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

OEWOD surfaces for biomolecular adsorption experiments

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

Dekan:

Prof. Dr. Bernd Becker

Prüfungskommission:

Vorsitz: Prof. Rühe

Beisitz: Prof. Stieglitz

Gutachter: Prof. Urban und Prof. Woias

Datum der Prüfung: 02.03.12

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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).

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.

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, brother, sisters, aunt, uncles and grandmothers in Mexico. For the education and life values received and their continuous spiritual, mystics support during all these years away from each other; Jose, Silvia, Clara, Alejandro, Cristina, Carlos, my mother; Magdalena and my father; Jose, who always were, are and will be in my mind. To my lovely family, Frederike Kienzle, my daughter Yaretzi, and my son Ollin, they are my treasure, who patiently and lovely accepted my long and late hours of work, without them I could not have the force to go ahead. Finally, though they have been long gone, but forever in my mind: Angeles Garza, Esperanza Portillo, Agapita Silva und Javier Martínez.

Workshop, Poster session and Literature

Martínez-Garza M., Herberth U., Urban G., Jobst G., Moser I. ―On avoiding and exploiting protein adsorption in Electrowetting Based Devices‖. Oral Presentation, Session: BioMEMS and Microfluidics. Second International Workshop on Multianalyte Biosensing Devices, Tarragona, Spain, 18–20 February, p. 61, 2004.

Herberth U., Martínez-Garza M., Jobst G., Moser I. ―Dynamics of Defined Volume Droplets Actuated with Open Electrowetting On Dielectrics” Poster Session: Microfluidics. Actuator 08 International Conference and Exhibition on New Actuators and Drive Systems, Bremen, Germany, 9–11 June, 2008

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Table of Contents Summary iv Zusammenfassung v Acknowledgements vi Workshop, Poster session, Contributions and Literature vii Table of Contents viii 1. Introduction 1 1.1 Overview of Biomolecular Adsorption in EWOD and OEWOD microfluidics platform 5 1.2 The state of the Art: EWOD and OEWOD microfluidics platform 10 1.3 Real Time Nucleic Acid Sequence Based Amplification (RT-NASBA) of Human 13 Papilloma Virus 16 (HPV16) on OEWOD microchip 1.4 Scope and Outline of this Thesis 15 2. Theoretical Background 20 2.1 Thermodynamics 21 2.2 Contact angle measurement 22 2.2.1 Static contact angles 22 2.2.2 Dynamic contact angles 24 2.2.3 Advancing angle 24 2.2.4 Receding angle 25 2.3 Biomolecular Adsorption: Fundamental Principles 25 2.3.1 The three-dimensional structure of globular proteins 26 2.3.1.1 Hydrophobic interaction. 26 2.3.1.2 Coulomb interaction 26 2.3.1.3 Lifshitz-van der Waals interaction 27 2.3.1.4 Hydrogen bonding 27 2.4 The adsorption process 28 2.5 Driving forces for the protein adsorption 31 2.5.1 Interaction between electrical double layers 32 2.5.2 Changes in the State of Hydration 34 2.5.3 Dispersion Interaction 35 2.5.4 Rearrangement in the protein Structure 36 2.5.5 Sorbent Surface Morphology 37 2.6 Chemiluminescence as biomolecular adsorption measurement technique 38 3. Experimental; Materials, equipment and methods 41 3.1 Teflon® AF 1600 41 3.1.1 Material Properties 43 3.2 Cellulose Acetate 44 3.2.1 Material Properties 45 3.3 Durimid 115A 45 3.3.1 Material Properties 45 viii

3.4 SU-8 46 3.4.1 Material Properties 47 3.5 Biomolecules ( GO, Bovine Serum Albumin, B-Phycoerithrin, Horseradish 47 Peroxidase, HRP-DANN, IgG-HRP) 3.5.1 B- Phycoerythrin (PE) 48 3.5.2 Glucose Oxidase (GOD) 48 3.5.3 Horseradish Peroxidase (HRP) 49 3.5.4 HRP5 isoelectric point 4.0 50 3.5.5 HRP4B isoelectric point 8.5 50 3.5.6 DNA- Horseradish Peroxidase 50 3.5.7 Peroxidase-Conjugated Rabbit Anti-Mouse Immunoglobulin (IgG-HRP) 52 3.6 HPV-NASBA 52 3.6.1 Molecular beacon 53 3.7 Equipment 54 3.7.1 Adsorption experiment set up 55 3.7.2 Contact angle device, Krüss G10 and the Drop shape analysis DSA10 56 3.7.3 Droplet Handling System (DHS) and Electronic Circuitry 57 3.7.4 OEWOD surfaces for adsorption experiment 58 3.7.5 OEWOD microchip for Real Time NASBA of HPV16 59 3.7.6 Heater and temperature control on OEWOD microchip 62 3.7.7 Luminescent image analyzer (LAS-3000) 62 3.7.8 Advanced Image Data Analyzer (AIDA), version 4.06 63 4. Result and Discussion 64 4.1 OEWOD surfaces characterization 64 4.1.1 Atomic Force Microscopy analysis of OEWOD surfaces 65 4.1.2 Laser Scanning Confocal Microscopy (ZEISS LSCM 510 UV) of 66 OEWOD surfaces annealed and unannealed 4.1.3 Reversibility and behaviour of electrolytes in comparison to HRP 67 under electrowetting effect (OEWOD conditions) 4.1.4 Reversibility and behaviour of biomolecules solution in water and in 68 PBS under electrowetting effect (OEWOD conditions) 4.1.5 Discussion and conclusions 71 4.2 Dependence on the electrode polarity and pH on OEWOD surfaces in the 75 biomolecular adsorption process on OEWOD surfaces 4.2.1 Discussion and conclusions 77 4.3 Biomolecular adsorption HRP under OEWOD conditions 79 4.3.1 Horseradish Peroxidase HRP5 and HRP4B 82 4.3.2 Discussion and conclusions 82 4.4 Peroxidase-Conjugated Rabbit Anti-mouse Immunoglobulin adsorption on 84 OEWOD surface

4.4.1 Dependence on the pH and time on the Peroxidase Conjugated 84 Rabbit Anti-mouse Immunoglobulin‘s adsorption 4.4.2 Dependence on the electrode polarity, concentration and pH on the 86 IgG- HRP‘s adsorption on OEWOD surfaces 4.4.3 Discussion and conclusions 87 4.5 DNA-HRP adsorption on OEWOD surfaces 89 4.5.1 Dependence on the electrode polarity, potential applied 89 and time adsorption on OEWOD surfaces 4.5.2 Discussion and conclusions 90 4.6 Applications on OEWOD Microfluidics-Tool Platform 91 4.6.1 OEWOD platform as a tool for kinetics 91 4.6.2 HRP kinetics enzyme on OEWOD surfaces 92 4.6.3 Discussion and conclusions 96 4.7 Real Time Nucleic Acid Sequence Based Amplification on OEWOD platform 97 4.7.1 Compatibility of NASBA components to the OEWOD based droplet 98 actuation platform 4.7.2 OEWOD Droplets Handling System (DHS) and Temperature 101 Controller 4.7.3 RT-NASBA of HPV16 Fluorescence measurements on OEWOD 102 microchip 4.7.4 Discussion and conclusions 103 4.8 General discussion and conclusions 104 4.9 Outlook 109 5. References 111 6. List of symbols and abbreviation 122 7. Figures and Tables 125 Annex A Measuring methods and Methods of evaluating the drop shape 132 Annex B Characterization of OEWOD surfaces 135 Annex C The double-layer charging current 144

In this modern and globalised world, the velocity of our time is accelerated and Disproportionate to our reasonability, besides our capacity to response, Our necessities and discoveries are overcoming our knowledge. The social milieu requirement for a high quality life, Support the Point of Care systems. Innovative medical devices for diagnostic diseases Are required to access efficient health care services

1. Introduction

The progressive and accelerated developments of biomolecular research and fluidic techniques have already reached the micro- and nano-world. The ―lab-on-a-chip‖ concept takes on a very important meaning in regards to the ―Point of care‖ systems. An efficient analysis system using micro/nano samples, analyzing with micro/nano volumes reagents and enable the simultaneous detection of several species, to diagnostics a specific disease or metabolic disorder are confirmed and demonstrated. Microsystems technology takes advantage of low energy consumption; combine to properly application of all biomolecules and material properties, forces and available energy at micro and nano level. These natural system properties have not been considered for a long time, even if they would make the systems more efficient.

In contrast to the high increment of energy consumed from our society, Microsystems technology represents and promises a pretty nice possibility to develop new diagnostic system devices with minimal power requirements and efficient application resources. The development of new micro/nano technologies, suitable for use in a new generation of biochips in combination with optical, biological and electrochemical detection methods or any mechanical application for every application field are the main challengers.

An ideal platform for microfluidics to perform a bioassay should fulfil many aims. Firstly, it requires two main processes being possible on the same surface: (1) fluidic assay protocol and (2) detection or analysis units, making the avoidance of cross-contamination possible. If this is accomplished then the area required infrastructure and the energy applied would be minimized. Besides this, the additional application of environmentally friendly materials and minimal generation of second contaminated products are required. On the other hand, the ability to control and manipulate the immobilization of biomolecules on a surface for bioassays is a

1 very important issue to developing new devices for bio-applications. Currently, the bio-techniques applied for this challenge need complex equipment or complicated surface treatments, which means high cost investments and, as a consequence, inefficient systems.

Electrowetting On Dielectrics (EWOD) platforms for microfluidics has gained considerable attention for its ability to transport micro/nano volumes of liquids (samples); this platform together with molecular diagnostics, has been developing rapidly over the last few years, especially in biochips and BioMEMS approaches. Because of the possibility to perform the main process unit at the same surface, its flexibility and low power requirements, it is a promising new microfluidics platform for bio-analytical devices [Yun, 2001; Srinivasan, 2003; Saeki, 2001; Lee, 2002; Fair, 2007]. An electrical polarization of the dielectric layer, which is the core of the EWOD system (due to a potential applied that causes an apparent increase in surface energy), switching in seconds their hydrophobic properties to hydrophilic. This system is then used for the flexible transport and manipulation of micro or nano droplets of biomolecules dissolved in electrolytes or any fluids, to perform any bioassay protocol and which is called ―actuation‖: the act of moving and manipulate droplets under the EWOD principle.

There are mainly two different system designs of this microfluidics platform: open and confined systems, both of them possess advantages and disadvantages [Lee, 2002; Chao-Yi Chen, 2003; Kim, 2006; Fouillet, 2006; Herbert, 2006; Fair, 2007]. The classical confined and most popular EWOD design used consists of two parallel plates, the top plate is pattern with a reference electrode, and the bottom plate is pattern with addressable driven electrodes. Both plates and electrodes are coating with a hydrophobic insulating layer. An electrical potential is apply between the top electrodes (reference) and the bottom electrodes (driven electrodes). In between, sit the droplet reagents or sample to be transport, depending on the bioassay protocol. Therefore in some EWOD devices with the classical design are frequently operated with oil as a surrounding medium to avoid contact to the surfaces (biomolecular adsorption), which creates the additional problem for forming emulsification or the interaction of the oil with the reagents involved [Pollack, 2000, Srinivasan, 2003, 2004].

Due to the design geometry, this system based in channels does not allows direct contact with the surface; which means including additional designing of reservoirs or inlet valves makes the system more complicated and less versatile.

―Open Electrowetting On Dielectrics‖ (OEWOD) is one of the several open system designs described in this thesis. In contrast to the typical EWOD mentioned above, in OEWOD the electrodes do not need a second subtract to contact the droplet on the top. The surfaces or subtracts are patterned with the corresponding electrode size designed and simulated by our colleagues Lienemann, et al (2002) and Herberth (2006). The droplets are transported on a planar hydrophobic surface using electric fields generated by underlying electrodes. The electrodes are arranged as paths; droplets reagents and a sample are manipulated individually or mixed with each other in a controlled manner and according to the bioassay protocol.

The flexibility of these open systems and its practical coplanar design allows the implementation of additional external optical or electrochemical sensing modules and enable the possibility for further application. Finally, the open system also simplifies the performance of any bioassay protocol by allowing manual and external complex steps (see chapter 4.7). OEWOD presents a practical and attractive technique because of the manipulation of the biomolecules droplets on a specific location to be mixed, incubated or adsorbed/immobilized. This platform does not need complicated infrastructure for bioassays microfluidics and at the same time has the flexibility necessary to implement any external sensing device to perform them.

Biomolecular adsorption in EWOD and OEWOD microfluidics platform is a critical issue and one that has been considered to decreases the functionality of the system ―EWOD effect‖, as well as causing the contamination of reagents or samples, due to the use of the same surfaces to performance microfluidics and analysis. Nevertheless, there are only a few studies about biomolecular adsorption and even fewer have been mentioned [Quinn, 2003; Yoon, 2003; Martínez-Garza, 2004; Wu, 2006; Bayiati 2007]. Biomolecular adsorption in EWOD and OEWOD microfluidics platform has been rarely evaluated; these studies are a contribution to resolving this critical issue or at least, intend to address a different approach, by understanding the behaviour and for the later manipulation of the parameter involved in the biomolecular adsorption on Electrowetting On Dielectrics surfaces. The approach to

3 functionalize the surface, through biomolecular adsorption, manipulating the parameter involved, and simultaneously taking advantage of the microfluidics options, represents a very practical and promising tool for bio-applications. The functionality of the surface through a biomolecular adsorption depends on several physical and chemical parameters of the system, as well on the bulk of properties of the biomolecules.

The principal idea is to provide an OEWOD microchip array with addressable, programmable sequence of the electrodes. OEWOD will allow the performance of basic unit operations as moving micro/nano droplets of samples and reagents, along the structure paths, depending on the bioassay protocol. Micro or nano droplets are deposited or dispensed, manually or mechanically, from an automatic dispenser or pipette; then droplets by means of EWOD are transported to specifically designed locations to be adsorbed or transported depending on the bioassay protocol. The droplets can be merged, mixed, or incubated to perform a chemical reaction or bioassay protocol. This thesis introduces an example of the OEWOD microchip as a platform for bioassays applications; 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.

OEWOD due to coplanar array and open surface allows the additional implementation of a dispensing system as well as some measurement system, as demonstrated in the biomolecular assay mentioned above. The few disadvantages of the OEWOD design have been resolved with this thesis: vapour pressure of water that is rather high at room temperature and thermal treatments are required for several bioassays, especially biomolecular assays, therefore liquid evaporation is a critical issue. This was overcome with an OEWOD adapted chamber frame and cover plate, placed on the top of the OEWOD microchip (see chapter 3.7.5 for details) and demonstrated with the performance of the biomolecular assay protocol above mentioned (see chapter 4.6 for details).

1.1 Overview of Biomolecular Adsorption in EWOD and OEWOD microfluidics platform

The ability to understand or manipulate the behaviour of biomolecules on electrowetting on dielectrics surfaces is very important for the development of possible microfluidics bio-applications based on this principle. The biomolecules tend to be absorbed on nearly every kind of surfaces. As a consequence in the case of OEWOD surfaces, this decreases the functionality of the system ―EWOD effect‖; ―actuation‖ which has already been mentioned. Therefore it is important to know the conditions of the system in order to avoid or to take advantage of this fact.

The electrowetting effect changes the surfaces properties from hydrophobic to hydrophilic while a potential is apply in the system during the ―EWOD effect‖. A hydrophobic surface is the core for the ―actuation principle‖ of the OEWOD platform and at the same time, this kind of surface tends to bind more biomolecules. Besides, the potential to the surfaces is apply for the same principle, which influences the distribution of ions in solution and their interactions with biomolecules. All these interactions combined between the biomolecules and material properties, raise additional questions on the interpretation of the complexity of the biomolecular adsorption phenomena. It is heavily debated which interactions (i.e., hydrophobic, electrostatic or, hydrogen-bonding, among others) are the most important affecting the biomolecular adsorption phenomena in EWOD and OEWOD systems [Yoon, 2003; Quinn, 2003;Wu, 2006; Cooney, 2006; Bayiati, 2007]. Some researchers have concluded that the hydrophobic interaction is the most important aspect in the biomolecular adsorption process, whereas others have claimed that electrostatic interaction and others factors are the most important aspect in systems based in EWOD principle.

In the last 8 years, the number of publications in the application field of Electrowetting, Electrowetting On Dielectrics, as well as different designs under this principle has increased. Unfortunately there are only a few studies that investigate or have been mentioning the biocompatibility of this platform of microfluidics with biomolecular adsorption (see table 1.1). A very considerable number of applications in this platform have already been demonstrated, but it is important to mention that most of them are developments in the EWOD confined design and only very few 5 applications have been demonstrated in the open or co-planar design, as in this thesis. In the state of art, (see chapter 1.2) is presented a chronologically review of the development of the EWOD and OEWOD platform, as well its applications relating to molecular diagnostics.

Preliminary experiments on surfaces available for electrowetting effect, have shown that the behaviour of biomolecules and electrolytes are different from what is expected in the theory (see chapter 2; Lippmann-Young´s equation) for conductive liquid as well for biomolecules [Yoon, 2003; Washizu, 1998; Quinn, 2003; Wu, 2006; Cooney, 2006] and some additional effect has to be considered; as saturation effect [Wang, 2005, Verheijen, 1999; Herberth, 2008]. Currently, it is known that biomolecules may be adsorbed by different electrode materials such as ITO, gold, silver, or pyrolytic carbon if a potential is applied [Holmström, 1998; Dominguez, 2002; Brusatori, 2001]. It is well known that biomolecules are readily and irreversibly adsorbed on hydrophobic surfaces such as Teflon [Rupp, 2002; Freitas, 2003] and that the biomolecular adsorption process is readily affected or influenced by biomolecular and surfaces properties such as: size, charge, structure stability, unfolding rate as well as topography, composition, hydrophobicity, heterogeneity, and potential [Horbett,1987; Andrade, 1985]. Yoon and Garrell (2003) in a confined EWOD system have performed experiments with BSA, hen egg white Lysozyme, and DNA from calf thymus. They showed that biomolecular adsorption can be avoided or minimized by limiting the ―actuation‖ time (potential applied), choosing the proper pH of the solution and the electrode polarity and they have suggested two mechanisms for biomolecular adsorption under confined EWOD conditions: (i) Passive adsorption arising from hydrophobic interactions and (ii) electrostatically driven adsorption when an external electric field is applied (see figures 1.1 and 1.2.). Wu, et al. (2006) in a similar system as in this study (they have called ―Open- configuration‖), have performed experiments with BSA and hen egg white Lysozyme. They have recognized that the most important aspect of biomolecular adsorption process in such OEWOD systems is mainly: the induced force between the biomolecules. They have assumed that the adsorption induced by hydrophobic interaction is inevitable. They have shown that electrostatic attraction is an important factor in the first minutes of the biomolecular adsorption and that adsorption can be minimized by limiting the duty-radio of the applied pulse voltage. The hydrophobic interaction is decreased through an innovative design in which the biomolecular drop

6 is immersed onto a suspended silicon-aluminium filament on the bottom plate [Wu, 2006]. In a different approach Fair, et al. (2007) and Srinivasam (2004) filled the channel in EWOD with oil avoiding in this manner the contact of the biomolecular drop with the surface and in this way, decreasing the biomolecular adsorption with the risk of forming an emulsion during the ―actuation‖ [Pollack, 2000, Srinivasan, 2003, 2004; Fair, 2007]. Mugele and Baret [Baret, 2005] mention in a very interesting review of electrowetting, ―From basic to application‖, that most of the studies mentioned here have not reported the important influence of the biomolecular adsorption due to the type or concentration of the electrolyte and biomolecules. Nevertheless, Quinn et al (2003) pointed out that an important dependency on the pH, ionic strength, and polymer composition has been found to influence electrowetting behaviour. The previous study was performed in a similar electrowetting design as this study and remarked the use of the electric field from DC voltage as in this study. Torkkeli [Torkkeli, 2003] has reported that in Electrowetting in superhydrophobic planar surfaces design, biological solutions (see table 1.1) were not enabled to ―actuate‖ and pointed out that this system cannot be used for a Lab- on-microchip purposes. Our colleagues Bayiati et al (2007) have reported that in films deposited by plasma polymerization on Si3N4, b-BSA adsorption have depended strongly on the chemical composition of the film but they emphasized that concentration, pH of the biomolecular solution, as well the polarity of the potential applied, could be neglected.

Table 1.1 sums up the chronological development of OEWOD systems, according to the system characteristics and biomolecules or electrolytes investigated and emphasizing investigations related to biomolecular adsorption. Table 1.1 shows only research studies, in a similar OEWOD design as in this study, with the exception of the study of Yoon et al (2003), which has been performed in confined EWOD system but with a very interesting and properly investigation about biomolecular adsorption in such platform. It is important to point out that there are other research studies in the same EWOD design as in this thesis, but without emphasis or mention of biomolecular adsorption and therefore these are not included in table 1.1.

Potential Reference EWOD design Biomolecules or electrolytes AC or DC Microfluidics system

Electrolytes: PBS, HEPES and KCl 1) Glucose Oxidase GOD (1-10-5 mg/ml) Method to determine adsorption in the OEWOD surfaces: Chemiluminescence. 2) Horseradish Peroxidase HRP (1- 10-5 mg/ml) in PBS/D.I. AC and DC System: Teflon®AF (~3.8 µm), Durimid 115A (~3.8 µm) –Teflon®AF (~100-150 nm) and This work Open Electrowetting On 3) Horseradish Peroxidase HRP p.I. 4.5 (1µg/ml) Cellulose Acetate(~ 400-500 nm)–Teflon®AF(~100-150 nm) spin coated films on Pyrex glass, [Martί nez -Garza, 2004, 2010] Dielectrics (OEWOD) 4) Horseradish Peroxidase HRP p.I. 8.5 (1µg/ml) with 10 nm titanium and 25 nm platinum electrodes, previously structured and deposited by 5) DNA-HRP (1µg/ml) EVD. 6) IgG-HRP (1µg/ml) 7) RT-NASBA components

[Bayiati, 2007] Co-planar 1) Biotinylated bovine serum albumin 2, 6 and 10µg/ml in No reference System: FC films 60 nm, plasma deposited on 100 nm Si3N4 acetate buffer, pH=7 and carbonated buffer, pH=9.5 Method to determine adsorption in the EWOD surface: Alexa Flour 546 labelled streptavidin System: Substrate silicon, electrode 5000Ǻ poly silicon doped with phosphorus, 1000 Ǻ high

[Wu, 2006] Open configuration EWOD 1) Mixture of 10µg/ml BSA with pH=7.4 phosphate buffer and DC performance thermally grown SiO2 film covered with a 300 Ǻ spin coated Teflon F1600. 10µg/ml hen egg white Lysozyme. Innovation to avoid hydrophobic adsorption, suspended silicon aluminium filament onto the droplet. No method mentioned for detection of biomolecular adsorption. [Cooney, 2006] Single planar surface 1) Dilution series of KCl 0, 1, 10, 100, 500 mM and 1M AC and DC System: 1.6 mm electrode in Indium Tin Oxide, 1µm Parylene, Chromium/gold electrical Grounding from below grounding line on the dielectrics, Teflon AF (601, 6%). SU-8, 50 nm Teflon

[Quinn , 2003] Co-planar 1) 0.1M KCl pH=5.6 DC System Teflon AF 0.1- 10µm, gold coated silicon 1) Bovine Serum Albumin (4µg/ml) Method to determine adsorption in the EWOD surface: Quartz Crystal Microbalance

[Yoon, 2003] Confined 2) Hen egg white lysozyme (4µg/ml) DC System 500 Å Teflon AF spin-coated onto 1200 Å SiO2 on silicon 3) Double-strand deoxyribonucleic acid from calf Thymus(4µg/ml) 1) Tris-HCL buffered NaCl (pH=7.8) 0.05% sodium azide, System; a different design with hydrophobic surfaces (Teflon AF, Inductively Coupled Plasma

[Torkkeli, 2003] Planar surface BSA bovine gamma globulins, Tween-40, AC and DC fluoropolymer by decomposing C4F8 gas in plasma, AKD is a wax-type material, plasma- etched diethylenettriaminepentaacetic acid and inert red dye Teflon AF and super hydrophobic surfaces. 2) 50 mM Tris-HCL (pH=8) 150 mM NaCl, 0.1% Tween-20, Method to determine adsorption in the EWOD surface: No mentioned, only it has mentioned no 100µM EDTA, 0.1% BSA, 0.05% NaN3 success actuation with the 3 different buffers solutions and it has noted the change of the 3) 50 mM Tris-HCL (pH=8) 150 mM NaCl, 0.01% Tween-20, wetting properties of water once after the intend to actuated the buffers. 100µM EDTA, 0.1% BSA, 0.05% NaN3

1) Deionised water System: Teflon AF; Photoresists polyamide UR5100 ~ 10µm Aluminium Electrodes. No method [Washizu, 1998] Planar surface 2) Ionic biological buffer solutions AC mentioned to recognized adsorption 3) Protein Solutions (0.5-5µl)

p.I. isoelectric point, D.I. Deionised water, KCl; potassium Chloride PBS; Phosphate buffered saline, HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, GOD; Glucose Oxidase, BSA, bovine serum albumin, HRP Horseradish Peroxidase, IgG-HRP; Peroxidase-Conjugated Rabbit Anti-Mouse Immunoglobulins and DNA-HRP; Deoxyribonucleic acid-Peroxidase. Table 1.1: Chronological development of OEWOD platform related to biomolecular adsorption

There are inclusively studies, which made a comparison and characterization of both designs: EWOD confined and OEWOD open systems [Chen, 2003; Kim, 2006; Fouillet, 2006; Fair, 2007] but also do not mention biomolecular adsorption.

The Electrowetting On Dielectrics platform for microfluidics requires a hydrophobic surface, which it switches frequently from hydrophobic to hydrophilic properties through electrical current for the actuation. Therefore in this case, the adsorption process takes different parameters than in a usual adsorption process. Here different interactions have to be considered, which are changing during the EWOD effect and in the next figures 1.1 and 1.2, Yoon and Garrell [Yoon, 2003] show the different interactions that could take place on the adsorption process under such conditions, and it shows that the sorbent surface is modified constantly; passive adsorption arising from hydrophobic interactions and electrostatically driven adsorption arising when an external electric field is applied.

Figures 1.1 and 1.2: Shows a schematic representation of the biomolecular adsorption mechanism on EWOD suggested for Yoon et al (2003): Hydrophobic interactions or passive adsorption (Teflon AF 1600); biomolecules adsorbs mainly through hydrophobic interactions while not potential is applied. Electrostatically interaction; biomolecules adsorbs mainly through electrostatic interaction during a potential is applied [Yoon, 2003]. Layout in CorelDraw, HRP image structure from RCSB PDB [Meno, 2000]

The confined configuration of EWOD microchip is a channel with consists of two parallel plates: the top plate patterned with a reference electrode and a bottom plate patterned with addressable driven electrodes; both plates and electrodes are coated with a hydrophobic insulating layer. An electrical potential is applied between the top electrodes (reference) and the bottom electrodes (driven electrodes), where the drops sit with air or oil in between [Pollack, 2000, Srinivasan, 2003, 2004]. This kind of system presents many advantages, as well as disadvantages; the implementing of

optical sensing devices and additional functions on the surfaces of the microchip are delimits in a typical EWOD system. This configuration of the electrowetting on dielectric (EWOD) is demonstrated to manipulate aqueous droplets in channels filled with air or oil [Srinivasam, 2004].

In this thesis, a different design of Electrowetting On Dielectrics is presented, one that we have called ―Open Electrowetting On Dielectrics‖. In contrast to the typical EWOD mentioned above, the electrodes do not need a second subtract to contact the droplet at the top. The surfaces or subtracts are patterned with the corresponding electrode size designed and simulated by our colleagues Lienemann, et al (2002) and Herberth (2006). The droplets are transported on a planar hydrophobic surface using electric fields generated by underlying electrodes. The electrodes are arranged as paths and droplets reagents and the samples are manipulated individually or mixed with each other in a controlled manner. Kim et al (2006) have characterized the electrowetting actuation of what they have called ―an addressable single-side coplanar electrode‖, which mainly is the same as what we have called OEWOD. There are some differences in the electrode pattern and in what they have called sub- electrodes. They have not mentioned anything about biomolecular adsorption in this system. Cooney et al (2006) have presented a study in what they have called ―grounded from below‖, an electrowetting design system in which the droplet can be electrically grounded from below, using thin conductive lines on top of the dielectrics layer that also do not require a top plate or wire. It is worth mentioning that a coplanar array exposes fewer surfaces to biomolecular adsorption due to the electrodes geometry mentioned before.

1.2 The State of the Art: EWOD and OEWOD microfluidics platform

This well-known phenomenon, frequently referred to as EWOD, became the basis of an increasing number of potential and realistic important microfluidics technologies as in lab-on-a-chip (Laboratory on a chip), Biomes (Bio-ElectroMechanical Systems), and µTAS (Micro Total Analysis System), and the EWOD applications in others fields are increasing rapidly. However, the faster development and the uncompleted understanding of the phenomenon EWOD, has currently been cause for a controversial polemic and confusion.

The phenomenon ―Electrocapillarity‖ was first described and basically formulated by Lippmann G. [Lippmann, 1875]. However, the term ―electro-wetting‖ was first introduced as a novel effect in 1981 by Hackwook et al (1981). They have described an effect for designing a new type of display device and they have defined electrowetting in a three-phase system (solid, liquid electrolyte, and a second fluid) as the change in solid electrolyte contact angle due to an applied potential difference between the solid and the electrolyte. Hackwook et al (1981) have recognized that Electro-wetting is akin to, but distinct from electrocapillarity. Although many research papers mentioned and observed this effect in different fields, such as Minnema et al (1980) in water treeing phenomenology in Polyethylene. Electrowetting On Dielectrics (EWOD) was first demonstrated by Berge [Berge, 1993] introducing a thin layer to separate the conductive liquid from the metal electrode in an open system design.

