is automatically generated, in the form of “snippets” to be employed in the arbiter modules of the platform, as required by the application. The resulting framework is applied in building an actual design solution for an MP3 stereo audio decoder application.

Studying the cytomechanic aspects of pollen tube growth behavior using Lab-On-Chip technology

L'élongation cellulaire de cellules cultivant bout comme hyphae fongueux, inculquez hairs, des tubes de pollen et des neurones, est limité au bout de la cellule, qui permet à ces cellules d'envahir l'encerclement substrate et atteindre une cible. Les cellules cultivant bout d'équipement sont entourées par le mur polysaccharide rigide qui régule la croissance et l'élongation de ces cellules, un mécanisme qui est radicalement différent des cellules non-walled. La compréhension du règlement du mur de cellule les propriétés mécaniques dans le contrôle de la croissance et du fonctionnement cellulaire du tube de pollen, une cellule rapidement grandissante d'équipement, est le but de ce projet. Le tube de pollen porte des spermatozoïdes du grain de pollen à l'ovule pour la fertilisation et sur sa voie du stigmate vers l'ovaire le tube de pollen envahit physiquement le stylar le tissu émettant de la fleur. Pour atteindre sa cible il doit aussi changer sa direction de croissance les temps multiples. Pour évaluer la conduite de tubes de pollen grandissants, un dans le système expérimental vitro basé sur la technologie de laboratoire-sur-fragment (LOC) et MEMS (les systèmes micro-électromécaniques) ont été conçus. En utilisant ces artifices nous avons mesuré une variété de propriétés physiques caractérisant le tube de pollen de Camélia, comme la croissance la croissance accélérée, envahissante et dilatant la force. Dans une des organisations expérimentales les tubes ont été exposés aux ouvertures en forme de fente faites de l'élastique PDMS (polydimethylsiloxane) la matière nous permettant de mesurer la force qu'un tube de pollen exerce pour dilater la croissance substrate. Cette capacité d'invasion est essentielle pour les tubes de pollen de leur permettre d'entrer dans les espaces intercellulaires étroits dans les tissus pistillar. Dans d'autres essais nous avons utilisé l'organisation microfluidic pour évaluer si les tubes de pollen peuvent s'allonger dans l'air et s'ils ont une mémoire directionnelle. Une des applications auxquelles le laboratoire s'intéresse est l'enquête de processus intracellulaires comme le mouvement d'organelles fluorescemment étiqueté ou les macromolécules pendant que les tubes de pollen grandissent dans les artifices LOC. Pour prouver que les artifices sont compatibles avec la microscopie optique à haute résolution et la microscopie de fluorescence, j'ai utilisé le colorant de styryl FM1-43 pour étiqueter le système endomembrane de tubes de pollen de cognassier du Japon de Camélia. L'observation du cône de vésicule, une agrégation d'endocytic et les vésicules exocytic dans le cytoplasme apical du bout de tube de pollen, n'a pas posé de problèmes des tubes de pollen trouvés dans le LOC. Pourtant, le colorant particulier en question a adhéré au sidewalls du LOC microfluidic le réseau, en faisant l'observation de tubes de pollen près du difficile sidewalls à cause du signal extrêmement fluorescent du mur. Cette propriété du colorant pourrait être utile de refléter la géométrie de réseau en faisant marcher dans le mode de fluorescence.

A flexible lab-on-a-chip for the synthesis and magnetic separation of magnetite decorated with gold nanoparticles

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)