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Characterization of the Adsorption of Biomolecules in Open Electrowetting on Dielectrics Microfluidics-Tool Platform for Bio-Analytical Applications

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Characterization of the Adsorption of Biomolecules in Open Electrowetting on Dielectrics Microfluidics-Tool Platform for Bio-Analytical Applications Characterization of the Adsorption of Biomolecules in Open Electrowetting On Dielectrics Microfluidics-Tool Platform for Bio-analytical Applications OEWOD surfaces for biomolecular adsorption experiments i Characterization of the Adsorption of Biomolecules in Open Electrowetting On Dielectrics Microfluidics-Tool Platform for Bio-analytical Applications Miguel Angel Martínez Garza Born in Mexico City A thesis presented in fulfilment of the requirement for the degree of Doctor of philosophy Institute of Microsystems Technology Department of Sensors Albert-Ludwig University Freiburg, Germany August 2011 ii Dekan: Prof. Dr. Bernd Becker Prüfungskommission: Vorsitz: Prof. Rühe Beisitz: Prof. Stieglitz Gutachter: Prof. Urban und Prof. Woias Datum der Prüfung: 02.03.12 iii Summary Open Electrowetting On Dielectrics (OEWOD) is one of several designs of the original Electrowetting On Dielectrics (EWOD) microfluidics platform. EWOD has gained considerable attention for its capacity of transporting smallest volumes of liquids especially in biochips and BioMEMS approaches. The electrical polarization of the dielectrics layer causing the apparent increase in surface energy is uses to transport microdroplets to carry out the basic operations fulfilling a bioassay protocol. This thesis was mainly focused to describe and characterize the behaviour of several electrolytes and biomolecules solution under OEWOD conditions. A general overview of OEWOD microfluidics platform will provide by (1) the characterization by Contact Angle (CA). (2) the atomic Force Microscopy (AFM) of OEWOD surfaces. (3) the Laser Scanning Confocal Microscopy (LSCM) of OEWOD surfaces, and (4) the relative quantitative biomolecular adsorption behaviour of several biomolecules as Horseradish Peroxidase [HRP], Peroxidase-Conjugated Rabbit Anti-Mouse Immunoglobulin [HRP-IgG] and Deoxyribonucleic acid-Peroxidase [HRP-DNA] under OEWOD ―actuation‖ and static conditions. The relative quantitative biomolecular adsorption in OEWOD surfaces will identified through a chemiluminescence method, which is a very practical and sensitive protocol where HRP and theirs labelled molecules are previously adsorbed at different OEWOD conditions. The adsorbed HRP catalyzed the reagent Luminol, emitting a signal by 430 nm and creating an image in a Charged Coupled Device (CCD)/Luminescent Image Analyser System (LAS- 3000). The luminescent image is then relative quantified with AIDA (Advanced Image Data Analyzer). The biomolecular adsorption in OEWOD depends on biomolecules properties like isoelectric point, pH of solution, polarity of the electric field, ―actuation‖ time and definitively of surface properties. Therefore in this thesis are mainly compared two surface systems: Durimid 115A–Teflon®AF and Cellulose Acetate–Teflon®AF. The manipulation of these properties: limiting the ―actuation‖ time- potential applied, choosing the proper pH of the solution and selecting the proper electrode polarity and surface, allow applying two options: (1) minimize biomolecular adsorption or (2) to intentional immobilization/adsorption of biomolecules to specific locations determined by the underlying actuation electrode structure. This thesis presents mainly two examples of the OEWOD microchip as a platform for microfluidic applications. OEWOD microchip as a tool for the research of enzymatic reaction inactivation or kinetics and the performance of a real time molecular biological isothermal protocol ―Nucleic Acid Sequence Based Amplification‖ (NASBA) for the amplification of Oligonucleotide of Human Papilloma Virus 16 (HPV16) in an integrated optical and temperature system, through Molecular Beacon (MB) technology allowing the real time measurement of the amplification products. However, the details, efficiency, or high-throughput of this bioassay was not covered herein in this thesis. The OEWOD microchip system shows the flexibility, potential and practical tools for bio- application and the availability to implement an additional external sensing device for additional measurements. Keywords: microfluidics platform, Electrowetting On Dielectrics (EWOD),Open Electrowetting On Dielectrics (OEWOD), Contact Angle (CA), biomolecular adsorption, Chemiluminescence, Horseradish Peroxidase (HRP), Peroxidase-Conjugated Rabbit Anti-Mouse Immunoglobulin (HRP-IgG) DNA-HRP; Deoxyribonucleic acid-Peroxidase (HRP-DNA), Durimid 115A– Teflon®AF, Cellulose Acetate–Teflon®AF, Luminescent Image Analyser System (LAS-3000), Nucleic Acid Sequence Based Amplification‖ (NASBA), Molecular Beacon (MB), Human Papilloma Virus 16 (HPV16), Advanced Image