Nowadays there are basically two different fundamental designs, described already in the previous chapter: co-planar or open EWOD and confined, enclosed, or two- dimensional array EWOD. The EWOD confined design has first demonstrated the successful unit operations of biomolecules, bioassays and many other applications by Richard Fair and co-workers at Duke University [Srinivasan, 2003, 2004, 2004]. Michael Pollack and Vamsee Pamula introduced for the first time the concept of ―Digital Microfluidics― in the engineering labs of Duke University. Several years later, they decided to found ―Advanced Liquid Logic Inc‖. Once this concept appeared, electrowetting on dielectrics began to appear in the background of this technology, although years earlier, Washizu et al (1998) and others had reported several applications with the same principle, but that was not in the fields of bio-application. Chang-Jin Kim UCLA Micromanufacturing Laboratory - Kim Group at UCLA in collaboration with Digital Microfluidics - Garrell Group at UCLA developed the first design. They demonstrated both designs: open and confined EWOD systems, several applications as well such Lab on a Chip applications. Besides, they have reported a very interesting study about preventing biomolecular adsorption in confined design EWOD system. Recently, Mugale F. and co-workers at the University of Twente [Malloggi and Mugele, 2007] have reported improvements in conventional electrowetting and proposed a combined system. The Wheeler Microfluidics Laboratory at the University of Toronto has developed a device based

on channels and digital microfluidics. They have reported hybrid methods for high- throughput "lab-on-a-chip" applications.

The transport of a variety of different fluids by EWOD means and not only electrolytes was frequently demonstrated but not only since the beginning. Cooney and Emerling [Cooney, 2006] demonstrated an alternative co-planar system called ―grounding from below‖ which should present more benefits for use as a lab on chip device.

Electrowetting On Dielectrics platforms for microfluidics have been developed in rapid succession over the past years, together with molecular diagnostics. There are mainly two different designs, which were already described in Chapter 1.1: OEWOD and Confined-EWOD. They possess their advantages and disadvantages and are well established [Lee, 2002; Chen, 2003; Kim, 2006; Fouillet, 2006; Fair, 2007].

Currently, there are EWOD based devices built for different molecular diagnostics such as PCR [Liao, 2005; Fair 2007; Zhang T 2004; Zhishan Hus, 2010] and complex analytical tasks such as sample preparation systems for MALDI in a confined design [Wheeler, 2005]. In addition, Yi-Ju Liu et al (2008) have demonstrated an automatic system using coplanar electrodes in EWOD for DNA ligation process and a parallel DNA-cloning system in the construction of an expression library.

The Open Electrowetting On Dielectrics (OEWOD) microchip is introduced in this thesis, as a platform for bioassays applications. The performance of a biomolecular isothermal protocol assay: ―Nucleic Acid Sequence Based Amplification‖ (NASBA) for the amplification of Oligonucleotide of Human Papilloma Virus 16 (HPV16) in an integrated optical and temperature system for real time detection through Molecular Beacon (MB) technology, is demonstrated here. There are other microfluidics platforms different from OEWOD, which have been successfully reported, the performance of NASBA technologies on Microsystems [Gulliksen, 2004, 2005; Glynn, 2008; Dimov, 2008].

1.3 Real-Time Nucleic Acid Sequence Based Amplification (RT- NASBA) of Human Papilloma Virus 16 (HPV16) on OEWOD microchip

Human Papillomavirus (HPV) is estimated to be one of the most common causes of sexually transmitted diseases in both men and women with the incidence of new infections ranging from 1- 5.5 million per year in the united states. HPV is associated with a wide variety of diseases ranging from benign cutaneous warts to cervical cancer [Ginocchio, 2004]. The incidence and death rates from cervical cancer in the U.S. have dropped almost 50% since the widespread use of Pap tests beginning in the early 1970s. Cervical cancer still remains the third most common gynaecological cancer in the U.S. Worldwide; it is the second most common cancer among women and the most common cause of death from a gynaecologic cancer [Gynecologists,

2003], apart from breast cancer.

Different HPV high risk types were identified over the years and different molecular biological methods were developed to support or in combination with the classical Pap tests to confirm their detection. For a specific age group and dependent of the initial cytology result, the combination of cervical cytology together with a HPV molecular diagnostics test is recommended [Gynecologists, 2003].

In an extended comparison study of HPV mRNA and HPV DNA related to cytological finding, the clinical data show that testing for HPV oncogene activity may be a more accurate clinical indicator for development of cervical cancer than testing viral DNA [Tor Molden, 2005].

Nowadays a HPV vaccine exists which prevents the infection with the most common types of "genital" HPV. However, HPV vaccine is only effective in specific conditions, and does not protect against all types of HPV that can cause cervical cancer. Therefore, a serious discussion about the vaccine campaigns started around the world, and only the next generation of young vaccinated girls, will prove the effectiveness of prevention.

Messenger RNAs are considered to be responsible to all biological activities in eukaryotic cells, bacteria, and virii [Gulliksen, 2007]. Therefore, clinical studies, microarray technology, and molecular amplifications methods are preferred as

reliable targets for disease diagnostics. Reverse transcriptase polymerase (PCR) and nucleic acid sequence-based amplification (NASBA) [Compton, 1991] are the amplification techniques more commonly used in routine analysis laboratories.

Microfluidics array technologies have enabled the automatization and miniaturization of such biomolecular techniques. PCR and array RNA analytical techniques are well known, but are cumbersome, time consuming, and cannot easily be miniaturized [Liao, 2005] due to the thermocycling step at high temperatures needed for each transcription step and required to achieve the same amplification rate as NASBA technology.

The isothermal amplification method for nucleic acids (NASBA) is a technology with the potential for broad applications in the field of RNA amplification systems [Compton, 1991]. The technology relies on the simultaneous activity of three enzymes: avian myeloblastosis virus reverse transcriptase, RNase H, and T7 RNA polymerase under isothermal conditions (41 °C), producing more than 109 copies in 90 min.

The ability of NASBA to homogeneously and isothermally amplify RNA (e.g., viral genomic RNA, mRNA or rRNA) extends its application range from decentralized viral diagnostics to the indication of biological activities such as gene expression and cell viability. Fluorescence molecular beacon probes enable real time monitoring of the amplification process [Gionata, 1998]. NASBA technology was successfully performed on Microsystems. However, the miniaturization needs sophisticated microfluidics and complicated measuring set-ups [Gulliksen, 2004].

This study introduces an Open Electrowetting On Dielectrics (OEWOD) microchip as microfluidics platform for the performance of Real Time Nucleic Acid Sequence Based Amplification (RT-NASBA) protocol for Human Papilloma Virus 16 (HPV16) detection, adapted for the basic operation, and an additional thermal element and temperature control sensor for the assay protocol and placed inside Luminescent Image Analyser System (LAS-3000). However, the details, efficiency or high- throughput of those are not covered herein. It was shown that RT-NASBA on OEWOD microchip was successfully accomplished with a larger time constant compared to PCR system, see chapter 4.6.3.

1.4 Scope and Outline of this thesis

This thesis describes and characterizes the behaviour of several electrolytes and biomolecules solutions under OEWOD conditions. A general overview of OEWOD microfluidics platform is provided 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 a biomolecular adsorption on OEWOD surfaces, and (4) the relative quantitative biomolecular adsorption behaviour of several biomolecules as Horseradish Peroxidase [HRP], Peroxidase-Conjugated Rabbit Anti- Mouse Immunoglobulins [HRP-IgG] and Deoxyribonucleic acid-Peroxidase [HRP- DNA], under EWOD effect (static conditions). The relative quantitative biomolecular adsorption in OEWOD surfaces is identified through a chemiluminescence method, which is a very practical and sensitive protocol where HRP and their labelled molecules are previously adsorbed at different OEWOD conditions.

The first experiments (see chapter 4.1.3) were performances to confirm the reversibility of such electrolytes and biomolecules under the EWOD principle. Biomolecular adsorption, as in every kind of such microfluidics platforms, represents for instance a functional problem, the so-called ―non-specific adsorption‖. Therefore, to address a different approach to this problem and to try to understand the behaviour of biomolecules adsorption on Electrowetting On Dielectrics surfaces, with the approach to functionalize the surface through the manipulation of the parameter involved in the biomolecular adsorption. On the other hand, in this study, biomolecular adsorption should be considered from the point of view of a new perspective. If the methodology used here to recognize it at this platform for microfluidics shows to be practical, sensitive, and faster than the traditional methodology used as well, it should not involve an additional and complex device, which is a requirement for the practical development of such devices.

This thesis introduces mainly two examples of possible applications. It does not concern the details, efficiency or high-throughput of those. OEWOD as a tool for the research of enzymatic reaction inactivation or kinetics in a luminescence imaging system, see chapter 4.6.1 and molecular biologics isothermal protocol: ―Nucleic Acid Sequence Based Amplification‖ (NASBA) for the amplification of artificial Oligonucleotide of Human Papilloma Virus 16 (HPV16) in an integrated optical and

temperature system, through Molecular Beacon (MB) technology which enable the real time measurement of the amplification products.

The knowledge acquired from previous biomolecular adsorption experiments and characterization of the surfaces, has provided a very important background for the development and performance of Real Time Nucleic Acid Sequence Based Amplification (RT-NASBA) protocol for Human Papilloma Virus 16 (HPV16) detection in OEWOD microchip. This study introduces an Open Electrowetting On Dielectrics (OEWOD) microchip as microfluidics platform for a bio-analytical device, adapted for the basic operation of the RT-NASBA protocol. The OEWOD microchip exhibits the feasibility for implementation, an additional thermal element and temperature control sensor, as well as the versatility to be placed inside an external optical sensing module, Charged Coupled Device (CCD)/Luminescent Image Analyser System (LAS-3000), or any other sensing devices, through the practical and flexible coplanar geometry, see chapter 4.7. The RT-NASBA protocol for HPV16 detection was performed and demonstrated on an OEWOD microchip and is described in chapter 4.7.3. That was possible thanks our project partner NorChip AS, Norway who provided and facilitated the consumables, advices, and very appreciated discussions.

The biomolecular adsorption parameters identified in previous studies, and in a similar OEWOD microfluidics platform to this thesis, are described in chapter 1.1 [Quinn, 2003; Yoon, 2003; Martínez-Garza, 2004; Wu, 2006; Bayiati, 2007]. Chapter 1.1 presents an overview of the development of the biomolecular adsorption in EWOD and OEWOD platforms. Chapter 1.2 introduces a State of the Art development of the applications field of OEWOD and EWOD devices Chapter 1.3 provides a brief introduction of cervical cancer development and the Real Time Nucleic Acid Sequence Based Amplification (RT-NASBA) protocol for Human Papilloma Virus 16 (HPV16) detection. Chapter 2 shows a general theory of thermodynamics in EWOD, the adsorption process principle, and forces involved in this phenomenon. In addition, it introduces the measurement techniques used in this study, such as contact angle principle and chemiluminescence methods. Chapter 3 describes the experimental setups, equipment‘s, protocols, and materials involves in this thesis.

The characterization of OEWOD surfaces were performed by means of Contact Angle (CA), Atomic Force Microscopy (AFM), and Laser Scanning Confocal Microscopy (LSCM) determinations; with the approach, first as a reference system for the adsorption experiments, and later, to try to recognize a third factor, that could influence the biomolecular adsorption processes, besides the optimization of the OEWOD surfaces, and provide a general overview of the OEWOD microfluidics platform, see chapter 4.1.

The ―reversibility‖ (EWOD effect) and ―actuation‖ behaviour of several electrolytes: PBS, HEPES, KCl, Horseradish Peroxidase (HRP) were performed and compared in two different OEWOD surfaces, see chapter 4.1.3. In chapter 4.1.4, the feasibility and compatibility of the EWOD effect principle of HRP in a single surface is described and compared to HRP and Glucose Oxidase (GOD) in hybrid surfaces available for OEWOD. The characterization of the biomolecular adsorption of Horseradish Peroxidase (HRP), Peroxidase-Conjugated Rabbit Anti-Mouse Immunoglobulins (IgG-HRP), and Deoxyribonucleic acid-Peroxidase (DNA-HRP) were analysed under OEWOD conditions and manipulating the parameters identified. In chapters 4.2, 4.3, 4.4, the comparison of mainly two OEWOD surfaces (Durimid 115A–Teflon®AF and Cellulose Acetate–Teflon®AF), through the biomolecular adsorption under different parameters are described. The proteins, enzymes, Oligonucleotide, and their conjugated uses in this study and previously mentioned, are described in chapter 3.5, and should be understood as biomolecules.

This thesis introduces chemiluminescence methods for determining the relative quantitative biomolecular adsorption in surfaces available for OEWOD. This is a very practical and sensitive protocol with a wide field of application, and is described in chapter 2.6, for the reaction mechanism see figure 2.6. This method was used to correlate the biomolecular adsorption to the parameters indentified and involved in the biomolecular adsorption process. Therefore, when HRP was adsorbed or biomolecules labelled with HRP, their properties catalyse the reagent Luminol, which was used to detect, 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 quantified with AIDA (Advanced Image Data Analyzer) see annex B, tables 4.3, 4.4 and 4.5.

It is important to mention that hypersensitive films were used for the chemiluminescence detection of the HRP and IgG-HRP experiments, see chapters 4.3 and 4.4. The experiments were realized in a dark room and the hypersensitive films were developed, fixed, washed and dried in an automatic X-ray film processor from Amersham Pharmacia Biotech Hyperprocessor. The HRP experiments with different p.I., potential applied, time, and the DNA-HRP were quantitative detected in the LAS-3000 imaging system already described, see chapter 4.5. The chemiluminescence detection in hypersensitive film ECL (standard X-ray films) was giving consistent autoradiograph quality and reproducible results. At that time, it had still not acquired the Luminescent Image Analyser System (LAS-3000). Therefore, the figures 4.12, 4.14, 4.17, 4.18, 4.19, 4.20, 4.21 and 4.22 were detected in this way and then scanned for this thesis. The quality of those can not to be improved; that will be mentioned again in chapter 4. The evaluation of the biomolecular adsorption in the hypersensitive films of OEWOD surfaces experiments were evaluated through the time necessary, or rinse steps necessary to de-adsorb the biomolecules absorbed, which will be mentioned with more detail later, see chapters 4.3 and 4.4.

The thesis has realized in OEWOD design, where the transport is performance in a co-planar array, the biomolecules are in direct contact with the hydrophobic actuated layer, which enables extremely flexible Lab-on-a-Chip devices that can be configured in software to execute virtually any assay protocol. The prototype OEWOD microchip was placed under or into a CCD device or Luminescence System Analyzer (LAS- 3000). In a typical laboratory, the complicated steps of any bioassay protocol could be performed, and in the OEWOD microchip, the measurement, detection or any sensing task could be performed. The performance of biomolecular adsorption experiments of HRP, IgG-HRP, and DNA-HRP were in (EWOD) statics condition, which means that the droplet was grounded by inserting a wire into the droplets.

Taking advantage of the biomolecules adsorption behaviour and the versatility of the coplanar microfluidics design, the possibility to adsorb/immobilization in OEWOD via EWOD tool was shown. Suitable micro-fabricated OEWOD microchip device structures into Luminescence system analysis chamber were assembled. By applying high voltage, specific polarization electrode, selected pH solution, surfaces, and time to specific locations during the ―actuation‖ or previously on the biomolecules, it was possible to minimize or increment biomolecular adsorption, for details see annex B, table 4.3, 4.4 and 4.5. 18

In this thesis, and according to Yoon et al. (2003), it is recognized that the biomolecular adsorption in OEWOD depends mainly on biomolecules properties like isoelectric point, pH of solution, polarity of the electric field, actuation time, and definitively of surface properties. Once the parameters were well known, the approach and its potential to optimize the adsorption process, in this case to functionalize/immobilize the OEWOD surface with any specific biomolecules and taking advantage of their properties, shall be considered for any assay. This allows to either minimize biomolecular adsorption (by considering the actuation time- potential applied, choosing the proper pH of the solution, and selecting the electrode polarity) or to intentionally immobilize/absorb biomolecules to specific locations determined by the underlying actuation electrode structure.

The physical or weak electrostatic biomolecular adsorption of HRP and their labelled biomolecules in Teflon AF 1600 surfaces, could be one step of the procedure on sensing protocol in OEWOD platform for bio-application therefore, here it will be mentioned mainly that immobilization before mentioned under EWOD condition is the simplest procedure and takes advantage of the EWOD protocol. It has to be considered, after or during the adsorption, if the biomolecules are un-oriented or their activity changes during the effect, or if an additional effect is presented according to the charge of the biomolecules in the cases of the classical electrowetting effect, for a possible realization of a bioassay protocol. This study do not presents any example of high throughput of the biomolecular adsorption (as immobilization technique) and such possible applications after the biomolecular adsorption. The details, efficiency or high-throughput of those are not covered herein. However, it is important to mention that after the biomolecular adsorption was performed, it was possible around a week later to detect it by chemiluminescence method.

In this thesis, biomolecular adsorption should be considered from the point of view of a new perspective besides the methodology (chemiluminescence detection) used to recognize it in this relative new platform for microfluidics, which has been confirmed to be practical, sensitive, and faster than the traditional methodology use. In addition, none involve an additional complex device, requirement for the practical development of BioMEMS devices.

2. Theoretical Background

Wetting is a phenomenon due to the contact between a liquid and a solid surface, giving by the intermolecular interactions properties. The amount of wetting depends completely on the surface energies like surface tensions of the interfaces involved, reaching a minimal energy state. The contact angle is a measure of the degree of wetting, the angle at which the liquid-vapour interface meets the solid-liquid interface; if the contact angle of wetting is 90° or greater, a surface is characterized as not-wettable, if the contact angle is below 90°, and the shape of a drop spreads to cover a larger area of the surface, a surface is characterized as wettable. The surface forces that control wetting are also responsible for other related effects, such as capillary effects, which are the cause of some confusing works between electrowetting and electrocapillarity [Berge, 1993].

In this thesis, the wetting properties are related to biomolecular adsorption, the contact angle analysis is extremely surface sensitive and is able to provide an insight in the dynamics process of the biomolecular adsorption. Surface tension is one of the dominant and significant forces in the micro scale and takes advantage of the strong physical stability of droplets in a surface with high surface tension properties like fluorocarbons. The principles are based on the fact that surface tension is a function of electric potential across the interface liquid-solid. Liquid handling and actuation by controlling surfaces tension have many advantages in micro scale applications and at this scale, electrostatic and/or Van der Waals and hydrogen bonding forces from the biomolecular fluidic become significant. Movement of the a biomolecular droplet to the lower surface tension area can also be interpreted as the tendency to minimize the energy by wetting the low surface tension region more than the higher one. If there is an external factor, for example electric field which changes the properties of the surfaces, the mathematical relationship between the applied electric potential and changed surface tension can be derived by thermodynamic analysis in the interface. The result is expressed in equation (1), called Lippmann‘s equation. 1 cV 2 o 2 (1)

where o is the surface tension when there is no applied electric potential across the interface, and c is the capacitance per unit area, should be considered as the surface tension in the interface between liquid metal and electrolyte, in equation

(1) needs to be interpreted as SL, surface tension in the solid-liquid interface. Young‘s equation (2) can be used to express the Lippmann‘s equation in terms of the contact angle as in equation (3). Let us call equation (3) as Lippmann-Young‘s equation.

SL SG LG cos (2)

1 1 2 cos cos o cV (3) LG 2

Where o is contact angle when the electric field across interfacial layer is zero, LG

Liquid-gas surface tension and SG solid-gas surface tension. Note that LG and SG are independent of the applied potential and remain constant [Kim, 2001].

The contact angle in equation (3) is a function of the applied voltage between liquid and electrode. As the contact angle changes due to applied voltage, surface wettability changes, and OEWOD system take advantages of the change in contact angle to induce liquid actuation.

2.1 Thermodynamics

The theoretical description of contact arises from the consideration of a thermodynamic equilibrium between the three phases: the liquid phase of the droplet (L), the solid phase of the substrate (S), and the gas/vapour phase of the ambient (V) (which will be a mixture of ambient atmosphere and an equilibrium concentration of the liquid vapour). The V phase could also be another (immiscible) liquid phase. At equilibrium, the chemical potential in the three phases should be equal. It is convenient to frame the discussion in terms of the interfacial energies. We denote the solid-vapour interfacial energy as SV , the solid-liquid interfacial energy as SL and the liquid-vapour energy (i.e. the surface tension) as simply , we can write an equation that must be satisfied in equilibrium (known as the Young equation):

0 SV SL cos (4)

where θ is the experimental contact angle. Thus the contact angle can be used to determine an interfacial energy (if other interfacial energies are known). This equation can be rewritten as the Young-Dupré equation (5):

(1 cos ) WSLV (5)

where ΔWSLV is the adhesion energy per unit area of the solid and liquid surfaces in the medium V [Gennes, 1985].

2.2 Contact angle measurement

A contact angle can be measured on static drops. The drop is produced before the measurement and has a constant volume during the measurement. A contact angle can be measured on dynamic drops. The contact angle is measured while the drop is being enlarged or reduced; the boundary surface is being constantly newly formed during the measurement. Contact angles measured on increasing drops are known as ―advancing angles‖; those measured on reducing drops as ―receding angles‖.

2.2.1 Static contact angles

In a static contact angle measurement the size of the drop does not alter during the measurement. However, this does not mean that the contact angle always remains constant; on the contrary; interactions at the boundary surface can cause the contact angle to change considerably with time. Depending on the type of time effect the contact angle can increase or decrease with time.

Figure 2.1: Alteration of the static contact angle as a function of time (Picture taken from Krüss website) [Krüss]

For example, these interactions could be: ● Evaporation of the liquid ● Migration of surfactants from the solid surface to the liquid surface ● Substances dissolved in the drop migrating to the surface (or in the opposite direction) ● Chemical reactions between the solid and liquid ● The solid being dissolved or swollen by the liquid

It may be a good idea to choose to measure the static contact angle when its variation as a function of time is to be studied as in the case of biomolecular adsorption. A further advantage of static contact angle measurement is that the needle does not remain in the drop during the measurement. This prevents the drop from being distorted (particularly important for small drops). In addition, when determining the contact angle from the image of the drop it is possible to use methods which evaluate the whole drop shape and not just the contact area. In the case of this work, static measurement were achieved with the needle remain in the drop during the measurement because the needle was used as electrode for simulating the EWOD condition. The size droplet in the static experiment are between 10 µm – 20 µm, the drop distorted due the needle were neglected.

However, changes with time often interfere with the measurement. There is also a further source of error: as the static contact angle is always measured at the same spot on the sample any local irregularities (dirt, inhomogeneous surface) will have a negative effect on the accuracy of the measurement. This error can be averaged out in dynamic contact angle measurements.

2.2.2 Dynamic contact angles

Dynamic contact angles describe the processes at the liquid/solid boundary during the increase in volume (advancing angle) or decrease in volume (retreating angle) of the drop, i.e. during the wetting and dewetting processes. A boundary is not formed instantaneously but requires some time before a dynamic equilibrium is established.

2.2.3 Advancing angle

During the measurement of the advancing angle the syringe needle remains in the drop throughout the whole measurement. In practice a drop with a diameter of about 3-5 μl (with the needle of 0.5 mm diameter which is used in Krüss measurement systems) is formed on the solid surface and then slowly increased in volume. At the beginning, the contact angle measured is not independent from the drop size because of the adhesion to the needle. At a certain drop size the contact angle stays constant; in this area the advancing angle can be measured properly (Fig. 2.2).

Figure 2.2: Measuring advancing angles (Picture taken from Krüss) [Krüss]

As a result of the wetting process, advancing angles always simulate a fresh surface for the contact angle; this is formed immediately after the creation of the contact between the liquid and the surface. This type of measurement is therefore the most reproducible way of measuring contact angles. As a result, advancing angles are normally measured in order to determine the surface free energy of a solid.

2.2.4 Receding angle

During the measurement of the receding angle the contact angle is measured as the size of the drop is reduced, i.e. as the surface is being de-wetted. By using the difference between the advancing and the receding angles it is possible to make statements about the roughness of the solid or chemical inhomogeneties; however, the receding angle is not suited for calculating surface energies. In practice a relatively large drop with a diameter of approx. 6 mm is deposited on the solid and then slowly reduced in size with a constant flow rate.

Figure 2.3: Measuring receding angles (Picture taken from Krüss) [Krüss]

The same guiding limits and conditions apply here as for the measurement of the advancing angle. For the measuring methods and methods of evaluating the drop shape applied for DSA Krüss G10, see Annex A.

2.3 Biomolecular Adsorption: Fundamental Principles

In this work biomolecular adsorption should be consider from the point of view, a new perspective, the methodology used here to recognized it at this relative new platform for microfluidics, should be practical, sensitive and faster than the traditional methodology used, as well, non involve an additional complex device, requirement for the practical development of BioMEMS devices.

Unfortunately the knowledge of biomolecular adsorption is not unified to predict the adsorption behaviour and mechanism of a specific system. The general theory is still only for determinate systems and conditions but something is true, the biomolecules could be consider as biopolymer, their specific biological function is related to its own

characteristics 3-D structure and therefore a continuation will be present merely theory about biomolecules, in special globular proteins which respect to practical applications, which are most relevant. There are several forces which may be involved in biomolecular adsorption and it process.

2.3.1 The three-dimensional structure of globular proteins [Norde, 2000]

Folding of a polypeptide into a compact globular structure occurs at the expense of the conformational entropy of the polypeptide chain. In particular, hydrogen bonds formed between the peptide units in α-helices and β-sheets largely reduce the rotational freedom of the bonds in the polypeptide chain. For instance, the folding of a polypeptide of molar mass 10,000 Da into a compact structure with 50% ordered (α-helices and/or β-sheet) structures involves a loss of conformational entropy of several hundreds of J K-1, which at room temperature, corresponds to a Gibbs energy increase of a few hundreds of kJ per mol. Hence, a stable globular structure exists only if this entropy loss is compensated by favourable interactions within the protein molecule and/or between the protein molecule and its environment. The major interactions for a protein molecule in an aqueous medium are indicated next.

2.3.1.1 Hydrophobic interaction. The favourable dehydration of apolar amino acid residues promotes association of theses residues in the interior of the globular protein molecule, thereby contributing to the stability of a compact structure. This contribution is estimated to amount to 9.2 kJ mol-1 per nm2 reduction of water accessible surface area. Hence, folding of a completely hydrated polypeptide of molar mass 10,000 Da into a compact globular structure of which the interior is 60% apolar residues lowers the Gibbs energy at room temperature by 500 kJ per mol.

2.3.1.2 Coulomb interaction. The majority of the ionic groups of a protein reside at the aqueous exterior of the molecule. When these charged groups are more or less homogeneously distributed their interactions may or may not stabilize a compact globular structure, depending on the pH relative to the isoelectric point of the protein. Under isoelectric conditions, when the protein has as many positive as negative charges, the overall intramolecular coulombic interaction is attractive and therefore favours a compact conformation. Away from the isoelectric point the excess of either positive or negative charge results in intramolecular repulsion, which promotes a

more expanded structure. Moreover, ionization of residues that are in the no ionized form buried in the low-dielectrics interior of the compact protein (e.g., histidine and tyrosine) stimulates the protein to unfold. When ionic groups are present in the interior of a compact protein molecule they usually occur as ion pairs. Disruption of such electrostatically favourable bonds, as would occur when the protein unfolds, is more or less compensated by hydration of the two ionic groups in the unfolded state.

2.3.1.3 Lifshitz-van der Waals interaction. These are interaction between fixed and/or induced dipoles. They are highly sensitive to the separation distance between the dipoles. When a highly hydrated polypeptide chain folds into a compact globular structure, dipolar interactions between the polypeptide and water are broken but a new dipolar interaction inside the protein molecule and between water molecules are formed. The net effect of dipolar interactions on the stability of the protein structure is difficult to estimated (it would be require accurate knowledge of the exact positions of the dipolar groups in the protein molecule), but it is generally assumed that because of the relatively high volume density in globular proteins dipolar interactions slightly favour a compact structure.

2.3.1.4 Hydrogen bonding. As with dipolar interactions, folding of the polypeptide chain implies a loss of hydrogen bonds between peptide units and water molecules on the one hand and the formation of intramolecular hydrogen bonds between peptide units (and possibly other groups) and among water molecules on the other hand. In the nonaqueous interior of a compact protein molecule, hydrogen bonds between peptide units stabilize secondary structures as α-helices and β-sheets. However, because of the compensating effects due to loss and creation of hydrogen bonds, it is not clear whether hydrogen bonding promotes a compact or an unfolded structure.

Under not too extreme conditions of pH, temperature, etc., hydrophobic interaction is major factor counteracting the loss of conformational entropy upon folding the polypeptide in an aqueous environment. Thus, a stable, low-entropy 3-D structure of the protein is achieved by virtue of entropy gain of water molecules that are released from contact with apolar amino acid residues. Because of these opposite effects that are of comparable magnitude, the folded compact structure is only marginally supported with Gibbs energy of stabilization of, typically, a few tens of kJ per mol of

protein. Hence, it should be realized that the other factors (i.e., Coulomb interaction, Lifshitz-van der Waals interaction, hydrogen bonding) may be decisive as to structure the polypeptide chain adopts. As a consequence, even mild changes in environment, such as changes in temperature, pH, ionic strength, addition of the other solutes, and exposure to an interface, may induce structural rearrangements in the protein molecule.

2.4 The adsorption process [Norde, 2000].

Protein adsorption comprises various aspects: kinetics, type of binding, adsorbed amount, and structure of the adsorbed layer and of the individual molecule therein. Figure 2.4 Schematically depicts the various step through which an adsorbing and desorbing protein molecule passes: transport toward the sorbent surfaces(1), deposition at the surfaces(2), relaxation of the adsorbed molecule(3), detachment from the surface(4), transport away from the surface(5), and possible restructuring of the desorbed protein molecule(6). The asterisks numbers indicate the degree of relaxation of the adsorbed molecule.

Figure 2.4: Schematic presentation of the protein adsorption process (Picture taken from Norde, 2000) [Norde, 2000].

The basic mechanisms of the transport toward the sorbent surface are diffusion and the convection by laminar or turbulent flow. In the absence of convection, the transfer of the protein molecules is a stochastic process. As adsorption proceeds the solution near the sorbent surfaces becomes progressively depleted, so that for the flux J toward a smooth sorbent surface

D J (c c )( )1/ 2 b s t (6)

Where cb and cs are the protein concentrations in the bulk solution and at the surface, respectively, D is the diffusion coefficient, and t is the incubation time. However, most transport processes take place under steady-state convective diffusion driven by a fixed concentration gradient.

J (cb cs ) (7)

Where k2 is a transport rate constant that depends on the hydrodynamics conditions and the diffusion coefficient. Deposition of the protein at the sorbent surface may be considered as the first order process d (k2cs ) dt (8)

Where is the adsorbed mass per unit sorbent surface area and k2 is the deposition rate constant. The value of k2 decreases with increasing coverage of the surface by the protein (i.e., with decreasing space available for the arriving protein to adsorb) and k2 is also lowered by any repulsive barrier for attachment. The origin of such a barrier might be electrostatic repulsion, a hydration effect, or the fact that a fraction of molecular collisions with the surface do not lead to attachment.

As the surface coverage increases, the rate of detachment increases. Eventually, equilibrium is reached, which implies that d k2 (cs ceq ) dt (9)

Where ceq is the concentration in bulk solution corresponding to the equilibrium value for , as given by the adsorption isotherm.

Relaxation of the Adsorbed Layer. Once the protein molecular has attached, it relaxes toward its equilibrium structure, which, because of the altered environment is as a rule different from the (native) structure in solution. Relaxation becomes more difficult as the protein-sorbent interaction is stronger because nonequilibrium states tend to become quenched. Furthermore, relaxation is retarded as the protein molecule has strong internal coherence. Structural relaxation implies optimization of

the protein-surface interaction and it normally involves a certain degree of ―spreading‖ of the protein molecule over the sorbent surface, developing a larger number of protein surfaces constant. As a consequence, after relaxation it becomes more difficult for the protein to detach from the surface. This may lead to a structural heterogeneity in the adsorbed layer: molecules arriving at an early stage of the adsorption process find sufficient area available for spreading, whereas this is not the case for the molecules that arrive when the surface is already (partially) covered with protein. Another consequence is that the outcome of the adsorption process depends on the rate of attachment and the rate of spreading relative to each other. When spreading occurs relatively quickly, the adsorbed molecules are more flattened.

Adsorption Isotherm. From the compilation of (t→∞) obtained at varios supply rates the adsorption isotherm (ceq), can be derived. Adsorption for a globular protein usually displays well-defined plateau values that are reached at ceq typically less than a few tenths of a g dm-3. Usually, the plateau adsorption is lower than or, maximally, comparable to the adsorbed amount in a closely packed monolayer of more or less native-like molecules. It will be understood that (ceq) depends on the mode of supply rate (the latter being higher at higher (ceq). Indeed, using various experimental techniques it has been concluded that the degree of structural rearrangements in adsorbed proteins decreases with increasing ceq. Hence, as at a given value ceq may depend on the history (i.e., the supply mode), (ceq) may not reflect true thermodynamic equilibrium. Just like other polymers, protein molecules attach via multiple contacts to a sorbent surface and one would therefore expect high-affinity adsorption, i.e. merging of the initial part of the isotherm with the axis. Such isotherms are indeed often observed, but isotherm showing a more gentle slope are not exceptional. It is remarkable that even in the case of non-high affinity character. Such a hysteresis between adsorption and desorption is a manifestation of (apparent) irreversibility of the protein adsorption process.

Adsorption and Desorption Rates. When the adsorption isotherm (ceq), is available, it can be inverted in (ceq) to give an explicit expression for d /dt. For the rising part of the isotherm cb is considerably larger than ceq resulting in a relatively high rate of adsorption but as soon as the plateau value is reached ceq rapidly

approaches cb and d /dt becomes very small. In the case of high-affinity isotherm ceq is almost zero up to reaching the (ceq) plateau value.

Reversibility of the Adsorption Process. Phenomenologically, a system is in equilibrium if no changes take place at constant surroundings. At constant pressure p and temperature T the equilibrium state of a system is characterized by a minimum value of the total Gibbs energy G. Any other state, away from this minimum is in nonequilibrium and there will be a spontaneous transition toward the equilibrium state provided that the Gibbs energy barriers along this transition are not prohibively large. By definition, a process is reversible if during the whole trajectory of the process the departure from equilibrium is infinitesimally small, so that in the reverse process the variables characterizing the state of the system return through the same value but in reverse order. Because a finite amount of the time is required for the system to relax to its equilibrium state, investigating the reversibility of a process requires that the time of observation exceeds the relaxation time.

2.5 Driving forces for the protein adsorption

The tendency of the proteins to accumulate at the interfaces is determined not only by properties of the protein molecules and the sorbent surface but also by the nature of the solvent, the present of the other solutes, pH, ionic strength, and temperature. Whatever the mechanism, at constant temperature T and pressure p adsorption proceeds spontaneously, if the Gibbs energy G of the system decreases.

adsG adsH T adsS 0 (10)

Where H and S are the enthalpy and entropy of the system and where ads indicates the change invoked by the adsorption process. The more negative the value adsG , the higher the adsorption affinity is.

One way to analyze the overall adsorption process is to establish how various interactions contribute to of course; such a thermodynamic approach can be successful only if the system is well characterized. Therefore, the forthcoming

discussion will be restricted to a systems composed of one type of protein and one type of sorbent in a well-defined environment.

2.5.1 Interaction between electrical double layers

Generally, both the protein molecule and the sorbent surface are electrically charged. In an aqueous medium charged sorbents and proteins are surrounded by counterions and coions, together accounting for the so-called counter charge that neutralizes the surfaces charge. A fraction of the counter charge may be bound to the sorbent surface and/or the protein molecule and the other part is diffusely distributed in the solution. The surface charge and the counter charge together form the electrical double layer. The Gibbs energy Gel to invoke a charge distribution can be calculated as the isothermal, isobaric reversible work.

0 ' ' Gel 0d 0 (11) 0

' ' Where 0 and 0 are the variable surface potential and surface charge density, respectively, during the charging process. Solving this equation requires knowledge of ( ) and this functionality can be derived from models for electrical double layer. For the bare sorbent surface the Gouy-Stern model may adopted. For the dissolved protein molecule a discrete-charge model, as developed by Kirwood, may be more appropriate and for the protein covered sorbent surface different model have been proposed. Charge distributions for the system before and after adsorption are schematically depicted in figure 2.5

Figure 2.5: Schematic representation of a charge distribution before (left) and after (right) protein adsorption. The charge on the sorbent surface and the protein molecule are indicated by +/-.The low-molecular-weight electrolyte ions are indicated by +/- inside the circle (Picture taken from Norde 2000) [Norde 2000]. 32

Under most (practical) conditions the Debye length, i.e., the separation distance over which charges interact, is considerably smaller than the thickness of the adsorbed protein layer. For instance, in a solution of 0.1 M ionic strength the Debye length is about 1 nm, whereas the thickness of the adsorbed protein layer in which the molecules retain a compact conformation is at least a few nanometers. Hence, such a compact protein layer shields the protein –sorbent contact region from electrostatic interaction with the solution is that the net charge density in that contact region is essentially zero. Using phenomenological (thermodynamic) linkage relations, this charge regulation can be derived from the electrolyte dependence of the protein adsorption isotherm. Charge neutralization has been confirmed experimentally by shift in the proton charge of the protein upon adsorption at a charge surface.

Adjustment of the sorbent surface charge may occur as well, as has been demonstrated for e.g., silver halide and clay particles. Apart from adjustments in the protein and the sorbent, charge neutralization may be further regulated by the incorporation of indifferent ions from solution into the protein-sorbent contact region. This has been demonstrated by electrokinetic data and, more directly, by tracing radiolabeled ions. Trends, derived from electrokinetics, they are clearly follow the charge antagonism between the protein molecules in solution and the bare sorbent surface.

As a consequence of the charge regulation adsGel is not very sensitive to the charge densities of the protein and the sorbent and it usually does not exceed a few tens of RT per mol of adsorbing protein. Its sign and value depend on the charge distributions and the dielectric constants of the electrical double layers before and after adsorption, respectively. It should be realized that the transfer of ions from the aqueous solution into the non aqueous protein sorbent environment is chemically unfavourable. In the other words, the chemical effect of ion incorporation opposes the overall adsorption process. This explains why maximum protein adsorption affinity is reached when the charge density on the protein just matches that on the sorbent surface so that no additional ions have to be incorporated.

2.5.2 Changes in the State of Hydration

In liquid water of not too high temperature, say <80°C, the water molecules are strongly hydrogen bonded, causing strong internal coherence.

Polar groups interact favourably with water molecules, mainly through electrostatic interaction, including hydrogen bonding. These interactions over compensate the strong cohesion between the water molecules, rendering the polar components readily soluble in water. Apolar groups, which do not offer the possibility for such favourable interactions with water, are expelled from an aqueous environment. This mechanism is at the basis of hydrophobic interaction. If the surfaces of the protein molecules and the sorbent are polar, their hydration is favourable. In that case it is probable that some hydration water is retained between the sorbent surface and the desorbed protein molecule. However, if (one of) the surfaces are (is) apolar, dehydration would be a driving force for adsorption.

Although the apolar residues of a globular protein in water tend to be buried in the interior of the molecule, the water-accessible surface of the protein may still constitute a significant polar fraction, even up to 40-50%. For the water-soluble, non- aggregating proteins the polar residues are more or less evenly distributed over the exterior of the molecules. The presence of pronounced apolar patches may lead to protein aggregation.

In several studies the influence of protein polarity on adsorption has been experimentally confirmed. In this context it should be realized that apart from the polarity of the outer shell the overall polarity of the protein could be relevant for its adsorption behaviour. The overall polarity influences the protein structural stability and, hence, the extension of structural perturbation upon adsorption. This in turn affects the adsorption affinity.

The effect of sorbent surface polarity is also difficult to assess, because modifying the potential usually involves a changes in the surfaces electrostatic potential as well. A fair estimate of contribution from hydration changes to Gibbs energy of the adsorption adsGhydr may be inferred from partitioning (model) components in water- nonaqueous two-phase systems. It has thus been estimated that, at room

temperature, dehydration of apolar surfaces involves a lowering of the Gibbs energy of about 10-20 mJ m-2. For a protein of molar mass 15,000 Da that adsorbs to about -2 1 mg m it results in adsGhydr ranging between –60 and –120RT per mole of adsorbed protein. It is obvious that apolar hydration dominates over the effects from overlapping electrical double layers and dispersion interaction.

2.5.3 Dispersion Interaction

Dispersion (or London-van der Waals) interaction results from attraction between electron ―clouds‖ that are in close proximity but do not overlap. This interaction is attractive. For a sphere interacting with a planar surface the contribution from dispersion interaction to the Gibbs energy of adsorption, adsGdisp , can be approximated by

A132 a a h G ln (12) ads disp 6 h h 2a h 2a

Where A is the Hamaker constant for the interaction between the flat sorbent(1) and the spherical protein molecule(2) across the (aqueous) medium(3), a the radius of sphere, and h the distance of closest approach between the sphere and the surface. Under most condition h<< a so that Eq. (8) simplifies to

a A132 G (13) ads disp 6h Values for Hamaker constants (of the individual components) are given in several references. The Hamaker constant for the system can be derived from the individual ones according to the followings rules.

1/ 2 1/ 2 1/ 2 1/ 2 A132 (A1 A3 )( A2 A3 ) (14) and

1/ 2 A132 (A131A232) (15)

In aqueous media usually A1>A3 and A2>A3, so that according to Eq. (14), A123 > 0 and, hence, < 0, which implies attraction. The Hamaker constant for that 35

interaction across water is about 6.6X10-21J for a globular proteins, (1-)X10-19 J for synthetic polymers such as polystyrene or Teflon. According to Eq. (13), adsGdisp varies proportionally with the dimensions of protein molecule and it drops off hyperbolically with increasing distance between the protein and the sorbent surface. Thus, for a globular protein molecule of 3 nm radius at 0.15 nm distances from the surface amounts to –(1-3) RT per mole at a synthetic polymer surface and to –(4-7) RT at a metal surface. Because of the various approximations involved, these values are only indicative. Deriving more accurate values is practically impossible because of the irregular shape protein molecules and, possibly, sorbent surfaces adopt in real systems. Moreover, rearrangements in the protein structure induced by adsorption may affect the Hamaker constant in an unknown way.

2.5.4 Rearrangement in the protein Structure

The three dimensional structure of a globular protein molecule is only marginally stable, so interaction with a sorbent may induce changes in the structure. However, as compared with flexible polymers the conformational changes in adsorbing protein molecules are usually small. It is generally observed that the thickness of a monolayer of adsorbed protein, determined, for example, by ellipsometry, light scattering, viscometry, scanning probe microscopy, or the surface force technique, is comparable to the dimensions of the native protein molecule. Structural rearrangements do not lead to unfolding into a loose, highly hydrated ―loop-and tail‖ structure. After adsorption, at one side of the protein molecule the aqueous environment is replaced by the sorbent material. As a consequence intramolecular hydrophobic interaction becomes less important as a structure-stabilizing factor; i.e., apolar parts of the protein that are buried in the interior of the dissolved molecule may become exposed to the sorbent surface without making contact with water. Hydrophobic interactions between amino acid residues support the formation of the α-helix and/or β-sheets content is indeed expected to occur if the peptide units released from the helices and sheets can form hydrogen bonds with the sorbent surface, as is the case at polar surfaces. Then a decrease in ordered (secondary) structure would result in an increased conformational entropy of the protein and, hence an increased adsorption affinity. The contribution from increased conformational entropy to a negative value for adsG may amount to some tens of RT per mol of protein. However, adsorption at an apolar, non-hydrogen-bonding 36

surface may stimulate intramolecular peptide-peptide hydrogen bonding, resulting in increased order in the protein´s structure. Whether or not extra hydrogen bonding within the protein molecule occurs depends on the outcome of opposing effects of the energetically favorable hydrogen bonds and the unfavourable change in the conformation entropy.

2.5.5 Sorbent Surface Morphology

Many surfaces are not completely smooth and rigid. For instance, at surfaces of polymeric materials polymers chains may protrude into the solution to some extent. Art natural surfaces such as those of, e.g., biological membranes and bacterial cell walls, natural surfaces such as proteins and/or polysaccharides are often present. When these surface polymers extend into the surrounding medium with some flexibility, the surface will respond dynamically to protein adsorption. On the one hand, this would offer the possibility of optimizing contact by confirming to the shape of the adsorbing protein molecule. On the other hand, squeezing the surface polymers between the sorbent and the adsorbed protein layer would cause steric repulsion because of increased osmotic pressure and decreased conformational entropy.

Grafting or preadsorbing water-soluble oligomers or polymers has been used to tune protein adsorption. In particular, polyethylene oxide (PEO) has been proved to be successful in producing protein-repelling surfaces. The protein resistance is determined by the length of the PEO chains and their density at the sorbent surface. Thus, relatively short chains of PEO, consisting of, say, less than 10 monomer units, do not severely hamper protein adsorption but they do prevent intimate contact between the protein and the underlying sorbent surface. As a consequence, less structural perturbation occurs so that the adsorbed molecules retain more biological activity. Longer PEO chains more effectively repel proteins [Norde, 2000].

It is to be expected that proteins do adsorb from an aqueous solution onto apolar surfaces, even under condition of electrostatic repulsion. With polar surfaces a distinction must be made between structurally stable (―hard‖) and structurally labile (―soft‖) proteins. The hard proteins adsorb at polar surfaces only if they are electrostatically attracted. The soft proteins undergo more severe structural

rearrangements (i.e., a decrease in ordered secondary structure) resulting in an increase in conformational entropy large enough to make them adsorb on a polar, electrostatically repelling surface.

The mutual influences of theses effects on protein adsorption are clearly demonstrated in a few systematic studies using well-defined proteins and sorbent materials [Baszkin, 2000].

The isoelectric point (p.I.) is the pH at which a particular molecule or surface has not net electrical charge. Amphoteric molecules called zwitterions contain both positive and negative charges depending on the functional groups present in the molecule. They are affected by pH of their surrounding environment and can become more positively or negatively charged due to the loss or gain of protons (H+). A molecule's p.I. can affect its solubility at a certain pH. Such molecules have minimum solubility in water or salt solutions at the pH which corresponds to their p.I. and are often seen to precipitate out of solution. Biological amphoteric molecules such as proteins contain both acidic and basic functional groups. Amino acids which make up proteins may be positive, negative, neutral or polar in nature, and together give a protein its overall charge. At a pH below their p.I., proteins carry a net positive charge. Above their p.I. they carry a net negative charge.

2.6 Chemiluminescence as Biomolecular Adsorption measurement technique

Many different protocols and techniques have been developed to measure the amount of biomolecular adsorption onto surfaces. The most commonly used ones are based on optical and spectroscopic principles such as: Optical Waveguide Light mode Spectroscopy, Total Internal Reflection, Scanning Angle Reflectometry, Ellipsometry, and Surfaces Plasmon Resonance. Techniques based on spectroscopy principles rely on the biomolecular adsorption on the interaction with photons in the interfaces such as: Infrared Adsorption, Raman Scattering, fluorescence emission, and circular dichroism. However, there are also non optical techniques such as Quartz Crystal balance based on weight measurement or chemiluminescence based on a chemical reaction. The Chemiluminesce technique has the speed and safety of chromogenic detection methods, at higher sensitivity

levels. The HRP substrate produces a high intensity emission with low spectral background, allowing the detection of minute quantities of a biomolecules. The most common use of chemiluminescence emission techniques is in the detection and assay of biomolecules in systems such as ELISA and Western blots. All of the techniques before mentioned present their respective advantages and disadvantages.

This thesis introduces the chemiluminescence emission technique, as a method that offers a great specificity, sensitivity, easily as well, suitable for detection of biomolecular adsorption in this kind of microfluidics platform, where polymer materials are involved. Horseradish Peroxidase (HRP) is widely used as an enzyme label for medical diagnostics, research applications, and availability substrates for colorimetric, fluorometric, and chemiluminescence, providing numerous detection options, therefore here it was used as a biomolecules (protein/enzyme) to adsorb on OEWOD surfaces, taking advantage of it is the enzyme which combined with peroxide catalyzed the oxidation of acridan substrate, generating thousands of acridinium ester intermediates per minute. These intermediates react with peroxide under slight alkaline conditions, as it decays to its ground state, producing a sustained, high intensity chemiluminescence with maximum emission at a wavelength of 430 nm, see reaction; figure 2.6. The resulting light is detected on hypersensitive film or a Luminescent image analyzer (LAS-3000).

Therefore adsorbed HRP and its labelled molecules in an OEWOD surface give a luminescent image. Additionally, contact angles were measured with a modified Krüss DSA10 system [Herberth, 2006] and used as a function of biomolecular adsorption [Daves, 1996]. The relative quantitative analysis of the biomolecular adsorption on the OEWOD surfaces was realized with the software Advanced image data analyzer Aida (raytest Isotopmessgeräte GmbH, Germany), see annex B, table 4.3, 4.4 and 4.5.

F F F F O O F Peroxide O H O F HRP

N H + N H CH 3 CH 3 Cyclic Diacylhidrazide Buffer Peroxide Acridinium ester

O F

+ F HO N H F

CH 3 C O 2 Exicted Product

Figure 2.6: Schematic presentation of the chemiluminescence reaction (Chemical structures layout in Symyx Draw) (ECL Plus western Blotting Reagents RPN2132, from Amersham Biosciences)

In theory, one photon of light emitted should be given off for each molecule of reactant. This is equivalent to Avogadro's number of photons per mole of reactant. In actual practice, non-enzymatic reactions seldom exceed 1% QC, quantum efficiency. Therefore, in this thesis the chemiluminescence emitted is interpreted as a function of biomolecular adsorption and was called: relative quantitative biomolecular adsorption.

The chemiluminescence method described above was chosen due to this practical and very sensitive protocol to identify absorbed or labelled biomolecules with HRP on surfaces available for OEWOD at different conditions and to indentify and manipulate the parameters involved in the biomolecular adsorption process in OEWOD microfluidics platform.

3. Experimental materials, equipment, and methods

In this thesis were performed mainly; characterization of OEWOD surfaces, biomolecular adsorption experiments in OEWOD surfaces, and the performance of bioassay protocol R-T NASBA for HPV16 detection in OEWOD microchip. The experimental protocols, methods, set up, materials and devices involved will be described in this chapter.

Hydrophobic and non sticking materials are needed to provide the basic requirements for the OEWOD microfluidics platform. A larger initial contact angle, a high dielectrics constant such as Teflon AF and Fluorocarbon polymers are the core of this principle according to the young-Laplace formulation. However, after the different approach of this thesis, other polymers were used in combination with Fluorocarbon polymers and with very similar properties for the system surfaces, to optimize the actuation and to decrease the biomolecular adsorption or to address, as is presented in this thesis.

3.1 Teflon®AF 1600 [DuPont]

Teflon®AF is the DuPont registered trademark and designation for its family of amorphous copolymers based on 2,2-bistrifluoromethyl-4,5-difluoro- 1,3-dioxole (PDD) with other fluorine-containing monomers. Tetrafluoroethylene (TFE) copolymers have the excellent chemical resistance and thermal stability of Teflon®PTFE, PFA, and FEP in addition to unique electrical and optical properties. Teflon®AF polymers have the lowest dielectric constants of any known solid polymer, extremely low refractive indices, high gas permeability, and low thermal conductivities. These copolymers are optically clear with high transmission values from the ultraviolet through the near infrared. They are soluble at room temperature in several fluorocarbon solvents. Properties may be varied by changing molecular weight, comonomer ration, and comonomer structure.

Polytetrafluoroethylene (PTFE) was discovered in 1937 by DuPont scientist, Dr. Roy J. Plunkett. This polymer is characterized by a high degree of crystalline order, a high melting temperature (327°C), and extreme chemical inertness. Molecular weight is very high, estimated to be between 5X106 and 100X106 Dalton. This high molecular weight leads to very high melt viscosities of the order of Pa sec at 380°C. 41

PTFE is also insoluble at temperatures below about 300°C. Thus, whereas PTFE possesses many desirable properties, it is difficult to process into desirable shapes by either thermal or solution methods. This problem is addressed by copolymers of TFE with hexaflouropropylene (HFP), introduced by DuPont in 1962 under the trade name Teflon®FEP. These copolymers have approximately 10 mol % HFP and estimated molecular weights in the range of (100-300) X106 Dalton. The presence of HFP in the polymer chain disrupts the ability of TFE to crystallize. The reduced crystallinity combined with lower molecular weight makes it possible to melt process FEP by many of the same methods used for other, more conventional polymers. The HFP comonomer reduces the melting temperature of the FEP copolymers to 265°C from the 327°C of TFE

F F

CF CF 2 2 O O

TFE F3C CF3

PDD

Figure 3.1: 2,2-bistrifluoromethyl-4,5-difluoro- 1,3-dioxole (PDD) and Tetrafluoroethylene (TFE) copolymers, (Chemical structure layout in Symyx Draw) picture taken from Resnick, 1993 [Resnick, 1993].

Homopolymer. Therefore, the upper use temperature of FED is reduced substantially below that of PTFE. In 1972, DuPont introduced copolymers of TFE with perfluoro (alkyl vinyl ethers) under the trademark Teflon®PFA. Because the perfluoro (alkyl vinyl ethers) are more effective than HFP in Disrupting chain order and reducing the degree of crystallinity, smaller amounts may be used to achieve melt processibility. The melting point of the copolymers is 305°C. The higher melting temperature gives properties to finish parts that are much closer to PTFE than FEP. The most recent advance in the DuPont family of fluoroplastics occurred in 1989 with the introduction of copolymers of 2,2-bistrifluoromethyl-4,5-difluoro-1,3-dioxole(PDD) with TFE. These are sold by DuPont under the trademark Teflon®AF.

The structure of the PDD monomer is shown in figure 3.1. It is made in a four-step synthesis starting with ethylene oxide and hexaflouroacetone. PDD readily 42

polymerizes with TFE and the other fluorine-containing monomers such as vinylidine fluoride, chlorotrifluoroethylene, vinylfluoride, and perfluoro (alkyl vinyl ethers). It can also homopolymerize to form an amorphous fluoropolymer with a glass transition

temperature (Tg) of 335°C, which is PDD homopolymer. This homopolymer is difficult to fabricate because it has limited melt flow below its decomposition temperature and is soluble to only a few tenths of a percent.

The two general-purpose grades of Teflon®AF currently available are AF 1600, with

a Tg of 160°C and 64 mol% dioxole, and AF 2400, with a Tg of 240°C and 87 mol% dioxole. In this thesis only Teflon ® AF 1600 is used. Teflon®AF copolymers share many characteristics of other types of DuPont fluoropolymer resins, but also have significant differences, see table 3.1. All of the polymers in the family of DuPont fluoropolymer resins have high temperature stability due to their perflourinated structure. This also accounts for their excellent chemical resistance, low surface energy, and low water adsorption. The limiting oxygen index (LOI) of all of the DuPont fluoropolymer is greater than 95, meaning that they require an atmosphere of greater than 95% oxygen to sustain combustion [Resnick, 1993] [DuPont].

3.1.1 Material Properties

Table 3.1 gives an overview of the material properties of Teflon®AF 1600. Material Properties

Young's modulus E 1.6 Gpa Melt Viscosity 2657 Pa s at 250°C, 100 s-1 Coefficient of thermal expansion 260 ppm/°C Thermal conductivity 0.18 W/m°C at 40°C[Resnick, 1999]

Glass temperature Tg 160 °C ±5 Refractive index n 1.31 Dielectric constant 1.93 Critical Surface Tension Wetting 13 mJ/m2[Sedev, 2004]

Table 3.1: Material properties of Teflon®AF 1600

Figure 3.2: Chemical structure of Teflon®AF 1600 (Chemical structure layout in ADC/ChemSketch) [Ho, 2003]

3.2 Cellulose Acetate

Cellulose Acetate is one of the most important esters of cellulose; see figure 3.3. It was first prepared by Paul Schützenberger in 1865. It took another 29 years before Charles Cross and Edward Bevan patented a process for its manufacture. At about the same time, Little in the US and Bronnert in Germany simultaneously produced cellulose acetate filaments, which were in actual fact cellulose triacetate, which differs in that it does not easily dissolve in common solvents. In 1904 George Miles found that partial hydrolyse Cellulose Acetate would dissolve in acetone. Brothers Henri and Camille Dreyfus exploited this fact to make Cellulose Acetate films and lacquers in 1910. During World War I, the technology was used for waterproofing and stiffening the fabrics covering aeroplane wings. In 1919, they introduced the first cellulose based yarn to the market, called Celanese. Commercially, Cellulose Acetate is made from processed wood pulp. The pulp is processed using acetic anhydride to form acetate flakes from which products are made. Coming from wood pulp means that unlike most man-made fibres, it comes from a renewable resource and is biodegradable. Another technique for producing Cellulose Acetate involved treating cotton with acetic acid, using sulfuric acid as a catalyst (http://www.azom.com/details.asp?ArticleID=1461).

O O CH CH O CH 3 O CH O CH 3 3 C 3 3 C O CH3 C C C O O C O O O O O O O O O O O O O O O O C C C O C O H C H C 3 O H C O C 3 O H C O 3 O 3 C H3C H C O 3

Figure 3.3: Chemical structure of Cellulose Acetate (Chemical structure layout in Symyx Draw).

3.2.1 Material Properties

Table 3.2 gives an overview of the material properties of Cellulose Acetate.

Material properties

Young's modulus E 55.9-70.7 Mpa [Longxiao, 1999] Coefficient of thermal expansion 298.5- 671.6 ppm/°C Thermal conductivity 0.59 – 1.34 W/m°C at 23°C

Glass temperature Tg 189.6°C[Longxiao, 1999] Refractive index n 1.49 Dielectric constant 6 (1kHz)

Table 3.2: Material properties of Cellulose Acetate

3.3 Durimid 115A

The Durimide 100 Series are a range of self priming, non-photosensitive polyamic acid formulations which become fully stable polyimide coatings after thermal curing. They can be photo-imaged using a positive photoresist mask. Softbaked polyimide films are coated with photoresistant, softbaked, exposed, and post exposure baked. When the photoresist is developed, the polyimide is etched, transferring the pattern from the photoresistant onto the polyimide. The photoresist is subsequently removed with a solvent rinse and the polyimide thermally cured. The minimum geometry which can be achieved by this method depends upon the thickness of the polyimide at softbake. The smallest resolvable feature is approximately four times the softbake film thickness [Fujifilm Electronic Material].

3.3.1 Material Properties

Durimide 100 is a polyamidic acid with the following structure, see figure 3.4:

Figure 3.4: Monomer structure of polyamidic acid (polyimide) (Chemical structure layout in ADC/ChemSketch)[Fujifilm Electronic Material]

Table 3.3 gives an overview of the material properties of Durimid 115A.

Material Properties

Density 1.49 g/cm³ Young's modulus E 3.3 GPa Viscosity (25°C) cS 6200 8300 Coefficient of thermal expansion CTE 32 ppm/°C Thermal Decomposition Temperature 597°C

Glass temperature Tg 371°C Refractive index n (cured) 1.81 Dielectric constant 1MHz; 0%-50% RH 3.1-3.4

Table 3.3: Material properties of Durimid 115A

3.4 SU-8

EPOXIES Photoresistants such as the SU-8, are based on epoxies. The term epoxy is a prefix referring to a bridge consisting of an oxygen atom bonded to two other atoms, very often carbon, already united in some way. Such a structure is called 1,2-epoxide, see figure 3.5. An epoxy resin is defined as a molecule containing one or more 1,2-epoxy groups. Such molecules are capable of being converted to a thermo set form or three-dimensional network structure, see figure 3.6. This process is called curing or cross linking.

Figure 3.5: Chemical structure of 1,2-epoxy ring (Chemical structure layout in ADC/ChemSketch)

The term curing or cross linking is used to describe the process by which one or more types of reactants, i.e., an epoxide and a curing agent, are transformed from a low-molecular-weight material to a highly cross linked network.