Data Analyzer (AIDA). iv Zusammenfassung Open Electrowetting On Dielectrics (OEWOD) ist eine von vielen Weiterentwicklungen der ursprünglichen EWOD (Electrowetting On Dielectrics) Mikrofluidikplattform. EWOD erreichte ein beachtliches Interesse aufgrund der Möglichkeit, kleinste flüssige Volumina auf einer Biochip Plattform zu transportieren. Die elektrische Polarisierung der dielektrischen Schicht, welche einen Oberflächenenergiezuwachs verursacht, wird verwendet, um Tröpfchen zu transportieren. Die vorliegende Arbeit fokussiert im Wesentlichen auf dem Verhalten verschiedener Elektrolyt- und Biomoleküllösungen unter EWOD Bedingungen. Sowohl die Charakterisierung der OEWOD Oberflächen mittels Kontaktwinkelmessung (CA), Atomkraft-Mikroskopie (AFM), Laser Scannning Confocal Microscopy (LSCM) als auch das relative quantitative biomolekulare Adsorptionsverhalten verschiedener Elektrolyte und Biomoleküle wie Horseradish Peroxidase (HRP), HRP-DNA, HRP-IgG unter OEWOD-Bedingungen verschaffen einen allgemeinen Einblick auf das Adsorptionsverhalten an Oberflächen unter elektrischer Feldeinwirkung. Die relative quantitative biomolekulare Adsorption auf OEWOD-Oberflächen wird mittels der Methode der Chemolumineszenz nachgewiesen. Diese basiert auf der Katalyse von Luminol, HRP wird als Biomolekül verwendet, das zuvor unter verschiedenen OEWOD Bedingungen für den späteren Nachweis adsorbiert wurde. Dies ist ein innovatives, praktisches und sehr sensitives Protokoll um relative quantitative biomolekulare Oberflächenadsorption nachzuweisen - im Besonderen für diejenigen Oberflächen, die für die biomolekulare Verwendung zur Verfügung stehen. Das adsorbierte HRP katalysiert das Reagens Luminol und emittiert ein Signal bei 430 nm, das ein Bild in einer CCD schafft. Das Luminiszenzbild wird mit Advanced Image Data Analizer (AIDA) relative quantifiziert. Die biomolekulare Adsorption in OEWOD hängt ab von den Eigenschaften der Biomoleküle; dies sind der isoelektrische Punkt, der pH der Lösung, die Polarität des elektrischen Feldes, die Wirkungsdauer und die Oberflächeneigenschaften. Dies ermöglicht entweder, die biomolekulare Adsorption zu minimieren (durch Limitierung der angewandten zeit-potentiellen Wirkung, durch Wahl des richtigen pHs der Lösung und der Elektrodenpolarität) oder absichtlich Biomoleküle an spezifische Orte, welche durch die zu Grunde liegenden wirkende Elektrodenstruktur bestimmt werden, zu immobilisieren bzw. zu adsorbieren. Die vorliegende Arbeit stellt hauptsächlich zwei Beispiele möglicher Anwendungen von OEWOD als einer mikrofluidischen Plattform für Bioassays vor: OEWOD als eine mikrofluidische Plattform für die Erforschung von Inaktivierung enzymatischer Reaktion oder Kinetiken in einem Lumineszenz bildgebendem System und die Durchführung eines Echtzeit-molekularen biologischen isothermen Protokolls ―Nucleic Acid Sequence Based Amplification‖ (NASBA), um Oligonukleide des Human Papilloma Virus 16 (HPV16) in einem integrierten optischen und Temperatursystem durch Molecular Beacon (MB) Technologie zu amplifizieren. Damit wird eine reale Zeitmessung der amplifizierten Oligonukleide von HPV16 ermöglicht. Deren Details, Effizienz oder Hoch-Durchlauf sind hierin nicht eingeschlossen. Das OEWOD Mikrochip-System zeigt die Flexibilität einer potentiellen und praktischen Plattform für Bioapplikation und erleichtert die Verwendung eines zusätzlichen externen Messgerätes für weitere Messungen. v Acknowledgements I would like to express my sincerely gratitude to my promoter Prof. Dr. Gerald Urban for giving me the technical support, suggestions and precious input to accomplish this scientific research work, as well Dr. Isabella Moser and Gerhard Jobst for giving me the opportunity, reliance, technical support and facilities to investigated in this fascinated and interesting field. I would like to express my sincerely gratitude to all my colleagues from the department of Microsystems Engineering, Sensors and all partners from European Project ―Micrometer scale patterning of protein and DNA- chips‖ G5RD-CT-2002-00744 for the financial and technical support, especially to my colleagues from NorChip AS, Norway for the discussions, advices and consumables facilities. In particular, Uwe Herberth, Anja Gulliksen, Jochen Kieninger, Paul Vulto, Gregory Dame, Hubert Flamm, Barbara Enderle, Thomas Weiss, Kuppusamy Aravindalochanan, Daniel Armbruster, Roland Hessler, Svetlana Santer, Christian Schlemmer for all discussions, ideas and technical support. For those, who was not mentioned here, but had contributed direct or indirectly to this thesis, my sincerely acknowledgements. I express my sincerely gratitude to Daniel Armbruster and especially to Clemens Kienzle for the grammar and spell checking of this thesis, as well as Cristina Martínez Garza for the appreciable suggestions and advices by the scientific writing. I would like to express my sincerely, invaluable and eternally love to my parents,
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