Figure 3.6: Chemical structure of SU-8. The basic SU-8 molecule, note the 8 epoxy groups (Chemical structure layout in ADC/ChemSketch)

3.4.1 Material Properties

Table 3.4 gives an overview of the material properties of SU-8 photoepoxies.

Material Properties Density 1.49 g/cm³ Young's modulus E 4.4 Gpa Viscosity 60% SU8-40% solvent : 1.5 Pa.s 70% SU8-30% solvent : 15 Pa.s Coefficient of thermal expansion CTE 186.5 ppm/°C Thermal conductivity 0.74 W/m°C

Glass temperature Tg 200°C Refractive index n 1.8 at 100 GHz / 1.7 at 1.6 THz Dielectric constant 3 at 10 MHz

Table 3.4: Material properties of SU-8 [Angelopolous, 2001]

3.5 Biomolecules (GOD, PE, HRP, IgG-HRP and DNA-HRP)

In this thesis, the following should be understood as biomolecules: B-Phycoerythrin (PE), Glucose Oxidase (GOD), Horseradish Peroxidase (HRP), Peroxidase- Conjugated Rabbit Anti-Mouse Immunoglobulins (IgG-HRP) and Deoxyribonucleic acid-Peroxidase (DNA-HRP). The electrolytes used: Phosphate buffered saline

(PBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and Potassium Chloride, (KCl) will not described in this thesis.

The electrolytes solutions PBS, HEPES, KCl and the biomolecules GOD and HRP were used in the preliminary microfluidic experiments on OEWOD surfaces, to confirm their availability to actuate by means of EWOD principle and later GOD and HRP for the optimization of OEWOD surfaces. HRP and their labelled molecules (DNA-HRP and IgG-HRP) were involved in adsorption experiments due the easy detection through chemiluminescence method. A continues a short description of their properties.

3.5.1 B- Phycoerythrin (PE)

B-Phycoerythrin (PE) was obtained from sigma, Germany. Phycoerythrin is one of the most common and brightest dyes and contains 25 flours. PE is a large protein with an approximate molecular weight of 240 kDa; it emits at about 570 nm and was excited by common Argon laser lines by the Zeiss LSCM devices. For the preliminary phase of OEWOD surfaces characterization, B-Phycoerythrin was adsorbed onto annealed and not annealed OEWOD surfaces, in order to identify by means of laser scanning confocal microscopy images, a physical change in Teflon®AF coated surfaces after the adsorption.

3.5.2 Glucose Oxidase (GOD)

The molar extinction coefficient of a 1% (wt/v) solution at 280 nm is 13.8 (in 0.1M potassium phosphate pH 7.0, yellow solution). The native protein is acidic having an isoelectric point (p.I.) of 4.2. The diffusion coefficient of the holo-enzyme in 0.1M NaCl is 4.94 x 10-7 cm2 s-1. The GOx native dimer has the molecular weight of 160 kDa (composed of two subunits of 80 kDa each). The GOx (native dimer) dimensions are 70 Å x 55 Å x 80 Å. Each subunit is a compact spheroid with approximate dimensions of 60 Å x 52 Å x 37 Å. The minimum distance between the flavin and the surface of the monomer is 13 Å. The two isoalloxazine moieties are separated by a distance of about 40 Å. Michaelis Constant (apparent) KM = 33 Molecular Activity (Turnover Number) = 2.28 104.

3.5.3 Horseradish Peroxidase (HRP)

Horseradish Peroxidase (HRP) is well investigated and one of the most important enzymes obtained from a plant source. It is an important heme-containig enzyme that utilizes hydrogen peroxide to oxidize a wide variety of organic and inorganic compounds through the follow catalytic reaction:

ROOR' + Electrons Donate (2 e−) + 2 H+ → ROH + R'OH

It continues to attract the attention of researchers from a variety of disciplines because of its practical and commercial applications. Advances in understanding the structure and catalytic mechanism of horseradish peroxidase have been made using protein engineering and other techniques. The physiological role of the enzyme is now being investigated in the context of new information on the plant peroxidase gene family of Arabidopsis thaliana [Veitch, 2004].

Figure 3.7: HRP Horseradish Peroxidase, picture taken from [Veitch, 2004]

Horseradish Peroxidase is widely used as an enzyme label for medical diagnostics, research applications and availability substrates for colorimetric, fluorimetric and chemiluminescent assays providing numerous detection options. In this work, it is introduced, as a biomolecules to adsorb and characterize the OEWOD surfaces, see chapter 2.6 for their detection.

Although the term Horseradish Peroxidase is used somewhat generically, the root of the plant contains a number of distinctive peroxidase isoenzymes of which the C isoenzyme (HRP C) is the most abundant [Veitch, 2004].

Horseradish peroxidase has six basic isoenzymes, E1 to E6 with different molecular weights due to the contents of carbohydrate and amino acid compositions. Of the six isoenzymes, four isoenzymes, E3 to E6, have a extremely high p.I. values of over 12 and appreciably low contents of carbohydrates, 0.8 to 4.2%, whereas the isoenzymes E1 and E2 contained relatively carbohydrates, 12.8 and 14.1%, respectively and lower p.I. values, 10.6 [Shigeo Aibara, 1981].

3.5.4 HRP5 isoelectric point 4.0

Horseradish Peroxidase, HRP-C E.C. number 1.11.1.7, CAS number 9003-99-0 was acquired from Biozyme Laboratories (product code HRP5). These isoenzymes can be distinguished by their isoelectric point 4.0, A403nm/A275nm (RZ~3,5), activity of the order of 80U/mg material (72-88U/mg material). In this work was used to confirm the pH dependence adsorption behaviour in OEWOD surfaces.

3.5.5 HRP4B isoelectric point 8.5

Horseradish Peroxidase, HRP-C E.C. number 1.11.1.7, CAS number 9003-99-0 was acquired from Biozyme Laboratories (product code HRP-4B). These isoenzymes can be distinguished by their isoelectric point 8.5, A403nm/A275nm (RZ 3,2), activity not less than 250U/mg material. This preparation has been confirmed to be isoenzyme C using isoelectric focussing (single band with p.I. 8.5) and the enzyme concentrations were determined spectrophotometrically [Hiner, 2002]. In this thesis was used to confirm the pH dependence adsorption behaviour in OEWOD surfaces.

3.5.6 DNA- Horseradish Peroxidase

DNA-Horseradish Peroxidase was from GE Healthcare Amersham ECL™ Direct Nucleic Acid Labelling and Detection Systems RPN3000. The ECL™ direct nucleic acid labelling and detection system from GE Healthcare is based on enhanced chemiluminescence reaction. The principle technique involves directly labelling probe DNA or RNA with the enzyme horseradish peroxidase. This is achieved by completely denaturing the probe so that it is in single-stranded form. Peroxidase, which has been complexed with a positively charged polymer, is added and it forms a loose attachment to the nucleic acid by charge attraction. These ionic interactions

will be interrupted by the presence of counter ions so it is important that the probe is in a low salt solution. Addition of glutarldehyde causes the formation of chemical cross-links so that the probe is covalently labelled with enzyme (see figure 3.8).

The control sample in ECL™ Direct Nucleic Acid Labelling and Detection Systems RPN3000, HindIII-Verdau of lambda-DNA, the size fragments are listed in table 3.5.

Fragment number Number of Base pair 1 23,130 (A) 2 9,416 3 6,557 4 4,361 (A) 5 2,322 6 2,027 7 564 8 125 (B) (A) cos ends are located on the bands, (B) very faint and often not visible. Table 3.5: DNA string size (ECL™)

Figure 3.8: Principles of the ECL direct nucleic acid labelling and detection system (Layout in CorelDraw) [GE Healthcare Amersham ECL™]

3.5.7 Peroxidase-Conjugated Rabbit Anti-Mouse Immunoglobulin (IgG-HRP)

Peroxidase-Conjugated Rabbit Anti-Mouse Immunoglobulin (IgG-HRP) was acquired from DakoCytomation Denmark A/S. It is the purified immunoglobulin fraction of rabbit antiserum conjugated with horseradish peroxidase of very high specific enzymatic activity. The coupling reaction is a modification of the two step glutaraldehyde method of Avrameas and Ternynck [Avrameas, 1971]. The reaction is gentle, efficient, and highly reproducible and gives conjugates molecules of predominantly 200 to 240 kDa (DakoCytomation Denmark A/S).

3.6 HPV-NASBA

The PreTec HPV Proofer Kit was supplied from NorChip AS, Norway. The NASBA reagents, primer set, molecular beacon probes, including the positive control sample artificial Oligonucleotide of human papilloma virus 16 (HPV16) were previously and strictly mixed and incubated as is recommended in the kit protocol and advices from colleagues of NorChip AS, (see table 3.6).

PreTec HPV-Proofer is real time nucleic acid amplification based qualitative assay to be used with a real time fluorescence reader for detection of E6/E7 mRNA, via molecular beacon from five different high-risk types in the human papilloma virus family in cervical specimens.

The isothermal amplification method for nucleic acids is a technology with the potential for broad applications in the field of RNA amplification system such as the PCR technique, the ability of NASBA to homogeneously and isothermally amplify RNA analytes (e.g., viral genomic RNA, mRNA or rRNA) extends it application range from viral diagnostics to the indication of biological activities such as gene expression and cell viability [Leone, 1998].

Nucleic acid sequence-based amplification (NASBA) is an isothermal method of RNA amplification that is carried out at a fixed temperature of 41°C, RNA is amplified by use of three enzymes, consisting of T7 RNA polymerase, AMV reverse transcriptase, and RnaseH and two target specific oligonucleotide primers. Primer P1, which contains T7 RNA polymerase promoter, hybridizes to the target RNA and

a cDNA is made by the AMV reverse transcriptase. This results in the production of a DNA/RNA hybrid. RnaseH digests the RNA, leaving a single strand of antisense DNA. Primer, P2 then binds to the DNA and is elongated, yielding a double stranded DNA molecule with an intact T7 RNA polymerase promoter sequence. From this double stranded product, many copies of single stranded antisense RNA are transcribed. This antisense RNA serves as the template for a new cycle of amplification, though the primers bind in reverse order [Compton, 1991; Kievits T. et al., 1991; Norchip AS], see diagram close to table 3.6.

NASBA reagents Content

Mastermix solution Lyophilized sphere containing nucleotides, dithiothreitol and MgCl2, TRIS/HCL, 45% DMSO; KCl and NASBA water.

Enzyme solution Lyophilized sphere containing AMV-RT, Rnase H, T7 RNA polymerase and BSA, Sorbitol in aqueous solution.

In vitro produced positive control NASBA reaction Oligonucleotide HPV16 in 5 mol/l Mixture guanidine thiocyanate, Synthetic Performance Control primers in water, master solution and enzyme solution and Molecular beacon

Table 3.6: NASBA reagents: PreTec HPV Proofer Kit and diagram of the real time amplification [NorChip AS]

3.6.1 Molecular beacon

The Molecular Beacon (MB) samples as mentioned before are included in the PreTec HPV-Proofer supplied from NorChip AS, Norway, and the so called real time fluorescence detection is possible. Molecular Beacon (MB) technology consists of a single stranded oligonucleotide stem with a specific size (bp), which is labelled with a dye and quencher at its 5' and 3' ends. In this thesis the MB samples are with 6- carboxy fluorescein (6-FAM) at its 5' end and quencher DABCYL at its 3' end. The oligonucleotide has a stem-loop structure. The loop sequence structure is complementary to the target nucleic acid expected to lead to a amplification. If the oligonucleotide loop structure is near to the target, it will be coupled and the quencher will separate from the dye and the sample which is frequently exited by 460 nm wavelength (6-FAM) will then fluoresce. In the opposite case, the oligonucleotide loop structure remains closed and does not fluoresce [Tyagi, 1996; Marras, 2003], see figure 3.9. The fluorescent signal emitted is measured at 530 nm

wavelength by the Y515Di filter in the LAS-3000 and the time at which it reaches a threshold of detection is measured every 10 or 15 min. The time it takes a sample to reach the threshold is a function of how much initial target mRNA/artificial HPV16 Oligonucleotide are in the sample. Nowadays there are several softwares: Beacon Builder from Premier Biosoft Int. (www.premierbiosoft.com) for the MB design or Biotechnology companies (Biomol) which supplied the required oligonucleotide (MB) with the adequate fluorophore.

Figure 3.9: Sketch of the Molecular beacon principle (picture taken from Marras et al.) [Marras, 2003]

3.7 Equipment

As has already been mentioned at the beginning of this chapter, there are mainly five different set ups, each of them involves specific material as well as equipment. Here, not all equipment will be mentioned in detail, only the most important. For example, all the dielectrics thin films were deposited by spin coating process: spin processor model WS-400 from Laurell, technologies. The thickness layers were measured with Tencor P11 profilometer and all the electrodes previously structured were achieved by EVD-process; 10 nm titanium and 25 nm platinum. The characterization of the layer experiments will be mentioned in chapter 4. Here mainly the equipment involved in adsorption experiments and RT-NASBA experiment will mentioned. 1) Contact angle device, Krüss G10 [Krüss] and the Drop shape analysis software DSA version 1.0, Krüss. 2), low and voltage power supplier, Droplet Handling system, and electronic circuitry for actuation [Herberth, 2006], the temperature control SC_interface TC-XX-PR-59, via USB virtual port and the program which is handling the communication with the Supercool Regulator Board from Supercool AB, Box 27, S-40120 Göteborg, Sweden. The setup of the experiments: Adsorption OEWOD surfaces design as well RT-NASBA OEWOD microchip design will mentioned in this chapter, as well the luminescence imaging system device from Fuji film (Luminescence analyser system LAS-3000). For details about the device, see references [Fujifilm]. The relative quantitative analysis of the adsorption experiment 54

in the OEWOD surfaces and the RT-NASBA on OEWOD Microchip were realized with the software: Advanced image data analyser Aida (raytest Isotopmessgeräte GmbH, Germany).

3.7.1 Adsorption experiment set up

Figure 3.10: Schematic setup of the static biomolecular adsorption experiments (layout in Corel draw)

The biomolecular adsorption experiment consists of a voltage power supplier and OEWOD surfaces previously structured (figure 3.10, 3.11, and 3.12).

Figure 3.11: Close up of set up of the statics experiments on Figure 3.12: Set up of statics experiments on OEWOD OEWOD surfaces surfaces and voltage power supplier

3.7.2 Contact angle device, Krüss G10 and the Drop shape analysis DSA 10

The contact angle measurement system and the drop shape analysis system DSA 10 from Krüss GmbH, Germany [Krüss] will be shown in figure 3.13. The DSA10 comprises the contact angle measuring system G10 which integrates a video system and the DSA software, developed by Young-Laplace. It is a powerful and user- friendly tool to determine the surface tension of liquids and solids, as well as, dynamic, static, advancing and receding contact angles. The DSA software, which runs on the Microsoft Windows operating system, offers 4 calculation algorithms, see annex A. Each method is the optimum solution depending on the absolute accuracy required, by measuring speed and/or the drop size. The method mainly used in this work is the dynamic sessile drop method. Drop images can be stored, printed, and newly treated whenever necessary. The user decides if the drop contour should be analyzed on-line or after the measurement is finished. Fully automatic calculation of the enlargement factor of the light intensive zoom optic and the focusing assistant are standard. The latter ensures the best image sharpness. Graphical display of measured values and results are on-line.

Figure 3.13: Dynamic Contact Angle System (DCAS) software [Herberth, 2006] and the DSA10 contact angle measuring system [Krüss]

The self-made Electronic circuitry for voltage control, the software Dynamic Contact Angle System (DCAS) and the Droplet Handling System (DHS) were developed by Herberth 2006 [Herberth, 2006] and they were used to determine the availability of NASBA compound in OEWOD platform. It allowed arbitrary excitation voltages and management of the contact angle device. The experimental data obtained enabled the evaluation of additional fitting and algorithms through the DSA software [Krüss].

The minimum reproducible time step is 25 ms. Voltage was controlled with a ±5V, 12bit digital-analog converter (DAC) from bmc Messtechnik GmbH.

The DCAS software enables running of a specific voltage sequence or ramp, simultaneously measuring the contact angle, as well as showing graphically the plot of contact angel versus voltage or time, which will be show in the chapter of results. The software provides the possibility to show real-time plots of contact angle versus time and contact angle versus voltage. All the above recorded data can be written to an ASCII data file for further evaluation.

3.7.3 Droplet Handling System (DHS) and Electronic Circuitry

The Droplet Handling System (DHS) was written in Visual basic 6 and operated the electronic circuitry, defining an electrode switching sequence or protocol for the basic assay operation in the OEWOD microchip. The electronic circuitry consists of hardware, voltage module and switch matrix. The mem-PIO digital I/O card from bmc Messtechnik GmbH is connected via USB to the unit processor and provides 24 digital I/O ports. The high generator of the voltage module supplied a voltage ranging from 0 to 600 Vdc, 1 mA and the output 200 Vdc, and implemented with AC module. Additionally, a video camera connected via USB to the unit processor gives a view of the actuation protocol; see details [Herberth, 2006]. Figure 3.14 shows the hardware for OEWOD experiments and DHS.

Figure 3.14: Electronic circuitry hardware and operation window of the actuation software (DSH). Picture taken from [Herberth, 2006]

3.7.4 OEWOD surfaces for adsorption experiment

The optimized OEWOD surfaces for adsorption experiments were structured previously by Electrochemical Vapor Deposition (EVD) technology, electrodes pathway on the array, 9 columns and 7 rows of electrodes with a total of 62 location for adsorption experiments which are contacted to each other, each of them are 7 mm width X 8 mm height, 3 electrodes for contact the actuation current, 15 mm width X 7 mm height, see Figure 3.15.

Figure 3.15: OEWOD surfaces for biomolecular adsorption experiments and array layout

For instance, two composite surfaces were compared, one them consisted of the following system: Durimid 115A (Fujifilm Electronic Material), 50% w/w 1-Methyl 2- Pyrolidone (from Arch Chemicals, Belgium) which was deposited by spin-coating (Spin processor model WS-400 from Laurell) on a Pyrex wafer (0.5 mm) at 6000 rpm during 30 sec. and 3000 rpm, 1 min. followed by curing at 96°C, 30 min., Durimid thickness reached ~3.8 µm. After the Durimid layer was cooled, a second layer of Teflon® AF 1600, 2% wt/wt (from DuPont, Wilmington, Delaware E.U.) dissolved in FC-75 (from 3M, Belgium) was deposited by spin coating at 2000 rpm during 30 sec. and 1000 rpm, 30 sec. followed by curing at 96°C, 4 hrs. Teflon thickness reached ~100-150 nm (TENCOR P11, profilometer). As actuation electrode (-) between the Pyrex wafer and the hybrid layer (Durimid-Teflon®), was previously deposited by EVD-process a structured pathway of 10 nm titanium and 25 nm platinum, see set up for electrowetting experiments, figure 3.10. A 0.3 mm platinum wire was used as electrode (+) inside the droplet during the voltage was applied.

The second composite surface consists of Cellulose Acetate, 3,5% w/w Acetone from Acros Organics, Geel, Belgium, which was deposited by spin coating at 2000 rpm during 30 sec. and 1000 rpm, 30 sec. followed by curing at 96°C for 30min.

Cellulose Acetate thickness reached ~ 400-500 nm (TENCOR P11), after the Cellulose Acetate layer was cooled, a second layer of Teflon® AF 1600, 2% wt/wt (from DuPont, Wilmington, Delaware E.U.) dissolved in FC-75 (from 3M, Belgium) was deposited by spin coating at 2000 rpm during 30 sec. and 1000 rpm, 30 sec. followed by curing at 96°C, 30 min. Teflon thickness reached ~100-150 nm (TENCOR P11, profilometer).

3.7.5 OEWOD microchip for Real Time NASBA of HPV16

The OEWOD microchip for Real Time NASBA of HPV16 detection has the following dimensions: 29.26 mm wide, 39.88 mm length, see figure 3.16.

The electrodes on the surface of the OEWOD microchip were previously deposited by EVD technology as mentioned before in the OEWOD surface for adsorption experiment, 10 nm titanium and 25 nm platinum, the electrode shapes and dimensions were simulated and optimized according to Lieneman et al [Lienemann, 2003] and [Herberth, 2006]. The minimum electrode size (Square shaped electrodes) for the optimum ―actuate force‖ required is: 1 mm and pitch 1.5 mm.

The microchip surfaces consists of Durimid 115A (from Fujifilm Electronic Material), 50% w/w 1-Methyl 2-Pyrolidone (from Arch Chemicals, Belgium) which was deposited by spin-coating (Spin processor model WS-400 from Laurell) on a pyrex wafer at 6000 rpm during 30 sec. and 3000 rpm, 1 min. followed by curing at 96°C, 30 min. Durimid thickness reached ~3.8 µm, after the Durimid layer was cooled, a second layer of Teflon® AF 1600, 2% wt/wt (from DuPont, Wilmington, Delaware E.U.) dissolved in FC-75 (from 3M, Belgium) was deposited by spin coating at 2000 rpm during 30 sec. and 1000 rpm, 30 sec. followed by curing at 96°C, 4 hrs. Teflon thickness reached ~400-500 nm (TENCOR P11, profilometer).

Figure 3.16: OEWOD microchip consisting of interdigitate electrodes with optimised shape for actuation.

Additionally accessories to the OEWOD microchip were designed to avoid evaporation and to keep it in a sterile environment inside of the luminescence system LAS-3000. The material selected for the adapter contact to the DHS and for the chamber reaction are Plexiglas and glass as cover. These materials do not emit fluorescence in the same wavelength range as the molecular beacon from the NASBA assay. The heat element and temperature sensor were cover with aluminium foil in order to avoid fluorescence background. The OEWOD microchip is located in a Petri disc and so as possible closed, see figures 3.17a, b, c, d.

b) a)

c) d) Figures 3.17: a) View of the OEWOD microchip, b) OEWOD microchip inside the Petri disc, heater and temperature sensor cover with aluminium foil to avoid fluorescence background, c) reaction window of Plexiglas and glass cover of the OEWOD microchip and d) close up view of the experimental set up

The chamber consists of the OEWOD chip which was covered by a 24.96 mm x 25.96 mm Plexiglas frame with 5.7 mm thickness and on the top of the Plexiglas a Pyrex glass cover plate with 5 mm thickness (see figure 3.18) was used. The reaction chamber and the glass cover are fixed to the OEWOD chip with a film of silicon oil. All materials and solutions used were previously tested for fluorescence background-noise in the same wavelength range as the molecular beacon of the NASBA assay.

T-Sensor Heating element

Frame Cover plate

Figure 3.18: OEWOD microchip implemented into the microfluidics system consisting of a heating element, temperature sensor, reaction chamber and cover plate for qualitative analysis of RT-NASBA.

3.7.6 Heater and temperature control on OEWOD microchip

SC_Interface (Sc_interface TC-XX-PR-59) is a program which handles the communication with the Supercool Regulator Board from Supercool AB, Box 27, S- 40120 Göteborg, Sweden, via serial port or USB virtual port. All data in the regulator can be saved to a file, and retrieved later for fast test of different settings. Runtime view helps the user to adjust the parameters for best result and it is also possible to save to runtime log file, for later analysis in other software. Drop a file in the view will open the file. The temperature selected in this case for all the NASBA experiments is 41°C. The Heater element sits below the OEWOD microchip and a temperature sensor is placed on the OEWOD surface, both are covered with aluminium foil to avoid fluorescence background and connected to the Supercool Regulator Board, see figure 3.19.

Figure 3.19: Temperature device control and software of temperature device control

3.7.7 Luminescent image analyzer (LAS-3000)

The Luminescent image analyzer (LAS-3000) system from Fujifilm [Fujifilm], combines a new CCD camera technology chip with 3.2M pixels and pixel size of 10.75 µm x 10.75 µm with a user interface software Image reader LAS-3000 via USB1.1 to provide significantly improved system sensitivity with a wide range of luminescence applications. The CCD imaging chip with a highly sensitive camera lens unit F0.85/43mm allows the capture of faint-light images of the OEWOD microchip surfaces, previously focusing adjusted and located inside in the intelligent

dark box with high sensitivity and resolution. See figure 3.20 and for more technical details [Fujifilm].

The LAS-3000 imaging system is supported with incident light source technology, blue excitation light source of 460 nm, filter changer for a specific wavelength (Y515Di) and EPI sample tray for Chemiluminescence mode or incident light source mode. The exposure mode enables several exposure types, a precision type which sets the time and creates an image, which was used for the adsorption experiments, an increment type which sets the exposure in determined intervals and creates multiple accumulated images, used for the Real Time NASBA experiment.

Figure 3.20: Luminescent Image Analyzer LAS-3000 and Image reader software

3.7.8 Advanced Image Data Analyzer (AIDA), version 4.06

The relative quantification of the biomolecules adsorption on OEWOD surfaces through chemiluminescence and the RT-NASBA amplification through the molecular beacon technology images were evaluated with the software Advanced Image Data Analyzer (AIDA) from raytest Isotopmessgeräte GmbH, Germany. AIDA enables the image performance in the LAS-3000 and saves in a TXT-file for later evaluation, based on the scale-grey evaluation. The software presents two types of evaluation, a one-dimensional (1D) or 1D densitometry and a two-dimensional (2D) or 2D densitometry.

4. Results and Discussion

The results of the surfaces characterization, the dependence of parameters as pH of the solution, dissolvent used, surface polarity, potential applied, concentration biomolecular, adsorption time and the comparison of those, in the biomolecular adsorption process of mainly two OEWOD surfaces Durimid 115A–Teflon®AF and Cellulose Acetate–Teflon®AF, will be described and discussed in this chapter. In addition a few previous experiments will be described as reference and for the set up and characterization of the biomolecular experiments protocol. The results of the LSCM images of the B-Phycoerythrin adsorbed on SU-8-Teflon®AF, the AFM analysis and the contact angle characterization, which showed a larger degree hysteresis and the irreproducibility of the EWOD effect presented in the work by Herberth [Herberth, 2006] and in previously experiments, has led to it being ruled out for SU-8-Teflon®AF surface and additional experiments. However, some of the results are shown partially and mentioned in the chapters 4.1.2, 4.2, figure 4.8, and table 4.2.

This thesis presents mainly two examples of the OEWOD microchip as a platform for bioassays applications: OEWOD microchip as a tool for the research of enzymatic reaction inactivation or kinetics and the compatibility, characterization of the NASBA components and the performance of a Real Time Nucleic Acid Sequence Based Amplification bioassay protocol on OEWOD microfluidics based droplet actuation platform will be described and presented in this chapter. The RT-NASBA application in OEWOD microchip is introduced as an example for the potentially development of the OEWOD platform for microfluidics bio-applications.

4.1 OEWOD surfaces characterization

The technique used for OEWOD surfaces characterization has provided information about the homogeneity, roughness, and some properties of the OEWOD surfaces. The measurement of dynamic contact angle profile analysis on the surfaces was used to characterize the homogeneities along all adsorbed location on the OEWOD surfaces. Dynamic and static contact angle measurements were used for the evaluation of biomolecular adsorption by EWOD means. Atomic force microscopy measurements were performed to confirm and evaluate the influence of roughness 64

on the biomolecular adsorption process. Laser Scanning Confocal Microscopy (ZEISS LSCM 510 UV) measurements were performed to evaluate the annealing process of the thin polymer layer involved when biomolecular adsorption takes place and is used for physical characterization changes of polymer coatings [Sung, 2003]. Profilometer measurements were performed to estimate the thickness of the layers, previously deposited on the OEWOD surfaces. Chemiluminescence and fluorescence images from the LAS-3000 were performed to identify and relative quantified the location exposed to biomolecular adsorption and the amplification of RT-NASBA products respectively. Aida Software was used to evaluate and relative quantified the LAS-3000 images.

The OEWOD surfaces for biomolecular adsorption experiment were described in chapter 3.7.4. Advancing and receding contact angles of a reference sample were obtained by means of the sessile drop method (for details see annex A), using a DSA Krüss G10 contact angle device implemented with a software and hardware self designed by the Sensors-IMTEK Department that made fully automated measurements of many points on a surface previously defined, see annex B, figures B1, B2, B3 and B4. During the measurement of the advancing contact angles the needle remained inside the drop. The droplet was monitored by a CCD-camera and analyzed by Drop Shape Analysis software (DSA Version 1.0, Krüss). The complete profile of the sessile droplet was fitted by the tangent method to a general conic section equation. The derivative of this equation at the baseline gives the slope at the three-phase contact point and thus the contact angle. In this way contact angles were determined on both OEWOD surfaces. The mean values are presented as follows: advancing and receding contact angle with D.I. water, 124.7° ± 0.34 and 112.12°± 4.24 for Durimid 115A-Teflon®AF 2% OEWOD surface and 124.6° ± 0.71 and 108.97°± 6.91 for Cellulose Acetate-Teflon®AF 2%, (see figures B1, B2, B3, B4 in annex B and table 4.1). Static contact angle as before mentioned were performed with HRP dissolved in PBS 0.1 M and adjusted to pH, 5, 7.2 and 10, see table 4.1.

The Atomic Force Microscopy (AFM) experiments in Durimid 115A-Teflon®AF 2% and Cellulose Acetate-Teflon®AF 2% shows non-significant surface roughness which could influence the adsorption process, see table 4.1.

4.1.1 Atomic Force Microscopy analysis of OEWOD surfaces

Surface roughness influenced contact angles, as well as biomolecular adsorption processes. Atomic force microscopy measurements were performed on Durimid 115A – Teflon®AF 2% and Cellulose Acetate–Teflon®AF 2% OEWOD surfaces. AFM measurements were performed to identify and determine the influence of the roughness of each component on the OEWOD surfaces. The subtract Pyrex, electrode structures (titanium, platinum), and polymers layer were independently analysed. The results showed that the roughness does not contribute significantly to the biomolecular adsorption process (see figures B5, B6, B7, B8, B9, B10 and B11 in annex B).

OEWOD surfaces Characterization

Open EWOD AFM analysis B Dynamics Contact angle Hysteresis Static Contact A c surfaces Rrms(nm) θ advancing θ receding Δθ angle θ( HRP)

Durimid 115A- 7 124.7± 0.7 112.12 ±4.2 12.5 HRP1 111.6 ± 7 Teflon®AF 2% 1600 HRP2 113.3 ±5.9 HRP3 113 ± 5.3

Cellulose Acetate- 9 124.6± 0.7 108.9 ±6.9 15.6 HRP1 117.3 Teflon®AF 2% 1600 HRP2 105 HRP3 114.7

ARms (Rq) B,D.I. (Deionized water) CHRP: Horseadish peroxidase 0.1M dissolved in PBS 0.1M. HRP1 adjusted to pH=5, HRP2 adjusted to pH=7.2, HRP3 adjusted to pH=10.

Table 4.1: OEWOD surfaces Characterization

4.1.2 Laser Scanning Confocal Microscopy (ZEISS LSCM 510 UV) of OEWOD surfaces annealed and un-annealed

Adsorption experiments were performed on three distinct annealed composite or hybrid layer surfaces; Durimid 115A-Teflon®AF, SU-8–Teflon®AF, and Cellulose Acetate–Teflon®AF to analyze the possible influence of the annealing process of the polymer layer in the biomolecular adsorption on OEWOD surfaces. The issue of Laser Scanning Confocal Microscopy (LSCM) analysis on OEWOD surfaces is of high importance for physical quantitative characterization changes of polymer coatings [Sung, 2003] and since biomolecules as proteins during the adsorption modify or change the surfaces depending on their physical and chemical properties.

The previous treatment of the polymers (annealing process) on the OEWOD surfaces could be a significant parameter for the biomolecular adsorption and EWOD effect. The modification of the OEWOD surface during the biomolecular adsorption was evaluated by this technique.

The two above mentioned layers were annealed under the following conditions: Durimid 115A, 170°C for 10 min., SU-8; 90°C, 20 min. and Cellulose Acetate; 90°C, 15 min., after the layers were cooled to room temperature the second layer (Teflon®AF) was spin-coated and annealed at 90°C for 30 min., similar composites were prepared without annealing process.

A 10 µl droplet of B-Phycoerythrin was dispensed on different locations of the surfaces. Surface morphology changes were analyzed after one and five min. of adsorption time. The changes on the surface morphology, as spots, holes formation, surface roughness and layers separation were identified, see figures B12, B13, B14, B15, B16 and B17 in annex B). Initially, the surface appeared to be smooth and featureless. The surface roughness increased along with the adsorption time. The appearance of spots, holes and roughness was evident through the fluorescence images. The size of the spots enhanced further and merged with its neighbour. The un-annealed sample of Durimid 115A-Teflon®AF OEWOD surface appeared to be very rough with apparent physical change. Similar is the case with un-annealed SU-8 –Teflon®AF after 5 min of adsorption time.

4.1.3 Reversibility and behaviour of electrolytes in comparison to HRP under electrowetting effect (OEWOD conditions)

An experiment was performed to verify the electrowetting behaviour of different electrolytes, HEPES, PBS, and KCl 10-5 M, in comparison to HRP. The following surface composition was studied: Teflon®AF 1600 (6% wt) in FC-75 was deposited by spin coating on a glass wafer (0.3 mm) at 3000 rpm during 1 min and 1500 rpm, 4 min, followed by annealing of the surfaces in air at 100°C, 10 min, Teflon®AF thickness ~ 3µm. Contact angles of 10µl droplets of HEPES, PBS, KCl, HRP (Isoenzyme C) 10-5 M were analysed. The droplet was deposited manually in the surface and a potential was applied between the conductive electrodes below the Teflon layer and the droplet. A 0.3 mm pt-wire was used as (+) electrode inside the

droplet and the electrodes below the layer were negatively (-) charged. Contact angles were measured as a function of the potential applied between 0 volts and 200 volts with intervals of 20 volts. Reversible contact angle was observed in all electrolytes as well in HRP, see below figure 4.1.

120 10-5 M HEPES 10-5 M PBS 110 10-5 M KCl 10-5 M HRP 100

ContactAngle (degrees) 70

60 20 40 60 80 100 120 140 160 180 200 Potential (volts)

Figure 4.1: Contact angle changes for 10 µl droplets of HEPES, PBS, KCl and HRP 10-5 M as a function vs. potential applied from 0 volts to 200 volts.

4.1.4 Reversibility and behaviour of biomolecules dissolved in water and in PBS solution under electrowetting effect (OEWOD conditions).

Experiments were performed to thesis the reversibility and behaviour of the EWOD effect in two biomolecular solutions (HRP and GOD) at the single surface Teflon®AF 6% and in hybrid surfaces: Durimid 115A-Teflon®AF 6%. Contact angle of 10 µl droplets of HRP and GOD dissolved in water (pH=6.3) and in PBS 0.1M (pH=7.4) with concentrations from 1 mg/ml to 10-5 mg/ml were measured as a function of the potential applied between 0 volts and 200 volts with intervals of 20 volts (see figures 4.2, 4.3, 4.4, 4.5, 4.6 and 4.7). Teflon®AF 1600 (6% wt/wt) was deposited by spin coating on a Pyrex wafer (0.5 mm) at 3000 rpm during 1 min. and 1500 rpm, 4 min. followed by annealing in air at 100°C, 5 min. Teflon®AF thickness ~3 µm. A counter electrode (-) between the Pyrex wafer and the Teflon®AF layer was previously deposited by EVD, a structured pathway of 10 nm titanium and 25 nm platinum (see chapter 3). 0.3 mm platinum was used as an electrode (+) inside the droplet while the potential was applied. The contact angles of 10 µl droplets of HRP and GOD at concentrations from 1 to 10-5 mg/ml dissolved in water were measured as a function

of the potential applied between 0 volts and 200 volts with intervals of 20 volts on both surfaces, see figures 4.2 and 4.3.

120 1 mg/ml HRP 10-1 mg/ml HRP 110 10-2 mg/ml HRP 10-3 mg/ml HRP 100 10-4 mg/ml HRP 10-5 mg/ml HRP

70 Contact angle (degrees) angle Contact

50 2 21.08 40.96 60.24 80.08 99.04 119.16 139.16 158.04 178.32 197.28 Potential (volts)

Figure 4.2: Contact angle (degrees) vs. potential applied (volts) for HRP at concentrations from 1 to 10-5 mg/ml dissolved in water on Teflon®AF 6% OEWOD surface.

120 1 mg/ml GOD 110 10-1 mg/ml GOD 10-2 mg/ml GOD 10-3 mg/ml GOD 100 10-4 mg/ml GOD 10-5 mg/ml GOD 90

Contact angle (degrees) angle Contact 60

40 1.84 21.28 41.04 60.12 80.2 98.84 118.96 139.04 158.44 178.4 197.04 Potential (volts)

Figure 4.3: Contact angle (degrees) vs. potential applied (volts) for GOD at concentrations from 1 to 10-5 mg/ml dissolved in water on Teflon®AF 6% OEWOD surface.

A hybrid surface consisting of Durimid 115A deposited by spin-coating at 3000 rpm during 1 min. and 1500 rpm, 4 min. followed by annealing in air at 250°C, 30 min in

the same substrate as in previous experiments, after the Durimid 115A layer was cooled, a second layer of Teflon®AF (6% wt/wt) was deposited by spin coating with the same procedure as before mentioned.

120

1 mg/ml HRP 110 10-1 mg/ml HRP 10-2 mg/m lHRP 10-3 mg/ml HRP 100 10-4 mg/ml HRP 10-5 mg/ml HRP

70 Contact(degrees) angle

50 1.84 21.32 40.96 60.04 79.92 99.32 119.2 139.5 158.3 178 197.4 217.3 Potential (volts)

Figure 4.4: Contact angle (degrees) vs. potential applied (volts) for HRP at concentrations from 1 to 10-5 mg/ml dissolved in water on Durimid 115A–Teflon®AF 6% OEWOD surface.

120 1 mg/ml GOD 10-1 mg/ml GOD 110 10-2 mg/ml GOD 10-3 mg/ml GOD 10-4 mg/ml GOD 100 10-5 mg/ml GOD

70 Contactangle (degrees) 60

50 1.72 21.12 41.04 60.04 80.08 99.16 119.44 139.04 158 177.88 197.32 217.24 Potential (volts)

Figure 4.5: Contact angle (degrees) vs. potential applied (volts) for GOD at concentrations from 1 to 10-5 mg/ml dissolved in water on OEWOD surface Durimid 115A–Teflon®AF 6%.

The next experiment was designed to identify the important role played by the electrolyte solution, the medium in which the biomolecules are dissolved. The contact angles of 10 µl droplets of HRP and GOD at concentrations from 1 mg/ml to 10-5 mg/ml dissolved in PBS were measured as a function of the potential applied

between 0 and 200 volts with intervals of 20 volts in both surfaces, see figures 4.6 and 4.7. The same experiments were performed as before mentioned, for 10 µl droplets of HRP and GOD at concentrations from 1 mg/ml to 10-5 mg/ml dissolved in water on hybrid surfaces, see figures 4.4 and 4.5.

120 1 mg/ml HRP 10-1 mg/ml HRP 110 10-2 mg/ml HRP 10-3 mg/m HRP 10-4 mg/ml HRP 100 10-5 mg/ml HRP

70 Contact angle (degrees) angle Contact 60

50 1.72 21.16 40.84 60.52 80.12 99.04 119.04 139.44 158.28 178.48 197.08 217.12 Potential (volts)

Figure 4.6: Contact angle (degrees) vs. potential applied (volts) for HRP at concentrations from 1 to 10-5 mg/ml dissolved in PBS 0.1 M on Durimid 115A–Teflon®AF 6% OEWOD surface.

1 mg/ml GOD 120 10-1 mg/ml GOD 110 10-2 mg/ml GOD 10-3 mg/ml GOD 100 10-4 mg/ml GOD 10-5 mg/ml GOD 90

Contactangle (degrees) 50

30 2 21.08 41.04 60.12 80.24 98.8 119.04 139.08 158.44 178.12 197.68 217.36 Potential (volts)

Figure 4.7: Contact angle (degrees) vs. potential applied (volts) for GOD at concentrations from 1 to 10-5 mg/ml dissolved in PBS 0.1 M on Durimid 115A – Teflon®AF 6% OEWOD surface.

4.1.5 Discussion and conclusions

The experimentally results confirm the feasibility of the OEWOD system and demonstrate the EWOD effect in biomolecules and electrolytes solutions. Reversible (EWOD effect) and reproducibility contact angle were observed in every electrolyte 71

concentration used, as well in HRP and GOD solutions. Which means automatically the possibility to perform the actuation protocol that simulated the potential sequence applied that is used to transport droplets by EWOD effect on OEWOD surfaces.

The advancing and receding contact angle with D.I. water, 124.7° ± 0.3 and 112.1°± 4.2 for Durimid 115A-Teflon®AF 2% OEWOD surface and for Cellulose Acetate- Teflon®AF 2%, 124.6° ± 0.7 and 108.9°± 6.9 showed a similar advancing contact angle with a deviation standard of less than 1%. The receding contact angle showed a significant deviation standard of approximately 4 and 7% respectively (see table 4.1). Cellulose Acetate-Teflon®AF 2% presented the larger hysteresis around 15° against 12° for Durimid 115A-Teflon®AF 2%, that showed the high roughness presented for Cellulose Acetate surfaces and confirmed for the AFM analysis parameter Rrms (9.3 nm), as well as the measured hysteresis of 15.6°. Additional, the static contact angle with HRP in PBS 0.1 M adjusted to pH 5, 7.2 and 10 was larger for the same surface; see table 4.1 and figures in annex A.

The laser scanning microscopy images of the previous annealed OEWOD surfaces do not show significant physical surface change during the adsorption time. Annealed OEWOD surface presented less biomolecular adsorption than its counterpart. The un-annealed surfaces showed more adsorption of B-Phycoerythrin, which increased proportional to the adsorption time. However, higher B- Phycoerythrin adsorption has been observed on Durimid 115A-Teflon®AF and SU- 8–Teflon®AF than on Cellulose Acetate–Teflon®AF surface. Physical surface change of Teflon®AF is slightly lower when it was spin-coated with SU-8. Cellulose Acetate–Teflon®AF annealed surface presented much less B-Phycoerythrin adsorption.

The changes in OEWOD surfaces morphology, identified as spots, holes formation, surfaces modification, and surface roughness increased proportional to adsorption time (see figures annex B12-17). The un-annealed Durimid 115A-Teflon®AF and unannealed SU-8–Teflon®AF OEWOD surface appeared to be very rough with apparent physical change after 5 min of biomolecular adsorption time, contrary to the Cellulose Acetate–Teflon®AF.

The un-homogeneities of the OEWOD surfaces due to the technology used to deposit the layers (spin-coating) are a factor to be considered. Nevertheless, no significant evident of surfaces roughness which could definitively influence the adsorption process was confirmed through the AFM analysis experiments; see table 4.1 and figures in annex A.

SU-8-Teflon®AF surface was finally ruled out for the AFM analysis measurements and contact angle characterization after the larger degree hysteresis and irreproducibility EWOD effect in certain experiment showed in the studies of Martί nez -Garza and Herberth [Martί nez, 2004; Herberth, 2006], and in this thesis. Additionally the LSCM images of the B-Phycoerythrin adsorption measurement on SU-8-Teflon®AF 2% showed higher biomolecular adsorption during EWOD conditions, which lead to them definitively to being ruled out for the future experiments in biomolecular adsorption. However, some results of SU-8-Teflon®AF 2% results will be shown in the next chapter: electrode polarity and pH dependence, due to the reliable results and as reference to the other surfaces.

The performed experiments compared the biomolecules dissolved in water and PBS 0.1M solution on single Teflon®AF 2% surface, Cellulose Acetate-Teflon®AF 2% and Durimid 115A-Teflon®AF 2% OEWOD surfaces. This gives an overview and confirmed the influences of the physical forces involved in the biomolecular solution and their interaction during the EWOD effect and emphasized the important role that play the OEWOD surfaces properties and the solvent medium of the biomolecules.

The single Teflon®AF surfaces showed for GOD and HRP reversibility in the contact angle (Electrowetting effect). Nevertheless, a threshold of 100 volts for every concentration biomolecular solutions was observed. Additionally, another pattern not expected following the theoretical behaviour (see Lippmann-Young´s equation, chapter 2, equation 3), was identified. The hybrid surfaces; Durimid 115A-Teflon®AF and Cellulose Acetate–Teflon®AF shifted lightly the initial contact angle for the same concentrations of HRP and GOD, this behaviour was more similar to the one expected. The contact angle was reduced around 45 degrees for some concentrations on this hybrid surfaces.

The hybrid surfaces, Durimid 115A-Teflon®AF and Cellulose Acetate–Teflon®AF presented the EWOD effect with their respective hysteresis as observed for the single surfaces Teflon®AF. The hybrid surfaces have shown more stability and reproducibility results. The single surfaces has shown an additional effect: the droplets vibrate and jump on the surface at a certain moment during the EWOD effect (for this effect see Herberth 2006 and Yoon et al., 2003) when a specific voltage was applied. In general more hysteresis and electrolysis were observed with single layer surfaces than in hybrid layer surfaces. GOD and HRP showed more electrolysis when they were dissolved in PBS than in water during the potential applied from 0 to 200 volts. GOD and HRP solutions decrease more in the net contact angle when they were dissolved in water than in PBS. However the biggest change in contact angle with around 60 degrees was measured with a concentration of 10-3 mg/ml of GOD dissolved in water after a change of 55 degrees by GOD concentration of 1 mg/ml dissolved in PBS. GOD and HRP solutions dissolved in PBS showed a threshold at 100 volts. After this potential was applied, no more significant change on the contact angle was observed; this phenomenon theoretically can be called saturation and it was thorough investigated and mentioned by several authors [Wang, 2005; Verheijen, 1999; Herberth, 2008]. Some other concentration of HRP and GOD showed a partial theoretical behaviour (see Lippmann-Young equation, chapter 2, and equation 3).

Experiments with different concentrations of GOD and HRP dissolved in water and PBS did not show a linear relation between the initial contact angle and the concentration. However the influence of the electrostatics interactions due to the ionic strength of the PBS in the OEWOD surface was confirmed.

4.2 Dependence on the electrode polarity and pH in the biomolecular adsorption process on OEWOD surfaces.

Experiments were performed on hybrid surfaces as previous described in order to analyze and identify the dependence on these parameters in the biomolecular adsorption process on OEWOD surfaces and their conditions. Several experiments were designed using HRP as adsorption biomolecules. The samples of HRP with isoelectric point (p.I.) equal to 7.2 were dissolved in three PBS solutions 0.1M; HRP1 with pH=7.4, HRP2 with pH ~ 5-6, HRP3 with pH ~ 9-10. The main idea was to manipulate the net charge of the biomolecular solution before they will be exposed to the OEWOD surface and observing how they behave while a potential is applied and whether the polarity of the electrode will change. The 10 μl sample of biomolecular solutions (HRP1, HRP2 and HRP3) were deposited in OEWOD surfaces and from 0 to 400 volts for Durimid 115A-Teflon®AF (6% wt/wt), SU-8-Teflon®AF (6% wt/wt) see figures 4.8 and 4.9 and from 0 to 180 volts for Cellulose Acetate–Teflon®AF (6% wt/wt) see figure 4.10. The polarity of the electrode was changed for every sample from positive (-) as counter electrode to negative (+). HRP1D- (pH=7.4 and counter electrode negative), HRP1D+ (pH=7.4 and counter electrode positive) and so on HRP2D- and HRP2D+, HRP3D- and HRP3D+, see curve notation in figures 4.8, 4.9 and 4.10. Additional contact angle were measured during the potential applied in every experiment, see also table 4.2.

HRP1D+ 120 HRP1D- HRP2D+ 110 HRP2D- HRP3D- 100 HRP3D+ KCl 10%D+ 90 KCl 10%D-

70 ContactAngle (degrees) 60

50 21 60 99 139 178 217 255 294 334 373 Voltage (volts)

Figure 4.8: Dependence on the electrode polarity and pH of HRP on SU-8-Teflon®AF 6%. HRP1D+: pH=7.4, counter electrode (+); HRP1D-: pH=7.4, counter electrode (-); HRP2D+: pH~5-6, counter electrode (+); HRP2D-: pH~5-6, counter electrode (-); HRP3D+: pH~9-10, counter electrode (+); HRP3D-: pH~9-10, counter electrode (-).

120 HRP1D+

110 HRP1D- HRP2D+ 100 HRP2D-

90 HRP3D+

HRP3D- 80

70 Contactangle (degrees)

50 0 50 100 150 200 250 300 350 400 450 Voltage (volts)

Figure 4.9: Dependence on the electrode polarity and pH of HRP on Durimid 115A-Teflon®AF 6%. HRP1D+: pH=7.4, counter electrode (+); HRP1D-: pH=7.4, counter electrode (-); HRP2D+: pH~5-6, counter electrode (+); HRP2D-: pH~5-6, counter electrode (-); HRP3D+: pH~9-10, counter electrode (+); HRP3D-: pH~9-10, counter electrode (-).

HRP1D+ 120 HRP1D-

110 HRP2D+ HRP2D- 100 HRP3D+ HRP3D- 90

60 Contactangle (degrees)

40 0 20 40 60 80 100 120 140 160 180 200 Voltage (volts)

Figure 4.10: Dependence on the electrode polarity and pH of HRP on Cellulose Acetate-Teflon®AF 6%. HRP1D+: pH=7.4, counter electrode (+); HRP1D-: pH=7.4, counter electrode (-); HRP2D+: pH~5-6, counter electrode (+); HRP2D-: pH~5-6, counter electrode (-); HRP3D+: pH~9-10, counter electrode (+); HRP3D-: pH~9-10, counter electrode (-).

4.2.1 Discussion and conclusions

The results summarized in the table 4.2 show that at the first 20 volts potential applied the initial contact angle has a strong dependence on the electrode polarities and HRP charge. The OEWOD surfaces used show also a significant difference, emphasizing the important role played by the surfaces system used.

Durimid115A- SU-8 – Cellulose Acetate Counter Biomolecule pH Teflon®AF Teflon®AF – Teflon®AF Electrode Adsorbed Initial contact Initial contact Initial contact HRP Angle (degrees) Angle (degrees) Angle (degrees) Polarity (Charge) 7.4 97 105 90 + p.I.(HRP1D+)

90 112 108 - p.I.(HRP1D-)

5-6 108 104 93 + Positive(HRP2D+)

103 112 105 - Positive(HRP2D-)

9-10 84 89 103 + Negative(HRP3D+)

91 102 107 - Negative(HRP3D-)

HRP1D+: pH=7.4 and counter electrode (+), HRP1D-: pH=7.4 and counter electrode (-), HRP2D+: pH~5-6 and counter electrode (+), HRP2D-: pH~5-6 and counter electrode (-), HRP3D+: pH~9-10 and counter electrode (+), HRP3D-: pH~9-10 and counter electrode (-).

Table 4.2 pH and polarity dependence

Biomolecular adsorption in OEWOD surfaces depends strongly on these parameters, inclusive before the potential is applied. The figures 4.8, 4.9, and 4.10 show the development of the contact angle while the potential is applied at the same conditions.

The Lippmann-young equation explains part of the behaviour of the curves 4.8, 4.9 and 4.10, until the saturation effect already mentioned appears, and the reason for an interesting polemic not described in this equation. More interesting is the development in the first ~100 volts of potential applied, which has kept almost constant until the threshold appeared. This behaviour depends mainly on system properties such as the dielectrics thickness and dielectrics properties. It should be the reason for the different threshold and change in the contact angle. The development and behaviour of the HRP with regards to biomolecular adsorption phenomenon on the OEWOD surfaces depends directly on the biomolecular solution charge (pH), electrode polarities and the time of the potential applied. However, it is important to mention that the saturation effect appeared earlier (~180 volts) in the

surfaces Cellulose Acetate–Teflon®AF and SU-8-Teflon®AF than in Durimid 115A- Teflon®AF for the same conditions.

The OEWOD system Durimid 115A-Teflon®AF was the most representative to confirm and demonstrate the contact angle behaviour of the biomolecular adsorption. When the biomolecular solution HRP was intentionally charged from positive (HRP2) to negatives (HRP3), the contact angle shifted some degrees above or below in regard to the contact angle of biomolecular solution HRP1 that was intentionally uncharged (charged equal or similar to the HRP p.I., pH=7.2) (see figure 4.9 and table 4.2). The polarity of the OEWOD surface was assumed to correspond to those from the counter electrode. When the HRP is positively charged and the polarity of the counter electrode is also positively charged (HRP2D+), the results confirmed that less biomolecular adsorption exist between the surface and the biomolecular solution due to the repulse forces. The largest contact angle was measured (108°) that is mean low biomolecular adsorption (less contact area).

In contrast to the case when both HRP and OEWOD surface were negatively charged (HRP3D-), the OEWOD surface Cellulose Acetate–Teflon®AF were more representative, the contact angle measured was 107° (see figure 4.10 and table 4.2).

The OEWOD system Durimid 115A-Teflon®AF was again confirmed that when the biomolecular solution HRP was intentionally negatively (HRP3) charged and the OEWOD surfaces was positively charged (HRP3D+), meaning, they have opposite charges, its expected attraction forces, the lower contact angle was measured at 84°(see figure 4.10 and table 4.2). When the biomolecular solution was intentionally uncharged (HRP1, pH~p.I.), the contact angles were measured in between the intentionally charged biomolecular solution. Here the electrostatically interactions between the biomolecular solution and OEWOD surfaces were reduced, however the contact angle change with the OEWOD surfaces polarity. That demonstrates how electrostatically interaction between biomolecules and the solvent depend on many other effects, which also have to be considered.

4.3 Biomolecular adsorption of HRP under OEWOD conditions

Experiments were performed to analyze the biomolecular adsorption on OEWOD conditions. HRP (POD) was used as biomolecules to adsorb on the OEWOD surfaces taking advantage of their known properties as label molecules, and for the posterior detection by chemiluminescence, which is needed to carry out the enzymatic generation of an acridinium ester/Luminol already mentioned and described in chapter 3. It is important to mention that some parts of the HRP and IgG-HRP experiments were quantitatively detected on a hypersensitive film, and some parts of the HRP with different p.I., potential applied and time as well the DNA- HRP experiments, were relative quantitatively detected in the LAS-3000 imaging system, already described chapter 3. The evaluation of relative quantitative biomolecular adsorption in the first OEWOD surfaces experiments were evaluated through the time necessary rinse or rinse steps necessaries to de-adsorb the biomolecules absorbed, which will be mentioned in more detail later. The biomolecular adsorption experiments with HRP4B and HRP5, IgG-HRP and DNA- HRP at different p.I., potential applied and adsorption time were % relative quantitatively evaluated with AIDA software.

A 1 mg/ml of HRP was dissolved in PBS 0.1 M with three different pH solutions; POD1 (pH=5), POD2 (pH=7.2) and POD3 (pH=10). The stock solution POD1, POD2 and POD3 were diluted at different concentration, 1 mg/ml (POD), 0.1 mg/ml (POD1´) and 0.01 mg/ml (POD1´´).

For the next experiment the followed system was used: Teflon®AF 1600 (6% wt) in FC-75 was deposited by spin coating on a glass wafer (0.3 mm) at 3000 rpm during 1 min and 1500 rpm, 4 min, followed by annealing in air at 100°C, 10 min, Teflon thickness ~ 3µm. Dry peel foil (0.1 mm) was used as electrode previously structured, obtaining an array with different a path to apply electrowetting, see figure 4.11.

10 µl droplets of POD2 where deposited on different locations of the array, see figure 4.11 and chapter 3, set up figures 3.11 and 3.12. Potentials from 60, 40, 20, 10 volts were applied for 5 min. on the droplet. In the same surfaces but in different locations 10 µl droplets were deposited during 5, 10, 15, 20 and 30 min. adsorption time, without potential applied, for reference. When the time adsorption was completed,

the droplets were carefully removed to avoid another contact with the surface. A sequence of chemiluminescence detection in hypersensitive film was performed, see below figure 4.12. Finally, after the adsorption time was finished. The surface was rinsed with PBS 0.1 M. For the experiment set up, see chapter 3.

Figure 4.11: Array for biomolecular adsorption experiment on Figure 4.12: Chemiluminescence detection on a OEWOD surface, 10 µl droplet of 1 mg/ml (HRP) POD are hypersensitive film deposited at different locations under different conditions of potential applied and time adsorption

A second experiment was performed with the OEWOD surfaces Durimid 115A - Teflon®AF 1600 6% wt, array and set up described in chapter 3. 10 µl droplets of POD2 with concentrations varying from 1 mg/ml to 10-5 mg/ml were deposited on different locations on the OEWOD surface, see figure 4.13. The adsorption time of POD2 on contact with the OEWOD surface was 10, 25 and 40 min. When adsorption time was finished, the droplets were carefully removed to avoid further contact with the surface. Chemiluminescence detection was performed, see figures 4.14.

Figure 4.13: Distribution of the samples. Adsorption time vs. Figure 4.14: Detection of chemiluminescence from the left (HRP) POD concentration. side of the OEWOD surface on a hypersensitive film.

The next experiment was performed to evaluate the relative quantification of the biomolecular adsorption on OEWOD surfaces depending on the pH of the biomolecular solution and polarity of the electrode.

Experiments were performed on OEWOD surfaces as previously described to analyze three different pH biomolecular solutions. POD1, POD2 and POD3 were prepared, each of them was dissolved in PBS with different pH values: POD1 was adjusted to a pH=7.4, which is approximately equal to the p.I. of the POD (7.2), to keep intentionally uncharged as in the previous experiment. The POD2 was adjusted to a pH~5-6 to intentionally charge positively and the POD3 was adjusted to a pH~ 9-10 to intentionally charge negatively. 10µl droplets of 1 mg/ml of POD1, POD2 and POD3 were deposited on different locations of the array and a potential was applied. The polarity of the electrode on the top of the droplet and beneath the polymer layer (counter electrode) was exchanged for the same pH of the biomolecular solution; finally contact angles were measured while potential was applied. Additionally conductivities of the PBS 0.1 M at pH 5, 7.2 and 10 were determined and not founded to be significantly different from 23.7 mS/cm at room temperature. Also, the evaporation rate and droplets area occupied during the adsorption time 1, 2 and 5 min. were not relevant for not applied potential experiments (see figures 4.15 and 4.16 in annex B).

The comparison of biomolecular adsorption in Cellulose Acetate–Teflon®AF and Durimid115A-Teflon®AF OEWOD surfaces for the same biomolecules HRP showed different biomolecular adsorption rates, which were unfortunately not relatively quantified due to the acquired method (hypersensitive film, see figures 4.15 and 4.16 in annex B). The HRP used for this experiment (see chapter 3.5.3) was a HRP C with contains a number of distinctive Peroxidase isoenzymes of which the C isoenzyme (HRP C) is the most abundant [Veitch, 2004] and therefore different isoelectric points properties lead to not establishing a specific behaviour to the above mentioned conditions. The assumption for this experiment that higher potential applied lead to higher biomolecular adsorption of a specific p.I. of the biomolecules solution, are relatively confirmed. However, doing only a visual evaluation based on the size spot of the chemiluminesce determination, the results could be confirming in general the previous assumption. The locations where a potential of 40 volts was applied, present a darker (grey scale value) spot than for the locations where a potential of 20 volts was applied. The higher the exposition time the higher the 81

biomolecular adsorption (spot area and grey scale value). The experiments for POD1 (uncharged) located on positive electrode polarity for both OEWOD surfaces (Cellulose Acetate–Teflon®AF and Durimid115A-Teflon®AF), shows an interesting result: the biomolecular adsorption was minimized for higher potential and longer time exposition, see figures 4.15A and 4.16A in annex B.

4.3.1 Horseradish Peroxidase HRP5 and HRP4B.

HRP5 and HRP4B with p.I.=4.0 and p.I.=8.5 respectively were adsorbed in Cellulose Acetate–Teflon®AF 2% and Durimid115A-Teflon®AF 2% surfaces (for HRP details, see chapter 3.5.4 and 3.5.5). The activities of HRP5 (80 units/mg) and HRP4B (250 units/mg) were adjusted to the same units/mg. HRP5 and HRP4B were dissolved in PBS 0.1M and adjusted with HCl to pH=4.0 and NaOH to pH=8.5 respectively to keep the charges deliberately to their p.I. The experiment protocol was the same as in previous experiments, for the distribution of the samples, conditions and % relative quantification of the biomolecular adsorption rates, see AIDA relative quantification figures 4.15A, 4.16A and 4.15B, 4.16B LAS-3000 chemiluminescence images and table 4.3 in annex B for details.

min HRP5 HRP4B HRP5 HRP4B min HRP5 HRP4B HRP5 HRP4B 1 0.97 0.97 10.59 10.30 2.39 10.99 1 2.14 0.73 6.41 5.87 0.17 1.65 + + 2 1.74 1.27 12.68 9.69 1.34 11.76 2 1.19 0.73 4.91 5.67 0.18 0.55

5 1.24 1.36 2.33 9.26 0.69 10.42 5 1.66 1.05 4.46 6.54 0.19 0.70

1 1.51 0.88 9.95 8.81 1 1.69 0.18 6.59 6.43 _ _ 2 1.23 3.38 12.53 8.38 2 2.31 0.26 7.26 6.42

5 0.75 2.53 0.53 10.13 5 0.54 0.99 6.46 3.05

40V 20V 40V 20V 40V 20V 40V 20V

Figure 4.15A: AIDA % relative quantification of Figure 4.16A: AIDA % relative quantification of biomolecular adsorption of chemiluminescence detection biomolecular adsorption of chemiluminescence detection of HRP5 and HRP4B on Cellulose Acetate-Teflon®AF of HRP5 and HRP4B on Durimid 115A-Teflon®AF OEWOD surface OEWOD surface

4.3.2 Discussion and conclusions

When the polarity of the counter electrode in both OEWOD surfaces is positive, the biomolecular adsorption for HRP5 (p.I.=4) in general showed low adsorption rates, which increased relative to the potential applied and time adsorption (see annex B,

table 4.3). However the contact angle changes (hysteresis) were most significant for Cellulose Acetate-Teflon®AF 2%. This supports again the suggestion that the same charge will be repulsive to each other.

The experiment results for HRP4B (p.I. =8.5) showed high rates of biomolecular adsorption for both surfaces, but higher ones for Cellulose Acetate-Teflon®AF 2%, where the surface was positively charged. In contrast to Durimid115A-Teflon®AF 2%, the contact angles changes were for 40 volts, not in the same range as for Cellulose Acetate-Teflon®AF 2%, which corresponds to the biomolecular adsorption rates.

When the surface was negative charged. HRP5 (p.I. =4) was slightly absorbed. The hysteresis in Cellulose Acetate-Teflon®AF 2% was higher than for Durimid115A- Teflon®AF 2%. The previous assumptions were not confirmed here: when the surface and biomolecules were negative charged, the results showed relative higher biomolecular adsorption rates for Cellulose Acetate-Teflon®AF 2% than for Durimid115A-Teflon®AF 2% for 40 volts potential applied. For 40 volts potential applied, Cellulose Acetate-Teflon®AF 2% presented more cases of electrolysis followed for dielectric breakdown than the Durimid115A-Teflon®AF 2% OEWOD surfaces and the adsorption rates apparently decreased or increased, depending on the availability to the surface electrode to absorb. The HRP4B showed higher biomolecules adsorption rates for Cellulose Acetate-Teflon®AF 2%. However, if it is supposed that initially the Teflon surface is negatively charged, which could indicate more extensive passive adsorption due to the hydrophobic surfaces [Yoon, 2003], which is the case if the biomolecular adsorption rates in the same order when not potential was applied (see annex B, table 4.3) confirming the suggestion from Wu et al. [Wu, 2006] which assumes that the passive adsorption during the first minutes are determining and important for the biomolecular adsorption process.

The sequence of chemiluminescence detections performed to evaluate the biomolecular adsorption of the experiments in chapter 4.3 showed that more rinse steps (time) were needed to wash the biomolecules from the Durimid 115A- Teflon®AF OEWOD surface than from the Cellulose Acetate-Teflon®AF OEWOD surface. The experiments with longer adsorption time (5 min. or more) shows that high biomolecular concentration (1 mg/ml) and high potential applied (40 volts or

more) needed around 30 min. to wash/deabsorbe all the biomolecules from the surface. While the experiments with short adsorption time (1 min.), low biomolecular concentration (10-3-10-5 mg/ml) and low potential applied (20 volts) needed around 5 min. to desorb all the biomolecules from the surface. As already mentioned, this was unfortunately not evaluated with LAS-3000 and AIDA. The chemiluminescence images sequence was determined in hypersensitive film; see figures 4.15B and 4.16B in annex B.

The higher the concentration of the biomolecular solution and adsorption time, the higher the biomolecular adsorption. Experiments showed that the higher the potential applied between the biomolecular solution and the counter under the insulating layer, the higher was the biomolecular adsorption on the OEWOD surfaces.

4.4 Peroxidase-Conjugated Rabbit Anti-mouse Immunoglobulin adsorption on OEWOD surface.

Experiments were performed to analyze the adsorption of IgG-HRP on Cellulose Acetate–Teflon®AF during the EWOD condition. Peroxidase-Conjugated Rabbit Anti-mouse Immunoglobulin (IgG-HRP) from DAKO, Denmark is a purified immunoglobulin fraction of rabbit antiserum, which is conjugated with horseradish peroxidase giving a very high specific enzymatic activity. The IgG-HRP adsorbed on OEWOD surfaces was determined as in previous experiments. HRP conjugated to the IgG was enabling the chemiluminescence detection.

4.4.1 Dependence on the pH and time on the IgG-HRP adsorption on Cellulose Acetate-Teflon®AF.

The OEWOD surfaces consist of Cellulose Acetate 3.5% wt/wt from Sigma Aldrich, diluted in 1-Methy-2-pyrolidone and deposited by spin-coating on a Pyrex wafer 0.5 mm at 2000 rpm during 30 sec. and 1000 rpm, 30 sec. followed by annealing in air at 90°C, 15 min.; after the Cellulose Acetate layer was cooled, a second layer of Teflon®AF 1600 1% wt/wt in FC-75 diluted was deposited by spin coating at 2000 rpm for 30 sec., 1000 rpm, 30 sec. followed by annealing in air at 90°C, 30 min. Teflon thickness is ~3 µm. As counter electrode (-) between the Pyrex wafer and the

layers (Cellulose Acetate-Teflon®AF) was previously deposited by EVD, a structured pathway of 10 nm titanium and 25 nm Platinum.

The first experiment consisted of the adsorption of IgG-HRP on Cellulose Acetate- Teflon®AF without potential applied. A 10 µl of IgG-HRP, 10-3 mg/ml were deposited on specific locations of the OEWOD surface. Adsorption times, from 5 to 30 min. with intervals of 5 min were measured. The IgG-HRP was diluted in PBS 0.1 M solution with pH=5, pH=7.2 and pH=8.5. (See figure 4.17 for samples distribution). The parameters analyzed were the biomolecular adsorption of IgG-HRP solution versus time and pH. When the adsorption time was finished, the surfaces were rinsed with PBS 0.1 M, pH=7.2 in a shaker for 20, 30, 45, 55 and 80 minutes. Figure 4.17 shows the first chemiluminescence detection immediately after the experiment. The figure 4.18 shows chemiluminescence detection after the OEWOD surface was washed for 20 min. in a shaker with PBS 0.1 M and the figure 4.19 chemiluminescence detection after the OEWOD surface was washed for 30 min at the same conditions before mentioned. The figures 4.20, 4.21, and 4.22 shows chemiluminescence detection for corresponding washing times of 45, 55 and 80 min. IgG-HRP was adsorbed stronger at higher adsorption time and pH=7.2 as well as, pH=8.5 it has needed longer washing time, around 80 min., while in the case of the IgG-HRP with pH=5 just 30 min. were needed to wash all the IgG-HRP from the OEWOD surface, see below, sequence of pictures from chemiluminescence detection.

pH=5 pH=7.2 pH=8.5 5 min.

10 min.

15 min.

20 min.

25 min.

30 min.

Figure 4.17: Chemiluminescence Figure 4.18: column left ; pH=5, 10 µl Figure 4.19: Chemiluminescence -3 -3 detection of IgG-HRP, 10 mg/ml IgG-HRP, 10 mg/ml after 20 min detection after 30 min. washing with at different time adsorption and washing with PBS PBS different pH solution

Figure 4.20: Chemiluminescence Figure 4.21: Chemiluminescence Figure 4.22: Chemiluminescence detection after 45 min. washing with detection after 55 min. washing with detection after 80 min. washing with PBS PBS PBS

The chemiluminescence experiments were realized in a dark room and detected on hypersensitive films, which were developed, fixed, washed and dried in an automatic X-ray film processor from Amersham Pharmacia Biotech Hyperprocessor. The hypersensitive films were scanned to digitalize, therefore the quality of the figures 4.17, 4.18, 4.19, 4.20, 4.21 and 4.22 cannot be improved.

4.4.2 Dependence on the electrode polarity, concentration and pH on the IgG- HRP’s adsorption on OEWOD surfaces

The Peroxidase-conjugated rabbit anti-mouse immunoglobulin stock solution 1.3 mg/ml was dissolved in PBS 0.1 M solution; 1) pH=5, 2) pH=7.2 and 3) pH=10 and dissolved at #: 1:10, #‘: 1:100, #‘‘: 1:1000 dissolutions. To simplify the same nomenclature, they were called 1# (0.13 mg/ml of IgG-HRP dissolved in PBS 0.1M, at pH=5), 1#‘ (0.013 mg/ml of IgG-HRP dissolved in PBS 0.1M, at pH=5), 1#‘‘ (0.0013 mg/ml of IgG-HRP dissolved in PBS 0.1M, pH=5), 2# (0.13 mg/ml of IgG- HRP dissolved in PBS 0.1M, at pH=7.2), 2#‘(0.013 mg/ml of IgG-HRP dissolved in PBS 0.1M, at pH=7.2), and so on.

10 µl droplets of IgG-HRP were deposited on specific locations on the OEWOD surface (see figures 4.23A and 4.24A). A potential from 40 and 20 volts was applied for 6 min. on the droplet. The set-up is the same used for previous experiments and 86

described in chapter 3.7.1. The same procedure was performed changing the polarity of the counter electrode from negative to positive. Both sequences of experiments were performed for both surfaces; Cellulose Acetate-Teflon®AF and Durimid 115A–Teflon®AF (see AIDA % relative quantification figures 4.23A, 4.24A and 4.23B, 4.24B LAS-3000 chemiluminescence images in annex B).

t/c 1min 2min 3min t/c 1min 2min 3min 3.34 0.96 8.40 0.19 1.88 1.64 2.27 0.93 5.28 0.11 1.08 4.44 + + 2.16 1.53 1.62 2.49 0.92 1.16 2.12 1.97 2.44 4.90 5.27 10.84

0.10 0.38 0.10 0.14 0.19 0.02 0.10 0.04 0.03 0.03 0.33 0.42

2.06 3.91 0.63 0.32 9.14 6.85 0.13 3.49 0.03 0.03 1.09 13.83 _ _ 0.06 2.30 0.51 1.47 1.84 1.50 0.09 0.91 0.03 0.19 0.01 3.00

0.04 0.00 0.00 0.00 0.00 0.10 0.01 0.02 0.01 0.00 0.00 0.00

40V 20V 40V 20V 40V 20V P/v 40V 20V 40V 20V 40V 20V P/v

Figure 4.23A: AIDA % relative quantification of Figure 4.24A: AIDA % relative quantification of biomolecular adsorption of chemiluminescence detection of biomolecular adsorption of chemiluminescence detection IgG-HRP on Cellulose Acetate-Teflon®AF OEWOD of IgG-HRP on Durimid 115A-Teflon®AF OEWOD surface surface

Figures 4.23B, 4.24B and table 4.4 in annex B shows chemiluminescence detections images of Cellulose Acetate-Teflon®AF and Durimid 115A–Teflon®AF OEWOD surfaces in LAS-3000 and AIDA % relative quantification of the images, respectively. To confirm that the chemiluminescence signal only corresponded to the biomolecular adsorption and not to the biomolecular solution accumulated on the surface, the OEWOD surfaces were washed initially for 10 min. with PBS 0.1M pH=7.2. Additional chemiluminescence detections after several washing steps in different intervals; 20, 30, 45, 55, and 80 minutes were performed. In annex B, table 4.4, the AIDA evaluation of the % relative quantification of biomolecular adsorption under specific conditions in both OEWOD surfaces was resumed.

4.4.3 Discussion and conclusions

The IgG-HRP adsorbed stronger at higher adsorption time, pH=7.2 and pH=8.5. It required longer washing time, around 80 min., while in the case of the IgG-HRP with pH=5 just 30 min. were needed to desorbed from the Cellulose Acetate-Teflon®AF OEWOD surface, see sequence of pictures from chemiluminescence detection, figures 4.17, 4.18, 4.19, 4.20, 4.21 and 4.22. The completely apparent desorption of

the IgG-HRP were reached at 80 min washing step protocol. The chemiluminescence detection was performed in hypersensitive films, which were then scanned to digitalize, therefore the quality of the figures 4.17, 4.18, 4.19, 4.20, 4.21, and 4.22 can not to be improved.

No significant % relative quantification biomolecular adsorption rates were observed in both OEWOD surfaces for that IgG-HRP diluted concentrations #‘‘ at different conditions, see figures 4.23A and 4.24A. However, the most of them presented a similar larger change contact angle after the potential was applied, than the other concentrations, see table 4.4 in annex B. It should be indicated as the best concentration for IgG-HRP to perform a bioassay, minimizing biomolecular adsorption in OEWOD surfaces under these conditions.

When the polarity of the bottom electrode in both OEWOD surfaces was negative, more electrolysis followed for dielectric breakdown was observed in general, see table 4.4 in annex B. The behaviour pattern of biomolecular adsorption rates increased over the potential applied in both surfaces, when the polarity of the bottom electrode in both OEWOD surfaces was positive, see annex B, table 4.4. However, the highest rate biomolecular adsorption was observed for Durimid 115A-Teflon®AF 2% by 3#, 20V bottom negative polarized 13.83 and in contrast to Cellulose Acetate- Teflon®AF 2% at the same conditions 6.85. This should be due to possible dielectric breakdown and followed for electrode biomolecular adsorption, although the biomolecular adsorption pattern follows the expected behaviour for both cases. This means that the adsorption rates decreased when the concentration decreased. The same behaviour was repeated by 2#, 40V for surfaces, 8.40 and 5.28, see figures 4.23A, 4.24A and table 4.4 in annex B.

The contact angle change showed no significant difference for both OEWOD surfaces and for any IgG-HRP concentration, nor the pH of the biomolecular solution. In general, under these conditions Durimid115A-Teflon®AF 2% showed less biomolecular adsorption rates than Cellulose Acetate-Teflon®AF 2%. It is difficult to assume a specific net charge of the biomolecules here, which suggested, that the charge net of the biomolecules IgG-HRP should be changed to positive charge or negative charge net due to the accumulation on the surfaces or charger layers formation. Some cases of electrolysis and dielectric breakdown on the layers

for 40 volts potential applied were observed, due mainly to the layer heterogeneities and respective roughness.

4.5 DNA-HRP adsorption on OEWOD surfaces

To measure the adsorption of DNA, HRP was linked as a marker to the DNA molecule. The following protocol, supplied from the ECL Direct Nucleic Acid Labelling System from Amersham Biosciences (see chapter 3.5.6 for details), was used for the labelling process: 100ng of the DNA to be labelled was diluted in 10µl of the water supplied. The DNA sample was denatured by heating for 5 minutes in a boiling water bath. After denaturation, the DNA was immediately cooled down for 5 minutes on ice. By a brief spinning in a microcentrifuge the contents were collected at the bottom of the tube. The equivalent DNA labelling reagent (10µl) was added to the cooled DNA and mixed gently but thoroughly. Glutaraldehyde solution was added, again the equivalent to the volume of labelling reagent (10µl), mixed thoroughly and spun briefly. The solution was incubated for 10 minutes at 37°C. The Hind III control DNA linked to the HRP has different size fragments number of base pair from 23,130 bp to 125 bp in length (see Chapter 3.5.6, table 3.5 DNA string size). To avoid some change of the DNA linked to HRP molecule to adsorb on the OEWOD surfaces, the pH of the solution for the posterior detection by chemiluminescence was not changed.

4.5.1 Dependence on the electrode polarity, potential applied and time adsorption of DNA-HRP on OEWOD surfaces

10 µl droplets of DNA-HRP where deposited on different locations of the Cellulose Acetate-Teflon®AF and Durimid115A-Teflon®AF OEWOD surfaces. A potential of 60, 40, 20 volts were applied during 1, 2, 5 min. on the droplet. The electrode polarity of the surface was positive. The same experiment was performed while the polarity of the surface was negative. Both sequences of experiments were performed for both surfaces. DNA-HRP (DNA) is 1 ng/ml and DNA-HRP (DNA‘) is 0.1 ng/ml diluted.

t/c `DNA-HRP DNA-HRP t/c `DNA-HRP DNA-HRP 1 1.70 0.59 0.05 5.01 3.06 5.85 1 0.34 0.03 0.51 6.66 2.98 0.09 + 2 1.42 1.07 0.03 7.56 4.54 2.41 2 0.60 0.04 0.13 9.81 9.75 7.13 +

5 0.10 0.19 1.20 9.86 4.30 2.43 5 0.00 0.02 0.54 10.6 3.90 0.04

1 0.31 0.53 0.06 1.06 3.30 2.33 1 0.01 0.00 0.37 2.99 0.04 0.03 _ 2 1.49 0.49 0.02 2.10 3.11 7.26 2 0.61 0.94 0.20 7.25 9.41 0.17 _

5 1.53 0.94 0.02 6.75 10.54 6.91 5 1.50 2.05 2.49 8.06 8.92 1.74

60V 40V 20V 60V 40V 20V P/v 60V 40V 20V 60V 40V 20V P/v

Figure 4.25A: AIDA % relative quantification of biomolecular Figure 4.26A: AIDA % relative quantification of adsorption of chemiluminescence detection of DNA-HRP on biomolecular adsorption of chemiluminescence detection of Cellulose Acetate-Teflon®AF OEWOD surface DNA-HRP on Durimid 115A-Teflon®AF OEWOD surface

4.5.2 Discussion and conclusions

Lower rates of biomolecular adsorption in both surfaces at concentration of DNA- HRP (DNA‘) 0.1 ng/ml were observed. However, Cellulose Acetate-Teflon®AF presented higher biomolecular adsorption rates than Durimid115A- Teflon®AF under the same condition.

When the OEWOD surfaces were positive polarized, the DNA-HRP (DNA) 1 ng/ml showed higher rates of biomolecular adsorption, which increased with the increment of the potential applied and time, see figures 4.25A, 4.26A and for the chemiluminescence pictures, see figures 4.25B and 4.26B in annex B. This suggested that DNA-HRP was negatively charged. When the OEWOD surfaces were negatively polarized, more electrolysis and dielectric breakdown were observed, see comments in table 4.5, annex B. Durimid 115A–Teflon®AF presented higher biomolecular adsorption rates by 60 volts and 1, 2, 5 minutes and increased over time, in contrast to Cellulose Acetate-Teflon®AF, which presented similar biomolecular adsorption rates under the same conditions. The different size fragments, number of base pair in lengths should play an important role: the smaller charger fragments could be first attached to the surfaces and polarize it, changing the charge of the surface. The repealing or attaching of DNA strings depends on charge and fragments size of the first minutes attached on the OEWOD surface. The DNA-HRP showed larger contact angles, increasing over the potential applied for Durimid 115A–Teflon®AF reaching for 60 V potential applied a contact angle change around 30 3 . In contrast, Cellulose Acetate-Teflon®AF under the same conditions, the contact angle changed around 10 2 . That confirmed the idea that 90

for larger contact angle changes, a the larger contact area was occupied, followed by high rates of biomolecular adsorption. Durimid 115A–Teflon®AF OEWOD surfaces presented higher rates biomolecular adsorption and larger contact angles for DNA- HRP (see figures 4.25A, 4.26A and 4.25B, 4.26B and table 4.5 in annex B).

4.6 Applications on OEWOD Microfluidics-Tool Platform

The microfluidics platform OEWOD has shown considerable availability and versatility for a wide range of applications. The feasibility design permits the additional adaptation of external equipment for several complementary tasks, such as a luminescent reader system, which is very important for fluorescence and chemiluminescence assays, especially in biochips or BioMEMs approaches. The manipulation of micro or nano volumes, depending on the electrode design and its programmable microfluidic protocols promises new perspectives in the BioMEMs future. The OEWOD microchip has already been used for several other tasks. However, in this thesis, it will only be used to demonstrate mainly two examples of applications. It is beyond the scope of this thesis to show their details, efficiency, or high-throughput.

Currently, there are many kinetic reactions on surfaces, which are complicated to follow with traditional methods because to the impracticability to measure the reaction, at the specific moment required as in the case when catalytic, enzymatic, or fluorescence reactions take place. The kinetics data are very important in order to talk about efficiency or kinetics enzyme inactivation investigation [Hiner, 2002]. The OEWOD platform permits the mixing and the immediately measurement of the resulting fluorescence or chemiluminescence through an easy protocol with an accuracy and adequate manner. In the same way, several bioassays protocol should be possible as RT-NASBA on OEWOD microfluidics platform.

4.6.1 OEWOD platform as a tool for kinetics

We have developed an OEWOD microchip system that located in an imagine system analyzer enables the detection of micro or nano amounts of reactant by fluorescence or any luminescence sensing protocol. OEWOD allows the manipulation of liquids in such a way that reactions can be triggered at an exact point in time and placed on a 91

substrate. Horseradish Peroxidase (HRP) in different pH solutions, while Luminol reactants are simultaneous merged by means of EWOD in OEWOD microchip. The luminescence signal can be followed from the beginning of the reaction to the end, and the kinetics during the reaction can be analyzed. The same principle can be applied for kinetics enzyme inactivation on surfaces investigation. Horseradish Peroxidase (HRP) is widely used as an enzyme label for medical diagnostics and research applications, the availability of substrates for colorimetric, fluorimetric and chemiluminescence assays provide numerous detection options [Akhaven-Tafti, 1994]. These intermediates react with peroxide under slight alkaline conditions to produce a sustained, high intensity chemiluminescence with maximum emission at the wavelength of 430 nm.

In this thesis, the resulting light from the reaction of Luminol with HRP are detected on a luminescent image analyzer (LAS-3000). The luminescent image analyzer followed the reaction, from the beginning until the reaction finalized. The quantification was performing with AIDA (Advanced Image Data Analyzer) software from raytest Isotopmessgeräte GmbH, Germany. The chemiluminescence detection from the reaction of Luminol with HRP in homogeneous solution causes intense chemiluminescence, which reaches peak intensity a few times. Luminol are based on the oxidation of the cyclic diacylhydrazide and the enzymatic generation of an acridinium ester, which produces an intense light emission by 430 nm. Such assays on surfaces can be followed at the beginning of the mixes process.

4.6.2 HRP kinetics enzyme on OEWOD surfaces

Experiments were performed with the followed parameters to analyse the kinetics of HRP and Luminol on OEWOD surfaces. A 10µl droplet, 0.01 mg/ml of HRP (Horseradish Peroxidase, Sigma Aldrich Chemie Gmbh P.O. 1120, 89552 Steinheim, Germany) was dissolved in PBS puffer 0.1M. The PBS 0.1 M was adjusted to different pHs: pH=6, pH=7.2 (Isoelectric point of HRP), and pH=10. A 10µl droplet of Luminol (Immobilon Western, Chemiluminescent HRP Substrate, Millipore Corporation, Billerica, MA 01821 U.S.A) were simultaneous merged to the 10 µl droplet by means of EWOD effect on OEWOD microchip (see figure 4.27 and 4.29).

Figure 4.27: Sketch of merging protocol and OEWOD microchip inside into LAS-3000 (Schematic picture left side was taken from Herberth 2006). Right side: sequence of pictures record from the beginning of the reaction in LAS-3000 and Droplet Handling System software.

The droplets were deposited into an OEWOD microchip placed inside the luminescence image analyser system (LAS-3000), see figures 4.28 and 4.29. Chemiluminescence signals were measured and quantified at the beginning of the reaction.

Figure 4.28: OEWOD microchip inside the luminescence imaging system LAS-3000.

The OEWOD microchip for kinetics experiments is basically the same OEWOD microchip for RT-NASBA, described in chapter 3.7.5. Nevertheless, they were coated with different size thickness of Teflon®AF layer, to avoid dielectrics breakdown and electrolysis during the ―actuation‖, merging protocol. The Teflon®AF layer reached a thickness of 250 nm.

Figure 4.29: Close up of OEWOD microchip and software of the luminescence imaging system LAS-3000.

The 3 droplets of 10µl of 0.1M HRP dissolved in PBS puffer 0.1M adjusted to pH=6, pH=7.2 and pH=10 were deposited manually by different pipettes, avoiding contamination of the OEWOD microchip surface, immediately the 3 droplets of 10µl Luminol were aligned and deposited in the same way.

The self-made circuitry and software, described in chapter 3.7.3, provided electrical control of the electrodes. The switching time of the electrodes were each approximately 3 ms. the measurement started at the same time, as the droplets were mixed by means of EWOD effect. The basic operation protocol was previous programmed to enable a homogenous mixing of the droplets and was consisted to move the droplets at the same location, once mixed, more than four times, towards the right to the left and coming back. The luminescence images system LAS-3000 made measurements for 210 second, in 15-second intervals. The chemiluminescence images detections were evaluated with the software AIDA, see figures 4.30, 4.31, 4.32, and 4.33.

pH=10 pH=7.2 pH=6

Time (s)

Figure 4.30: Sequence of pictures from the luminescent image analyzer (LAS-3000), reads the emitted chemiluminescence every 15 sec. from the HRP and Luminol reaction on Durimid 115A-Teflon®AF surface at different pH´s HRP solution. 94

40 pH 6 35 pH 7,2 pH 10 30

10 intensity - background [LAU] background - intensity 5

0 0 20 40 60 80 100 120 140 160 180 200 -5 time [s]

Figure 4.31: HRP and Luminol: intensity-Background [LAU] vs. Time [s] of chemiluminescence detection on Durimid 115A- Teflon®AF surface at different pHs HRP solution, evaluated with AIDA.

pH=10 pH=7.2 pH=6

Time (s)

Figure 4.32: Sequence of pictures from the luminescence image analyser (LAS-3000), reads the emitted chemiluminescence every 15 sec. from the HRP and Luminol reaction on Cellulose Acetate-Teflon®AF surface at different pHs HRP Solution.

45 pH 6 40 pH 7,2 35 pH 10

10 intensity - background [LAU] 5

0 0 20 40 60 80 100 120 140 160 180 200 -5 time [s]

Figure 4.33: HRP and Luminol. Intensity-Background [LAU] vs. Time [s] of chemiluminescence detection on Cellulose Acetate-Teflon®AF OEWOD surface at different pH´s HRP solution evaluated with AIDA.

4.6.3 Discussion and conclusions

The biomolecular adsorption plays an important role in the kinetics of the reaction on surfaces. Biomolecules undergo a conformational change during the adsorption process, which changes the kinetics of the reaction according to the different biomolecules-surfaces interaction forces.

The chemiluminescence detection of HRP and Luminol on Durimid 115A-Teflon®AF surface showed higher intensity (Arbitrary units) than for Cellulose Acetate- Teflon®AF. The different behaviour of intensity rates according to the pH solution of the HRP and OEWOD surfaces confirmed and demonstrated that kinetic reactions depend on the surfaces where they take place. The figures 4.30, 4.31, 4.32, and 4.33 showed the kinetics behaviors (intensity) of the chemiluminescence reaction at those OEWOD surfaces. Table 4.6 shows the linear equation (the behaviour of the intensity light) depending on the parameter mentioned above.

Durimid115A-Teflon®AF Cellulose Acetate–Teflon®AF PH

6 y = -0.0371x + 5.98 Y = -0.0698x + 10.591

7.2 Y = -0.0738x + 10.74 Y = -0.1093x + 15.874

10 y = -0.123x + 17.28 Y = -0.1542x + 23.846

Table 4.6 Linear equation of the chemiluminescence intensities of HRP on OEWOD surfaces

The analysis of the chemiluminescence intensity behaviour on surfaces should be supporting the kinetics enzyme inactivation research, as well the design of specific experiment related to the kinetics field.

OEWOD is a practical and attractive tool to evaluate kinetics reactions, as well as kinetic reactions on surfaces. It allows small volumes of liquid to be manipulated and controlled on-chip. This is an enabling technology, which in combination with the luminescence image analyser system and the temperature control, makes feasible the thesis of diverse and interesting problems on surfaces chemistry, biology and provides the possibility to a wide variety of applications including high throughput lab on a chip, as well as kinetics analysis of the mixes process through the measurement of a reaction at the initial point of the mixing process and at specific intervals. The properties and kinetic behaviour of the chemiluminescence emission on surfaces, the adsorption of HRP on OEWOD surfaces, as well the mixing protocol could contribute to explaining the kinetics in the adsorption process at surfaces.

4.7 Real Time Nucleic Acid Sequence Based Amplification on OEWOD platform

Real Time Nucleic Acid Sequence Based Amplification (RT-NASBA) is a relatively new biomolecular technology: a short description, as well the state of art was already introduced in chapter 1.3 and 3.6. A continuation will introduce the microfluidics suitability of Open Electrowetting On Dielectrics based droplet actuation platform for RT-NASBA application. The characterization of the components of RT-NASBA on

OEWOD platform will be shown in the table 4.7. The compatibility of the RT-NASBA was confirmed, meanings the actuation of the reagents via EWOD and the performance of the bioassay protocol on the OEWOD platform. The preliminary characterization was carried out on the two OEWOD surfaces, Cellulose Acetate– Teflon®AF and Durimid 115A–Teflon®AF.

4.7.1 Compatibility and characterization of NASBA components to the OEWOD based droplet actuation platform

Table 4.7 shows the preliminary contact angle and pH characterization of the components of the PreTec HPV Proofer Kit (henceforth called, NASBA reagents solution) on the OEWOD surfaces: Cellulose acetate–Teflon®AF 2% and Durimid 115A–Teflon®AF 1600 (2% wt/wt).

NASBA reagents PH~ Contact angle Contact angle Content (degrees)1 (degrees)2 Lyophilized sphere containing 10 105 105 nucleotides, dithiothreitol and MgCl2, Mastermix solution TRIS/HCL, 45% DMSO; KCl and NASBA water. Lyophilized sphere containing AMV-RT, Enzyme solution 8 80 89 Rnase H, T7 RNA polymerase and BSA, Sorbitol in aqueous solution. In vitro produced Control RNA in 5 NASBA reaction 8 84 82 mol/l guanidine thiocyanate, Synthetic Mixture Performance Control primers in water, master solution and enzyme solution

1 Durimid 115A (50% wt/wt) – Teflon®AF 1600 (2% wt/wt), 2 Cellulose Acetate (3.5% wt/wt) – Teflon®AF 1600 (2% wt/wt)

Table 4.7: Shows the contact angles and pH characterization of the PreTec HPV Proofer Kit components on OEWOD surfaces.

The contact angles of 5 µl droplets of each NASBA reagents solutions were measured with the DCA-system as a function of the potential applied from 0 to 30 V. Two optimized OEWOD surfaces according to the technical requirement and biomolecular adsorption properties determined and described in previous experiments were used. The ―actuation‖ experiments to optimize the potential applied in Cellulose Acetate–Teflon®AF 1% and Durimid 115A–Teflon®AF 1% lead experimentally to verification that droplets containing NASBA reagents could be transported, with the exception of the mRNA and NASBA reaction mixture samples, which have shown electrolysis and dielectrics breakdown after 17 volts potential applied, see table 4.8. The NASBA standard reagents showed that some of the 98

standard solutions were relatively able to transport, mix and dispense. It was confirmed that the contact angle of most of the standard NASBA reagents change when a voltage was applied, which is the basic characteristic required of OEWOD microfluidics platform. The electrolysis and consequently dielectrics breakdown in the mRNA samples, as well as NASBA reaction solution, could be due to the high concentration of 5 mol/l ions guanidine thiocyanate.

Initial contact Contact angle after 30 volts applied Hysteresis NASBA reagents angle (degrees) Cellulose Acetate – Teflon®AF (degrees)

Sphere diluents 95 83 - KCl stock / NASBA water 104 83 - Primer 88 74 - Enzyme diluents 105 80 - mRNA 85 Electrolysis after 17 volts -

NASBA reagents

Master mix solution 92T1 77 T1 4 Enzyme solution 73 T2 58 T2 5 NASBA reaction mixture 75 69 or electrolysis -

T1: Tangent method 1, T2: Tangent method 2.

Table 4.8: Contact angles characterization of NASBA standard reagents under EWOD effect on Cellulose Acetate–Teflon®AF surface.

In order to find the optimal OEWOD surfaces for RT-NASBA application, a second sequence of experiments was performed. Experiments were performed on Cellulose Acetate–Teflon®AF and Durimid 115A–Teflon®AF as before described and to avoid electrolysis and increase the efficiency of the actuation protocol (microfluidics), the thickness of the actuated layer (Teflon®AF) was increased. Teflon®AF 2% wt/wt was used with the same spin-coating program for this objective, which gives a layer around 400-500 nm thick. The figures 4.34, 4.35 and 4.36 show the behaviour of the NASBA mixture reagents of the EWOD effect on Cellulose Acetate–Teflon®AF and Durimid 115A–Teflon®AF.

Cellulose acetate - Teflon AF 1600 (2%w/w) Durimid 115A - Teflon AF 1600 (2%w/w)

Mastermix solution Mastermix solution

) )

degrees degrees

degreesdegrees

Angle ( ( Angle Angle

Contact Contact Contact

(Volts) (Volts)

(Volts)(Volts)

Voltage Voltage Voltage

VoltageVoltage

Time (sec.) Time (sec.)

Figure 4.34: Show the contact angles of Mastermix solution as a function of the potential applied on OEWOD surfaces: Cellulose Acetate–Teflon®AF 2% and Durimid 115A–Teflon®AF 2%

Cellulose acetate - Teflon AF 1600 (2%w/w) Durimid 115A - Teflon AF 1600 (2%w/w)

Enzyme solution Enzyme solution

) )

degrees degrees

degreesdegrees

Angle ( ( Angle Angle

Contact Contact Contact

(Volts) (Volts)

(Volts)(Volts)

Voltage Voltage Voltage

VoltageVoltage

Time (sec.) Time (sec.)

Figure 4.35: Shows the contact angles of the enzyme solution as a function of the potential applied on OEWOD surfaces: Cellulose Acetate–Teflon®AF 2% and Durimid 115A–Teflon®AF 2%

Cellulose acetate - Teflon AF 1600 (2%w/w) Durimid 115A - Teflon AF 1600 (2%w/w)

NASBA reaction NASBA reaction

) )

degrees degrees

Angle ( ( Angle Angle

Contact Contact Contact

(Volts) (Volts)

(Volts)(Volts)

Voltage Voltage Voltage

Time (sec.) Time (sec.)

Figure 4.36: Shows the contact angles of NASBA reaction as a function of the potential applied on OEWOD surfaces: Cellulose Acetate–Teflon®AF 2% and Durimid 115A–Teflon®AF 2%.

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The potential applied for the actuation protocol in both surfaces was between 30 and 50 volts during at least 280 cycles. The reproducibility of the ―actuation‖ protocol decreased during the cycles more for Durimid 115A–Teflon®AF 2% surface than for Cellulose Acetate – Teflon®AF 2% surface.

4.7.2 OEWOD Droplets Handling System (DHS) and Temperature Controller

The Droplets Handling System (DHS) software-system (Driven Control) and the Supercool Regulator Board (Temperature Controller) were already described in chapter 3.7.3 and 3.7.6. The DHS software-system was programmed with a sequence of steps to realize the basic operation: mixing the droplets of RT-NASBA assay, see sequence of pictures figures 4.37, which were previously mixed as recommended in the PreTec HPV Proofer Kit protocol. The droplets were manually deposited on the OEWOD microchip. The Mastermix solution 10µl consisted of the mixture of 5µl enzyme solution and 5µl NASBA reaction (Primers, Oligonucleotide HPV16 (mRNA) and Molecular Beacon). The first step of the protocol was to mix the NASBA reaction to the Mastermix solution on the middle of the OEWOD microchip, and the second step of the protocol was to move the enzyme solution to the rest of the NASBA reaction to be mixed. The Supercool Regulator Board program controlled the temperature of the heater element placed below the OEWOD microchip. The temperature was kept constant at 41° 0.5 on the OEWOD microchip surface, necessary for the RT-NASBA assay. A temperature sensor was positioned on the OEWOD microchip surfaces to control and register any temperature change, see figure 3.18. The OEWOD microchip was placed inside in luminescence imaging system for the real time fluorescence measurements, see figure 4.28 and 4.38.

a) b)

c) d) Figures 4.37: Shows mixing protocol: a sequence of the basic microfluidics operations on OEWOD microchip for RT-NASBA for HPV16 detection. 101

4.7.3 Fluorescence Measurements of the RT-NASBA for HPV16 detection on OEWOD microchip

The NASBA amplification of the artificial oligonucleotide sequence HPV type 16 was quantitatively detected through the fluorescence signal of the Molecular beacon in a luminescence image analyzer system from Fujifilm (LAS-3000, see figure 4.38).

Figure 4.38: the OEWOD microchip inside the luminescence image analyzer system from Fujifilm (LAS-3000).

The measurement of the fluorescence signal was evaluated for 200 min. and intervals of 5 and 10 min. Images of the OEWOD microchip were performed with specific sensitivity and resolution, described in chapter 3.7.7. The quantitative analysis of the amplification in the OEWOD microchip was realized with the software Advanced Image Data Analyzer (AIDA) from raytest Isotop Messgeräte GmbH, Germany, see figure 4.39, described in chapter 3.7.8.

On the OEWOD microchip two assays were realized: negative control (NASBA water) and positive control (artificial oligonucleotide HPV16). The NASBA reaction was performed on OEWOD microchip and merged by EWOD mean (50 volts) and consisted: 5µl of 0.1 M HPV16, 10 M Primers, and 20 M Molecular Beacon, 5µl enzyme solution and 10µl Mastermix reagent solution. The NASBA reaction was incubated by 41°C during 3.3 hrs. (200 min.) on OEWOD microchip. The real time detection of the HPV16 amplification (0.1 M) was followed through the molecular beacon fluorescent probe technology, see chapter 3.6.1. The emitted fluorescence from the molecular beacon was detected at 460 nm every 10 min. The RT-NASBA assay was parallel performed in Lightcycler 1.2 PCR system device during 160

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cycles 2.86 hrs. (172 min.), see figures 4.40 and 4.41(The curve corresponding to the negative sample was normalized to the curve of the Amplification of HPV16 by Real-Time NASBA protocol on OEWOD microchip system*).

Figures 4.39: Right side: LAS-3000 analysis system software show the sequence of steps programmed to measure the molecular beacon every 5 or 10 min. Left side: quantification analysis of the fluorescence NASBA amplification of HPV16 with AIDA analyzer software.

4 RT-NASBA_HPV16_Positive Control

Fluorescence (arbitry unit ) unit Fluorescence(arbitry 2 RT-NASBA_HPV16_Negative Control

0 00:00 00:28 00:57 01:26 01:55 02:24 02:52 Time(h)

Figures 4.40 and 4.41: Right side: Normalized results of the amplification of HPV16 by Real-Time NASBA protocol on OEWOD microchip system. Left side: Amplification of HPV16 by Real-Time NASBA protocol in Lightcycler 1.2 PCR system device.

4.7.4 Discussion and Conclusions

Real Time Nucleic Acid Sequence Based Amplification (RT-NASBA) of oligonucleotide Human Papilloma Virus 16 (HPV16) on Open-Electrowetting On Dielectric (OEWOD) microchip system has been demonstrated. The main components of NASBA standard reagents solutions were previously prepared as recommended for NorChip PreTect HPV-Proofer kit protocol and manually (pipette) placed on the OEWOD microchip located in a luminescence image analyzer system (LAS-3000) combining a CCD camera technology with an epifluorescence module

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for fluorescence technology detection. Two RT-NASBA reactions (negative and positive controls) have been successfully performed on OEWOD microchip by means of simple unit operations as moving and merging under electrowetting effect.

Biomolecular adsorption was observed during the RT-NASBA reaction on OEWOD microchip, which could be contributed to the low yielding of the amplified NASBA products compared to the results obtained in PCR system device for a similar assay. Additional biomolecular adsorption was decreased the actuation on the OEWOD microchip. BSA and PEG were adsorbed previous at the NASBA assay locations to avoid the adsorption of NASBA products, without significant difference. It was unsuccessfully attempted to influence the biomolecular adsorption through the selection of electrode polarity during the NASBA assay performance. The complex enzyme mix needed for the NASBA amplification was influenced by surface electro- adsorption avoiding at certain points the enzyme activity, which could explain the partial exponential development of the amplification products (HPV16).

4.8 General discussion and conclusions

The biomolecular adsorption on OEWOD surfaces is a very complex process, which is not yet fully determined. It is well-known that it is driven by several forces and interactions between the biomolecules, medium and OEWOD surfaces, depending on several physical and chemical parameters of the system, as well as on the bulk of properties of the biomolecules. In addition, these forces and interactions change during the ―EWOD effect‖. This thesis presented two different studies. In the first one, introduces the chemiluminescence protocol as very sensitive tool in OEWOD platform. Identified the parameter involves in the biomolecular adsorption of HRP, IgG-HRP and HRP-DNA on Durimid 115A-Teflon®AF and Cellulose Acetate- Teflon®AF surfaces. The high biomolecular adsorption rates under manipulated parameter such as electrode polarity, concentration, pH, OEWOD surface and potential applied confirmed the approach to functionalize the surface, through biomolecular adsorption. The second one demonstrated: a Real Time Nucleic Acid Sequence Based Amplification (RT-NASBA) of oligonucleotide Human Papilloma Virus 16 (HPV16) on Open-Electrowetting On Dielectric (OEWOD) microchip system and confirmed the microfluidics platform as a very practical and promising tool for bio-applications.

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Jeong-Yeol Yoon and Robin L. Garrel [Yoon, 2003] suggested that the biomolecular adsorption process in such systems, is mainly driven by hydrobophic forces (passive adsorption), due to the core of the OEWOD system (Teflon®AF) and electrostatically forces, due to the potential applied for the ―actuation protocol‖. In contrast, the experiment results of this thesis confirmed: 1) Biomolecular adsorption shows a significant dependence on OEWOD surfaces. The OEWOD surfaces Durimid 115A- Teflon®AF and Cellulose Acetate-Teflon®AF presents different biomolecular adsorption rates under the same conditions. 2) The ionic strength of the solvent influences the behaviour of the EWOD effect (see experiments with water and PBS 0.1M as solvent medium for biomolecules). 3) In the interpretation of the estimated capacitances and double-layer charging currents should be considered the distance between electrodes, droplets contact area to the bottom electrode and the resistivity of biomolecular solution, see discussion later. 4) The energy stored in the electrostatic field influences the behaviour of the biomolecular adsorption process in OEWOD surfaces.

The interactions and forces identified can be influenced from the different parameters involved on the biomolecular adsorption process such as isoelectric point and pH from the biomolecular solution, polarity of the surface electrode, potential applied, time exposed to biomolecular adsorption, concentration and surface properties. It has been shown that minimizing the ―actuation‖ time and potential applied, choosing the proper pH and concentration of the biomolecular solution, and selecting the proper electrode polarity and surface, enables the OEWOD system to: (1) minimize biomolecular adsorption or (2) to intentionally immobilize/absorb biomolecules to specific locations determined by the underlying actuation electrode structure.

Electrolytes can be considered as a homogeneously charged unit, and therefore present a described behaviour (net effect). This is not the case for biomolecules, which frequently change the net charge, due to the different interactions (between surface, biomolecules, and solvent); influencing the stability of a biomolecular compact structure. They are also affected by the pH of their surrounding environment and can become more positively or negatively charged due to the loss or gain of protons (H+). One of the intention of this thesis was to favour a compact conformation of the biomolecules at their isoelectric conditions, to describe their behaviour on OEWOD surfaces and consequently take advantage to minimize or 105

increases the biomolecular adsorption on OEWOD surfaces. The experiments with different pH solutions to the isoelectric point of the HRP solution (pH=7.2) offered a possibility to understand the electrostatic forces and charge involved in the biomolecular adsorption process on OEWOD systems and already suggested from Jeong-Yeol Yoon and Robin L. Garrel [Yoon, 2003].

The experiment with both surfaces revealed the importance of the selectivity of the electrode polarities and OEWOD surfaces. While the polarity in the bottom electrode was negative, several experiments showed electrolysis and bubble formation. When the bottom polarity was positively charged, longer adsorption times and high potential were able to absorb HRP, IgG-HRP and DNA-HRP on both surfaces. However, different behaviours and biomolecular adsorption rates of DNA HRP, IgG- HRP and DNA-HRP were determined; see tables 4.3, 4.4, 4.5 in annex B.

The experiments performed with water and PBS as a biomolecular solvent medium confirmed the influence regarding to the interaction (biomolecules-electrolyte) of the ionic strength due to the PBS solution in comparison with water in single or in hybrid OEWOD system. In contrast to Jeong-Yeol Yoon and Robin L. Garrel [Yoon, 2003] we found significant differences between the behaviour of the biomolecular solutions dissolved in water versus dissolved in PBS solution. The contact angles for HRP concentration of 1 and 0.1 mg/ml were shifted to several degrees below to the same concentration for HRP dissolved in water. HRP was more attracted due to the PBS solution to the OEWOD surfaces. That should be the cause to attract the biomolecules to the positive polarized surface, see discussion and conclusion chapter 4.1.4.

Experiments showed in general that the higher the voltage applied between the biomolecular solution and the electrode embedded under the insulating layer the higher the biomolecular adsorption on the Teflon®AF layer on the hybrid layer, which is the layer involved on the actuation and in direct contact with the biomolecular solution.

Decreasing the time during which the potential is applied, low concentration, selecting the electrode polarity properly and - if possible - selecting a pH of the

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biomolecular solution as well as the OEWOD surface should minimize the biomolecular adsorption.

Biomolecules (HRP/DNA/IgG) showed different behaviour as well as different biomolecular adsorption rates depending on the OEWOD surfaces and the conditions already mentioned, presenting a hysteresis coming back in different degrees than the electrolytes. The increased hysteresis observed due to biomolecular adsorption and possible charged distribution could avoid or be addressed with the polarity of the electrodes and the electrical charge as a function of the pH in the solution of the biomolecules.

Durimid 115A-Teflon®AF 1600 and Cellulose Acetate-Teflon®AF surfaces showed different rates of adsorption at the same conditions, leading to think about some dependency of the lower layer on the actuating layer (Teflon®AF). The adsorption amount was relative quantified as a spot on the surface through chemiluminescence reaction, however it was also possible to identify the locations, where more biomolecules were absorbed through the time necessary to rinse it from the surface and a sequence of chemiluminescence detection was performed. After the surfaces were rinsed with PBS on a shaker (0.1 M, pH=7,4), it was observed that in locations with longer biomolecular adsorption time and higher biomolecular (HRP/DNA/IgG) concentration, more time to rinse the biomolecules from the surface was needed, in contrast to locations with shorter adsorption time and lower concentrations.

By constant HRP concentration, changing the potential applied and time adsorption, was observed that the higher the potential applied between the biomolecular solution and the electrode embedded under the insulating layer the higher was the biomolecular adsorption on the surfaces.

Based only on the contact angle (the lower the contact angle the higher the biomolecular adsorption) measured on the OEWOD surface that depends on the pH of the HRP solution and on the polarity of the actuation electrode, it was observed that a specific pH and polarity of the actuation electrode minimize the biomolecular adsorption. Table 4.2 shows the contact angle measured with different pH biomolecular solutions while 20 volts were applied. If it is taken into account that the p.I. of the HRP is 7.2 then it is possible to talk about a relative charge depending on the pH solution in which the HRP was dissolved.

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The adsorption process depends on the surface charge, surface roughness, surface energetic and solution/biomolecules properties. Most surfaces acquire some charge when exposed to ionic solution, in such cases the electrostatic interaction dominates. Although a charged biomolecules is expected to prefer adsorption onto an oppositely charged surface, if the net charge of the biomolecules is zero, i.e. at isoelectric point, the electrostatic interaction between biomolecules and surface should be influenced by the medium where it has dissolved, because the charge distribution on the biomolecules surface is not uniform. However this thesis dealt with biomolecules bigger than BSA and labelled to HRP, which could show a more representative distributed net charge.

In contrast to Jeong-Yeol Yoon et al., this thesis considers the behaviour of the system as a capacitor in cases, when the polymer layer has a considerable thickness to store energy. When the polymers layer is thinner and lower potential is applied, the system could behave as an insulator until the energetic barrier goes through the limits.

In several experiments with applied high potential, high concentrations of the biomolecules, and high adsorption time, dielectric breakdown and electrolysis were observed, which indicated that the OEWOD system behaved as a capacitor. By a potential difference across the biomolecular solution a static electric field was developed across the dielectric layer. This caused the polarization of the surface, which depends on the electric charge on each electrode to the potential difference. The energy stored in the electrostatic field could influences in the behaviour of the biomolecular adsorption process. However, the estimated current capacitances and double-layer charging current suggested by Jeong-Yeol Yoon and Robin L. Garrel [Yoon, 2003] and calculated for this thesis show that the system does not behave as capacitor.

The estimated current capacitances and double-layer charging current calculated in this thesis for the OEWOD system: Durimid 115A-Teflon®AF and Cellulose Acetate- -7 Teflon®AF are: CD-T = 0.076 nF, CCA-T = 0.8476 nF, ID-T = 5.58x10 A and ICA-T =1.3x10-6 A, respectively. The magnitude of current capacitances and double-layer charging current for this system, above calculated suggested corresponds to the

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behaviour as an ―insulator‖. Nevertheless, it is very important to mention here that Jeong-Yeol Yoon et al. [Yoon, 2003], for the earlier calculation, assumed for practicability the distance between electrodes to be the thickness of the layers, the resistivity of the D.I. water and did not consider the electrode shape (droplets contact area to the bottom electrode).

This thesis completely agrees with the suggestion from Sally L. Gras et al. [Gras, 2007], who believe that electrode shape (droplets contact area to the bottom electrode) and distance between electrodes have a significant effect on the distribution of the electric field and degrees of electrowetting, which unfortunately where not taken into account for the estimation of most of the experiments in this thesis. Additionally, the resistivity of the biomolecular solution should be considered. If these factors would have been strictly considered in this thesis, we could explain part of the different patterns as a capacitor with variable capacitances and the influence on the biomolecular adsorption process observed in the experiments.

4.9 Outlook

Further experiments with biomolecules (HRP/DNA/IgG) adsorbed in OEWOD surfaces under the conditions above mentioned should be realized. To confirm the functionalization/immobilization of those biomolecules, corresponding antibodies or target should be bonded to the previous locations, where biomolecules were adsorbed to demonstrate any protocol assay.

It is still not clear, what the influence of the adsorption process of biomolecules in OEWOD by the polymer layer beneath the actuated polymer layer is (Teflon®AF). It is possible to find some energetically answer due to the interface between the layers. This interface should be investigated in a more advanced and detailed manner.

OEWOD is a practical and very attractive tool to perform bioassays. It allows small volumes of liquid to be manipulated and controlled on the microchip. OEWOD is an enabling technology that enables - in combination with the luminescence image analyzer system and the temperature system or any external measurements device - the studies of diverse and interesting problems on surfaces chemistry and biology.

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This includes the development of high throughput ―lab on a chip‖ devices for a wide variety of applications.

To improve the yielding of the NASBA amplification product in this platform, further fundamental investigation of NASBA standard reagents composition tailored to the properties of Teflon®AF is recommended, especially for the enzyme solution. The complex enzyme mix for the amplification could influence the surface adsorption, avoiding at a certain point the enzyme development and consequently induce enzyme inhibition.

To further improve an isothermal RNA analysis assay, different surfaces with tailored surface polarities have to be evaluated, as well as further experiments to optimize the stoichiometric enzyme concentration ratio, taking in account the biomolecular adsorption of the surface will be recommended.

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6. List of abbreviation and symbols

OEWOD Open Electrowetting On Dielectrics EWOD Electrowetting On Dielectrics Lab-on-a-Chip Laboratory on a Chip CCD Charged Coupled Device PBS Phosphate buffered saline HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid KCl Potassium Chloride PE B-Phycoerythrin BSA Bovine Serum Albumin GOD Glucose Oxidase HRP (POD) Horseradish Peroxidase IgG-HRP Rabbit Anti-Mouse Immunoglobulin Horseradish Peroxidase DNA-HRP Deoxyribonucleic acid-Horseradish Peroxidase NASBA Nucleic Acid Sequence Based Amplification MB Molecular Beacon ITO Indium Titanium Oxide BSA Bovine Serum Albumin pH Co-logarithm of the activity of dissolved hydrogen ions (H+) D.I. Deionised water DC Direct current AC Alternating current EVD Electrochemical Vapor Deposition BioMEMs Bio-ElectroMechanical Systems µTAS Micro Total Analysis System LSCM Laser Scanning Confocal Microscopy FC-75 Fluorinate liquid (perfluoro-2-buthyltetrahydrofuran) DSA1 Drop Shape Analysis programs 1 DSA2 Drop Shape Analysis programs 2 rmp Revolutions per minute E1-E6 Isoenzymes M Molar wt (w) Weight cm Centimetre s Second ms Milisecond u/mg Units per milligram Ǻ Anstrong v Volumen L Liquid phase of the droplet S Solid phase of the substrate V gas/vapor phase of the ambient µm Micrometer F Faraday mm2 Square millimetre l Micrometer nm Nanometer ml/min Flow rate mm Milimeter Km Michaelis Contants mRNA Messenger Ribocinucleic Acid

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p.I. Isoelectric Point 3-D Three dimensional 2-D Two dimensional kDa kiloDaltons J Joules K Kelvin mS/cm Milisiemens/centimeter (conductivity) m cm Megaohms/centimetre (resistance) nF Nanoamperes mA Milliamperes N Newton t Time m Meter T Temperature G Gibbs Energy A Ampere (intensity) v Volts P Pressure H Enthalpy S Entropy PEO Polyethylene oxide bp Base pair DMSO Dimethyl sulfoxide FAM 6-carboxy –fluorescein AMV-RT Avian myeloblastosis virus reverse transcriptase Dabcyl 4-(4‗-dimethylaminophenylazo) benzoic acid PEG Polyethylene glycol mRNA messenger RNA PCR Polymerase chain reaction DCAS Dynamic Contact Angle System DAC Digital Analogical Converter ELISA Enzyme-Linked ImmunoSorbent Assay DHS Droplet Handling System LAS-3000 Luminescent Image Analyzer AIDA Advanced Image Data Analyzer Surface tension

o Surface Tension when there is no applied electric Ɛ o Permittivity of a free space Ɛ i Dielectric constant C Capacitance V Potential

SL Solid-liquid interface surface tension

SG Solid-gas interface surface tension

SV Solid-vapour interfacial energy

SL Solid-liquid interfacial energy

LG Liquid-gas interface surface tension Contact angle

o Contact angle when the electric field across interfacial layer is zero ΔWSLV Energy per unit area of the solid and liquid surfaces Reversibility

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ads Change invoked by the adsorption process

Gel Gibbs energy Gel to invoke a charge distribution surface potential

adsGel Gibbs energy of the change of charge distribution surface potential

adsH Enthalpy change invoked by the adsorption process

adsS Entropy change invoked by the adsorption process G Gibbs energy adsorption of the changes hydration ads hydr G Gibbs energy adsorption of the changes dispersion interaction ads disp A Hamaker constant

A132 Hamaker constant for the interaction between the flat sorbent(1) and the spherical protein molecule(2) across the (aqueous) medium(3) H+ Protons ' Surface potential 0 ' 0 Surface charge density Adsorbed mass per unit sorbent surfaces area (ceq) Concentration of the bulk solution cb Protein concentration cs Protein concentration k2 Transport rate constant RT R Gas constant, Temperature H Distance d Distance between electrodes A Electrode area Rs Solution resistance Cd Double layer capacitance I Double-layer charging current Tg Glass Temperature g/dm-3 Gram / decimetre Qc Quantum efficiency Pa Pascal M Mega PTFE Polytetrafluoroethylene TFE Tetrafluoroethylene PDD 2,2-bistrifluoromethyl-4,5-difluoro-1,3-dioxole

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7. Figures and Tables

Cover: OEWOD surfaces for biomolecular adsorption experiments.

Table 1.1: Chronological development of OEWOD platform related to biomolecular adsorption.

Figure 2.1: Alteration of the static contact angle as a function of time (Picture taken from Krüss website) [Krüss].

Figure 2.2: Measuring advancing angles (Picture taken from Krüss) [Krüss].

Figure 2.3: Measuring receding angles (Picture taken from Krüss) [Krüss].

Figure 2.4: Schematic presentation of the protein adsorption process (Picture taken from Norde, 2000) [Norde, 2000].

Figure 2.6: Schematic presentation of the chemiluminescence reaction (Chemical structures layout in Symyx Draw) (ECL Plus western Blotting Reagents RPN2132, from Amersham Biosciences).

Figure 3.2: Chemical structure of Teflon®AF 1600 (Chemical structure layout in ADC/ChemSketch) [Ho, 2003].

Table 3.1: Material properties of Teflon®AF 1600.

Figure 3.3: Chemical structure of Cellulose Acetate (Chemical structure layout in Symyx Draw).

Table 3.2: Material properties of Cellulose Acetate. Figure 3.4: Monomer structure of polyamidic acid (polyimide) (Chemical structure layout in ADC/ChemSketch) [Fujifilm Electronic Material].

Table 3.3: Material properties of Durimid 115A.

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Figure 3.5: Chemical structure of 1, 2-epoxy ring (Chemical structure layout in ADC/ChemSketch).

Figure 3.6: Chemical structure of SU-8. The basic SU-8 molecule, note the 8 epoxy groups (Chemical structure layout in ADC/ChemSketch).

Table 3.4: Material properties of SU-8 [Angelopolous, 2001].

Figure 3.7: HRP Horseradish Peroxidase, picture taken from [Veitch, 2004].

Table 3.5: DNA string size (ECL™).

Figure 3.8: Principles of the ECL direct nucleic acid labelling and detection system (Layout in CorelDraw) [GE Healthcare Amersham ECL™].

Table 3.6: NASBA reagents: PreTec HPV Proofer Kit and diagram of the real time amplification [NorChip AS].

Figure 3.9: Sketch of the Molecular beacon principle (picture taken from Marras et al.) [Marras, 2003].

Figure 3.10: Schematic setup of the static biomolecular adsorption experiments (layout in Corel draw).

Figure 3.11: Close up of set up of the statics experiments on OEWOD surfaces.

Figure 3.12: Set up of statics experiments on OEWOD surfaces and voltage power supplier.

Figure 3.13: Dynamic Contact Angle System (DCAS) software [Herberth, 2006] and the DSA10 contact angle measuring system [Krüss].

Figure 3.14: Electronic circuitry hardware and operation window of the actuation software (DSH). Picture taken from [Herberth, 2006].

Figure 3.15: OEWOD surfaces for biomolecular adsorption experiments and array layout.

Figure 3.16: OEWOD microchip consisting of interdigitate electrodes with optimised shape for actuation.

Figures 3.17: a) View of the OEWOD microchip, b) OEWOD microchip inside the Petri disc, heater and temperature sensor cover with aluminium foil to avoid fluorescence background, c) reaction window of Plexiglas and glass cover of the OEWOD microchip and d) close up view of the experimental set up.

Figure 3.18: OEWOD microchip implemented into the microfluidics system consisting of a heating element, temperature sensor, reaction chamber and cover plate for quantitative analysis of RT-NASBA.

Figure 3.19: Temperature device control and software of temperature device control.

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Figure 3.20: Luminescent Image Analyzer LAS-3000 and Image reader software.

Table 4.1: OEWOD surfaces Characterization.

Figure 4.1: Contact angle changes for 10 µl droplets of HEPES, PBS, KCl and HRP 10-5 M as a function vs. potential applied from 0 volts to 200 volts.

Figure 4.2: Contact angle (degrees) vs. potential applied (volts) for HRP at concentrations from 1 to 10-5 mg/ml dissolved in water on Teflon®AF 6% OEWOD surface.

Figure 4.3: Contact angle (degrees) vs. potential applied (volts) for GOD at concentrations from 1 to 10-5 mg/ml dissolved in water on Teflon®AF 6% OEWOD surface.

Figure 4.4: Contact angle (degrees) vs. potential applied (volts) for HRP at concentrations from 1 to 10-5 mg/ml dissolved in water on Durimid 115A–Teflon®AF 6% OEWOD surface.

Figure 4.5: Contact angle (degrees) vs. potential applied (volts) for GOD at concentrations from 1 to 10-5 mg/ml dissolved in water on OEWOD surface Durimid 115A–Teflon®AF 6%.

Figure 4.6: Contact angle (degrees) vs. potential applied (volts) for HRP at concentrations from 1 to 10-5 mg/ml dissolved in PBS 0.1 M on Durimid 115A– Teflon®AF 6% OEWOD surface.

Figure 4.7: Contact angle (degrees) vs. potential applied (volts) for GOD at concentrations from 1 to 10-5 mg/ml dissolved in PBS 0.1 M on Durimid 115A – Teflon®AF 6% OEWOD surface.

Figure 4.8: Dependence on the electrode polarity and pH of HRP on SU-8- Teflon®AF 6%. HRP1D+: pH=7.4, counter electrode (+); HRP1D-: pH=7.4, counter electrode (-); HRP2D+: pH~5-6, counter electrode (+); HRP2D-: pH~5-6, counter electrode (-); HRP3D+: pH~9-10, counter electrode (+); HRP3D-: pH~9-10, counter electrode (-).

Figure 4.9: Dependence on the electrode polarity and pH of HRP on Durimid 115A- Teflon®AF 6%. HRP1D+: pH=7.4, counter electrode (+); HRP1D-: pH=7.4, counter electrode (-); HRP2D+: pH~5-6, counter electrode (+); HRP2D-: pH~5-6, counter electrode (-); HRP3D+: pH~9-10, counter electrode (+); HRP3D-: pH~9-10, counter electrode (-).

Table 4.2 pH and polarity dependence.

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Figure 4.11: Array for biomolecular adsorption experiment on OEWOD surface, 10 µl droplet of 1 mg/ml (HRP) POD are deposited at different locations under different conditions of potential applied and time adsorption.

Figure 4.12: Chemiluminescence detection on a hypersensitive film.

Figure 4.13: Distribution of the samples. Adsorption time vs. (HRP) POD concentration.

Figure 4.14: Detection of chemiluminescence from the left side of the OEWOD surface on a hypersensitive film.

Figure 4.15A: AIDA % relative quantification of biomolecular adsorption of chemiluminescence detection of HRP5 and HRP4B on Cellulose Acetate-Teflon®AF OEWOD surface.

Figure 4.16A: AIDA % relative quantification of biomolecular adsorption of chemiluminescence detection of HRP5 and HRP4B on Durimid 115A-Teflon®AF OEWOD surface.

Figure 4.17: Chemiluminescence detection of IgG-HRP, 10-3 mg/ml at different time adsorption and different pH solution.

Figure 4.18: column left; pH=5, 10 µl IgG-HRP, 10-3 mg/ml after 20 min washing with PBS

Figure 4.19: Chemiluminescence detection after 30 min. washing with PBS.

Figure 4.20: Chemiluminescence detection after 45 min. washing with PBS.

Figure 4.21: Chemiluminescence detection after 55 min. washing with PBS.

Figure 4.22: Chemiluminescence detection after 80 min. washing with PBS.

Figure 4.23A: % relative quantification of biomolecular adsorption of chemiluminescence detection of IgG-HRP on Cellulose Acetate-Teflon®AF OEWOD surface.

Figure 4.24A: % relative quantification of biomolecular adsorption of chemiluminescence detection of IgG-HRP on Durimid 115A-Teflon®AF OEWOD surface.

Figure 4.25A: % relative quantification of biomolecular adsorption of chemiluminescence detection of DNA-HRP on Cellulose Acetate-Teflon®AF OEWOD surface.

Figure 4.26A: % relative quantification of biomolecular adsorption of chemiluminescence detection of DNA-HRP on Durimid 115A-Teflon®AF OEWOD surface.

Figure 4.27: Sketch of merging protocol and OEWOD microchip inside into LAS- 3000 (Schematic picture left side was taken from Herberth 2006). Right side:

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sequence of pictures record from the beginning of the reaction in LAS-3000 and Droplet Handling System software.

Figure 4.28: OEWOD microchip inside the luminescence imaging system LAS-3000.

Figure 4.29: Close up of OEWOD microchip and software of the luminescence imaging system LAS-3000.

Figure 4.31: HRP and Luminol: intensity-Background [LAU] vs. Time [s] of chemiluminescence detection on Durimid 115A-Teflon®AF surface at different pH´s HRP solution, evaluated with AIDA.

Figure 4.32: Sequence of pictures from the luminescence image analyser (LAS- 3000), reads the emitted chemiluminescence every 15 sec. from the HRP and Luminol reaction on Cellulose Acetate-Teflon®AF surface at different pH´s HRP Solution.

Figure 4.33: HRP and Luminol. Intensity-Background [LAU] vs. Time [s] of chemiluminescence detection on Cellulose Acetate-Teflon®AF OEWOD surface at different pH´s HRP solution evaluated with AIDA.

Table 4.6: Linear equation of the chemiluminescence intensities of HRP on OEWOD surfaces.

Table 4.7: Shows the contact angles and pH characterization of the PreTec HPV Proofer Kit components on OEWOD surfaces.

Table 4.8: Contact angles characterization of NASBA standard reagents under EWOD effect on Cellulose Acetate–Teflon®AF surface.

Figure 4.34: Show the contact angles of Mastermix solution as a function of the potential applied on OEWOD surfaces: Cellulose Acetate–Teflon®AF 2% and Durimid 115A–Teflon®AF 2%.

Figure 4.35: Shows the contact angles of the enzyme solution as a function of the potential applied on OEWOD surfaces: Cellulose Acetate–Teflon®AF 2% and Durimid 115A–Teflon®AF 2%.

Figure 4.36: Shows the contact angles of NASBA reaction as a function of the potential applied on OEWOD surfaces: Cellulose Acetate–Teflon®AF 2% and Durimid 115A–Teflon®AF 2%.

Figures 4.37: Shows mixing protocol: a sequence of the basic microfluidics operations on OEWOD microchip for RT-NASBA for HPV16 detection.

Figure 4.38: the OEWOD microchip inside the luminescence image analyzer system from Fujifilm (LAS-3000).

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Figures 4.40 and 4.41: Right side: Amplification of HPV16 by Real-Time NASBA protocol on OEWOD microchip system. Left side: Amplification of HPV16 by Real- Time NASBA protocol in Lightcycler 1.2 PCR system device.

Figure B1: Advancing contact angle characterization of the OEWOD surface Cellulose Acetate-Teflon® AF 2%with D-I water for Adsorption experiment.

Figure B2: Receding contact angle characterization of the OEWOD surface Cellulose Acetate-Teflon® AF 2% with D-I water for Adsorption experiment.

Figure B3: Advancing contact angle characterization of the OEWOD surface Durimid 115A-Teflon® AF 2% with D-I water for Adsorption experiment.

Figure B4: Receding contact angle characterization of the OEWOD surface Durimid 115A-Teflon® AF 2% with D-I water for Adsorption experiment.

Figure B5: Section analysis 1 of subtract Pyrex glass structured with 10 nm titanium and 25 nm Platinum.

Figure B6: Section analysis 2 of subtract Pyrex glass structured with 10 nm titanium and 25 nm Platinum.

Figure B7: Roughness analysis 1 of subtract Pyrex glass structured with 10 nm titanium and 25 nm Platinum.

Figure B8: Roughness analysis 2 of subtract Pyrex glass structured with 10 nm titanium and 25 nm Platinum.

Figure B9: 3D scale of the roughness Analysis of subtract Pyrex glass structured with 500nm Titanium and 20 mm Platinum.

Figure B10: Roughness Analysis of subtract Pyrex glass structured with 10 Titanium, 25 mm Platinum and Cellulose Acetate-Teflon® AF.

Figure B11: Roughness Analysis of subtract Pyrex glass structured with 10 nm Titanium, 20 mm Platinum and Durimid 115A-Teflon® AF.

Figure B12: Confocal microscopic image. Adsorption experiment, a) 1 min. adsorption time of B-Phycoerythrin on annealed OEWOD surface, b) 5 min. adsorption time.

Figure B13: Confocal microscopic image. Adsorption experiment, a) 1 min. adsorption time of B-Phycoerythrin on unannealed OEWOD surface, b) 5 min adsorption time.

Figure B14: Confocal microscopic image. Adsorption experiment, a) 1 min. adsorption time of B-Phycoerythrin on unannealed OEWOD surface, b) 5 min adsorption time. 130

Figure B15: Confocal microscopic image. Adsorption experiment, a) 1 min. adsorption time of B-Phycoerythrin on unannealed OEWOD surface, b) 5 min. time adsorption time.

Figure B16: Confocal microscopic image. Adsorption experiment, a) 1 min. adsorption time of B-Phycoerythrin on annealed OEWOD surface, b) 5 min. adsorption time.

Figure B17: Confocal microscopic image. Adsorption experiment, a) 1 min. adsorption time of B-Phycoerythrin on unannealed OEWOD surface, b) 5 min adsorption time.

Table 4.3: AIDA evaluation, % relative quantification of the biomolecular adsorption of HRP4B and HRP5 on Cellulose Acetate-Teflon® AF and Durimid- Teflon® AF surfaces.

Table 4.4: AIDA evaluation, % relative quantification of the biomolecular adsorption of IgG-HRP on Cellulose Acetate-Teflon® AF and Durimid- Teflon® AF surfaces.

Table 4.5: AIDA evaluation, % relative quantification of the biomolecular adsorption of DNA-HRP on Cellulose Acetate-Teflon® AF and Durimid- Teflon® AF surfaces.

Figure 4.15B: Chemiluminescence detection of HRP5 and HRP4B absorbed on OEWOD Cellulose Acetate-Teflon®AF surface.

Figure 4.16B: Chemiluminescence detection of HRP5 and HRP4B absorbed on OEWOD Durimid115A-Teflon®AF surface.

Figure 4.23B: LAS-3000 Chemiluminescence detection of IgG-HRP on Cellulose Acetate-Teflon®AF OEWOD surface.

Figure 4.24B: LAS-3000 Chemiluminescence detection of IgG-HRP on Durimid 115A-Teflon®AF OEWOD surface.

Figure 4.25B: % relative quantification of biomolecular adsorption of chemiluminescence detection of HRP-DNA on Cellulose Acetate-Teflon®AF OEWOD surface.

Figure 4.26B: % relative quantification of biomolecular adsorption of chemiluminescence detection of HRP-DNA on Durimid 115A-Teflon®AF OEWOD surface.

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Annex A Measuring methods and Methods of evaluating the drop shape

Measuring methods The Krüss contact angle device and the software Drop Shape Analysis applied basically four algorithms. The sessile method and dynamic Wilhelmy method were used in this work for the determination of the contact angle of biomolecules in OEWOD surfaces.

Sessile method: Sessile drop method is an optical contact angle method. This method is used to estimate wetting properties of a localized region on a solid surface. Angle between the baseline of the drop and the tangent at the drop boundary is measured. Ideal for curved samples or where one side of the sample has different properties than the other side.

Dynamic Wilhelmy method: A method for calculating average advancing and receding contact angles on solids of uniform geometry. Both sides of the solid must have the same properties. Wetting force on the solid is measured as the solid is immersed in or withdrawn from a liquid of known surface tension.

Single Fiber Wilhelmy method: Dynamic Wilhelmy method applied to single fibers to measure advancing and receding contact angles.

Powder Contact Angle method: Enables measurement of average contact angle and adsorption speed for powders and other porous materials. Change of weight as a function of time is measured.

Methods of evaluating the drop shape

The basis for the determination of the contact angle is the image of the drop on the surface. In the DSA1 program the actual drop shape and the contact line (baseline) with the solid are first determined by the analysis of the grey level values of the image pixels. To describe this more accurate, the software calculates the root of the secondary derivative of the brightness levels to receive the point of greatest changes 132

of brightness. The found drop shape is adapted to fit a mathematical model which is then used to calculate the contact angle. The various methods of calculating the contact angle therefore differ in the mathematical model used for analyzing the drop shape. Either the complete drop shape, part of the drop shape or only the area of phase contact are evaluated. All methods calculate the contact angle as tang at the intersection of the drop contour line with the solid surface line (base line).

Tangent method 1 The complete profile of a sessile drop is adapted to fit a general conic section equation. The derivative of this equation at the intersection point of the contour line with the baseline gives the slope at the 3-phase contact point and therefore the contact angle. If dynamic contact angles are to be measured, this method should only be use when the drop shape is not distorted too much with the needle. This method was used as standard to evaluate the biomolecular adsorption in this work. As before mentioned the droplet shape was not distorted from the needle (electrode).

Tangent method 2 That part of the profile of a sessile drop which lies near the baseline is adapted to fit a polynomial function of the type (y=a + bx + cx0,5 + d/lnx + e/x2) The slope at the 3- phase contact point at the baseline and from it the contact angle are determined using the iteratively adapted parameters. This function is the result of numerous theoretical simulations. The method is mathematically accurate, but is sensitive to distortions in the phase contact area caused by contaminants or surface irregularities at the sample surface.

As only the contact area is evaluated, this method is also suitable for dynamic contact angles. Nevertheless, this method requires an excellent image quality, especially in the region of the phase contact point.

Height-width method In this method the height and width of the drop shape are determined. If the contour line enclosed by a rectangle is regarded as being a segment of a circle, then the contact angle can be calculated from the height-width relationship of the enclosing

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rectangle. The smaller drop volume, the more accurate the approximations for smaller drops are more similar to the theoretically assumed spherical cap form. As the drop height cannot be determined accurately when the needle is still in the drop, the height-width method is not suitable for dynamic drops. This method also has the disadvantage that the drops are regarded as being symmetrical, so that the same contact angle is obtained for both sides, even when differences between the two sides can be seen in the actual drop image.

Circle fitting method As in the height-width method, in this method the drop contour is also fitted to a segment of a circle. However, the contact angle is not calculated by using the enclosing rectangle, but by fitting the contour to a circular segment function. The same conditions apply to the use of this method as to the height-width method with the difference that a needle remaining in the drop disturbs the result far less.

Young- Laplace (sessile drop fitting) The most complicated, but also the theoretically most exact method for calculating the contact angle is the YOUNG-LAPLACE fitting. In this method the complete drop contour is evaluated; the contour fitting includes a correction which takes into account the fact that it is not just interfacial effects which produce the drop shape, but that the drop is also distorted by the weight of the liquid it contains. After the successful fitting of the YOUNG-LAPLACE Equation the contact angle is determined as the slope of the contour line at the 3-phase contact point.

If the magnification scale of the drop image is known (determined by using the syringe needle in the image) then the interfacial tension can also be determined; however, the calculation is only reliable for contact angles above 30°. Moreover, this model assumes a symmetric drop shape. There for it cannot be used for dynamic contact angles where the needle remains in the drop.

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Annex B Characterization of OEWOD surfaces

Advancing and Receding contact angles of OEWOD surfaces for biomolecular adsorption

Figure B1: Advancing contact angle characterization of the Figure B2: Receding contact angle characterization of the OEWOD surface Cellulose Acetate-Teflon® AF 2%with D-I OEWOD surface Cellulose Acetate-Teflon® AF 2% with D-I water for Adsorption experiment. water for Adsorption experiment.

Figure B3: Advancing contact angle characterization of the Figure B4: Receding contact angle characterization of the OEWOD surface Durimid 115A-Teflon® AF 2% with D-I water OEWOD surface Durimid 115A-Teflon® AF 2% with D-I for Adsorption experiment. water for Adsorption experiment.

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Atomic Force Microscopy analysis of OEWOD surfaces

Figure B5: Section analysis 1 of subtract Pyrex glass Figure B6: Section analysis 2 of subtract Pyrex glass structured with 10 nm titanium and 25 nm Platinum. structured with 10 nm titanium and 25 nm Platinum.

Figure B7: Roughness analysis 1 of subtract Pyrex glass Figure B8: Roughness analysis 2 of subtract Pyrex glass structured with 10 nm titanium and 25 nm Platinum. structured with 10 nm titanium and 25 nm Platinum.

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Figure B9: 3D scale of the roughness Analysis of subtract Pyrex glass structured with 10nm Titanium and 25 mm Platinum.

Figure B10: Roughness Analysis of subtract Pyrex glass Figure B11: Roughness Analysis of subtract Pyrex glass structured with 10 Titanium, 25 mm Platinum and Cellulose structured with 10 nm Titanium, 25 mm Platinum and Durimid Acetate-Teflon® AF. 115A-Teflon® AF.

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Laser Scanning Confocal Microscopy of OEWOD surfaces annealed and unannealed

B-Phycoerythrin on Durimid 115A- Teflon® AF

a) a)

Figure B13: Confocal microscopic image. Adsorption Figure B12: Confocal microscopic image. Adsorption experiment, a) 1 min. adsorption time of B-Phycoerythrin on experiment, a) 1 min. adsorption time of B-Phycoerythrin on unannealed OEWOD surface, b) 5 min adsorption time. annealed OEWOD surface, b) 5 min. adsorption time.

B-Phycoerythrin on SU-8 - Teflon® AF

a) a)

b) b)

Figure B14: Confocal microscopic image. Adsorption Figure B15: Confocal microscopic image. Adsorption experiment, a) 1 min. adsorption time of B-Phycoerythrin on experiment, a) 1 min. adsorption time of B-Phycoerythrin on unannealed OEWOD surface, b) 5 min adsorption time. unannealed OEWOD surface, b) 5 min. time adsorption time.

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B-Phycoerythrin on Cellulose Acetate- Teflon® AF

a) a)

b) b) Figure B16: Confocal microscopic image. Adsorption Figure B17: Confocal microscopic image. Adsorption experiment, a) 1 min. adsorption time of B-Phycoerythrin on experiment, a) 1 min. adsorption time of B-Phycoerythrin on annealed OEWOD surface, b) 5 min. adsorption time. unannealed OEWOD surface, b) 5 min adsorption time.

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% Chemiluminescence Counter Biomolecule Time Intensity- biomolecular Electrode Adsorbed Adsorpt Comments Adsorption*** (bottom) HRP5 and ion HRP4B (min.) Potential Contact angle Applied C.A.I * – C.A.F** Cellulose Acetate Durimid115A polarity (volts) - Teflon® AF 2% -Teflon® AF 2% CA-T D-T 20 118-110 112-99 0.97 0.73 + HRP5 1 20 118-105 117-114 1.27 0.73 + HRP5 2 20 116-106 108-102 1.36 1.05 + HRP5 5 40 115-87 116-107 0.97 2.14 + HRP5 1 40 117-85 105-101 1.74 1.19 + HRP5 2 40 116--- 95-83 1.24 1.66 + HRP5 5 Electrolysis/ 20 113-111 108-106 10.30 5.87 + HRP4B 1 20 112-103 103-101 9.69 5.67 + HRP4B 2 20 94-85 107-101 9.26 6.54 + HRP4B 5

40 110-85 106-105 10.59 6.41 + HRP4B 1 40 114-98 107-103 12.68 4.91 + HRP4B 2 40 113--- 109-101 2.33 4.46 + HRP4B 5 Electrolysis/ 20 117-86 86-83 0.88 0.18 - HRP5 1 20 117-106 110-106 3.38 0.26 - HRP5 2 20 117-106 114-105 2.53 0.99 - HRP5 5 40 114-84 103-93 1.51 1.69 - HRP5 1 Electrolysis/ Electrolysis 40 119---- 1.23 2.31 - HRP5 2 Electrolysis/ Electrolysis 40 120---- 107-94 0.75 0.54 - HRP5 5 Electrolysis/ Electrolysis 20 110-103 104-102 8.81 6.43 - HRP4B 1 20 110-107 105-103 8.38 6.32 - HRP4B 2 20 115-109 109-104 10.13 3.05 - HRP4B 5 40 104---- 109-104 9.95 6.59 - HRP4B 1 40 110--- 106-103 12.53 7.26 - HRP4B 2 40 ------101-94 0.53 6.46 - HRP4B 5 Electrolysis/ 111-110 112-111 10.99 1.65 HRP4B 1 107-106 109-108 11.76 0.55 HRP4B 2 111-107 114-111 10.42 0.70 HRP4B 5 113-113 116-115 2.39 0.17 HRP5 1 117-116 120-119 1.34 0.18 HRP5 2 116-113 118-115 0.69 0.19 HRP5 5

*C.A.I; Initial contact Angle (degrees), ** C.A.F; final contact Angle (degrees), *** Evaluation of the chemiluminescence image with AIDA software, CA-T; Cellulose Acetate-Teflon® AF surface, D-T; Durimid- Teflon® AF surface

Table 4.3: AIDA evaluation, % relative quantification of the biomolecular adsorption of HRP4B and HRP5 on Cellulose Acetate- Teflon® AF and Durimid- Teflon® AF surfaces.

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% Chemiluminescence Counter Biomolecule Intensity- biomolecular Electrode Adsorbed Comments Adsorption*** (bottom) IgG-HRP Potential Contact angle Applied C.A.I * – C.A.F** Cellulose Acetate Durimid115A Polarity *** /****123 (volts) - Teflon® AF 2% -Teflon® AF 2% CA-T D-T 20 116-110 117-112 0.96 0.93 + 1 20 120-115 120-114 1.53 1.97 + 1 20 120-115 120-113 0.38 0.04 + 1 20 115-110 116-112 3.91 3.49 - 1 20 119-115 116-112 2.30 0.91 - 1 S. Electrolysis 20 120-117 120-115 0.00 0.02 - 1 S. Electrolysis 40 118-92 118-96 3.34 2.27 + 1 S. Electrolysis 40 120-99 119-98 2.16 2.12 + 1 40 121-95 125-95 0.10 0.01 + 1

40 118-97 114-97 2.06 0.13 - 1 /Electrolysis 40 119-100 118-98 0.06 0.09 - 1 /Electrolysis 40 119-95 118-107 0.04 0.01 - 1 /Electrolysis 20 117-115 117-111 0.19 0.11 + 2 20 120-114 117-112 2.49 4.90 + 2 20 117-116 110-104 0.14 0.03 + 2 20 118-112 115-98 0.32 0.03 - 2 Electrolysis 20 119-114 118-97 1.47 0.19 - 2 Electrolysis 20 120-114 119-95 0. 0 0.00 - 2 Electrolysis 40 116-92 118-99 8.40 5.28 + 2 Electrolysis/ 40 119-92 118-98 1.62 2.44 + 2 Electrolysis 40 121-101 120-98 0.10 0.03 + 2 40 119-94 119-99 0.63 0.03 - 2 Electrolysis 40 119-92 119-90 0.51 0.03 - 2 Electrolysis 40 120-90 119-96 0.0 0.01 - 2 Electrolysis 20 117-108 115-110 1.64 4.44 + 3 20 119-114 119-113 1.16 10.84 + 3 20 116-108 120-115 0.02 0.42 + 3 20 119-114 120-115 6.85 13.83 - 3 /Electrolysis 20 119-113 118-113 1.50 3.00 - 3 /Electrolysis 20 120-116 120-115 0.10 0.00 - 3 /Electrolysis 40 117-97 118-102 1.88 1.08 + 3 40 119-98 119-100 0.92 5.27 + 3 40 120-100 121-99 0.19 0.33 + 3 40 116-90 119-95 9.14 1.09 - 3 /Electrolysis 40 119-95 119-95 1.84 0.01 - 3 /Electrolysis 40 121-96 120-95 0.00 0.00 - 3 /Electrolysis 0 (6min) 1.06 2 0 (10min) 1.24 2

*C.A.I; Initial contact Angle (degrees), ** C.A.F; final contact Angle (degrees), *** Evaluation of the chemiluminescence image with AIDA software after 10 min. the washing step. *** concentration, the sock solution IgG-HRP were diluted at #: 1:10, #‘: 1:100, #‘‘: 1:1000. ****123 pH of the PBS 0,1M solution, 1 (pH=5), 2 (pH=7.2) and 3 (pH=10). CA-T; Cellulose Acetate-Teflon® AF, D-T; Durimid - Teflon® AF

Table 4.4: AIDA evaluation, % relative quantification of the biomolecular adsorption of IgG-HRP on Cellulose Acetate-Teflon® AF and Durimid- Teflon® AF surfaces.

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% Chemiluminescence Counter Time Biomolecule Intensity- biomolecular Electrode Exposed Adsorbed Comments Adsorption*** (bottom) (Min.) DNA-HRP Potential Contact angle Applied C.A.I * – C.A.F** Cellulose Acetate Durimid115A Polarity DNA/DNA (volts) - Teflon® AF 2% -Teflon® AF 2% CA-T2% / D-T2% CA-T D-T

20 116-113 118-113 5.85 0.09 + 1 DNA 20 114-108 118-113 2.41 7.13 + 2 DNA 20 118-115 117-115 2.43 0.04 + 5 DNA 20 116-109 117-110 2.23 0.03 - 1 DNA 20 116-111 117-110 7.26 0.17 - 2 DNA 20 120-117 118-110 6.91 1.74 - 5 DNA 40 116-108 117-97 3.06 2.98 + 1 DNA 40 117-107 118-95 4.54 9.75 + 2 DNA 40 117-102 116-98 4.30 3.90 + 5 DNA 40 113-104 116-92 3.30 0.04 - 1 DNA Electrolysis 40 117-104 117-99 3.11 9.41 - 2 DNA Electrolysis 40 118-103 116-94 10.54 8.92 - 5 DNA Electrolysis 60 118-106 118-85 5.01 6.66 + 1 DNA 60 117-109 116-84 7.56 9.81 + 2 DNA 60 116-105 115-87 9.86 10.65 + 5 DNA 60 116-105 117-84 1.06 2.99 - 1 DNA Electrolysis 60 114-105 116-85 2.10 7.25 - 2 DNA Electrolysis 60 117-105 118-86 6.75 8.06 - 5 DNA Electrolysis 20 114-111 118-101 0.05 0.51 + 1 DNA 20 117-114 118-109 0.03 0.13 + 2 DNA 20 116-113 119-117 1.20 0.54 + 5 DNA 20 116-110 118-113 0.06 0.37 - 1 DNA 20 118-112 119-113 0.02 0.20 - 2 DNA 20 118-111 119-111 0.02 2.49 - 5 DNA 40 116-95 118-103 0.59 0.03 + 1 DNA 40 118-101 117-103 1.07 0.04 + 2 DNA 40 118-94 119-102 0.19 0.02 + 5 DNA 40 118-92 117-94 0.53 0.00 - 1 DNA 40 118-92 117-97 0.49 0.94 - 2 DNA 40 118-94 119-94 0.94 2.05 - 5 DNA 60 116-90 117-83 1.70 0.34 + 1 DNA 60 118-89 118-80 1.42 0.60 + 2 DNA 60 118-85 117-86 0.10 0.00 + 5 DNA 60 118-80 119-81 0.31 0.01 - 1 DNA Electrolysis 60 119-84 119-81 1.49 0.61 - 2 DNA Electrolysis 60 118-82 118-82 1.53 1.50 - 5 DNA Electrolysis

*C.A.I; Initial contact Angle (degrees),** C.A.F; final contact Angle (degrees), *** Evaluation of the chemiluminescence image with AIDA software, CA-T; Cellulose Acetate-Teflon® AF surface, D-T; Durimid- Teflon® AF surface. DNA-HRP (DNA) is 1ng/ml and DNA-HRP (DNA‘) is 0,1 ng/ml diluted.

Table 4.5: AIDA evaluation, % relative quantification of the biomolecular adsorption of DNA-HRP on Cellulose Acetate-Teflon® AF and Durimid- Teflon® AF surfaces.

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Figure 4.15B: Chemiluminescence detection of HRP Figure 4.16B: Chemiluminescence detection of HRP absorbed on OEWOD Cellulose Acetate-Teflon®AF absorbed on OEWOD Durimid115A-Teflon®AF surface surface These pictures were performed in hypersensitive films, which were scanned to digitalize. Therefore the quality of those figures can not to be improved.

Figure 4.23B: LAS-3000 Chemiluminescence detection of IgG- Figure 4.24B: LAS-3000 Chemiluminescence detection of IgG- HRP on Cellulose Acetate-Teflon®AF OEWOD surface HRP on Durimid 115A-Teflon®AF OEWOD surface

Figure 4.25B: % relative quantification of biomolecular Figure 4.26B: % relative quantification of biomolecular adsorption of chemiluminescence detection of HRP-DNA on adsorption of chemiluminescence detection of HRP-DNA on Cellulose Acetate-Teflon®AF OEWOD surface Durimid 115A-Teflon®AF OEWOD surface

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Annex C The double-layer charging current

Assuming the OEWOD system as capacitor as proposed from Jeong-Yeol Yoon et al. [Yoon, 2003]. The double-layer charging current for practicability can be estimated from the capacitance of Cellulose Acetate–Teflon®AF and Durimid 115A– Teflon® AF systems as follows: A C 0 i d And 1 1 i C Ci Where: C, Capacitance -12 -1 Ɛ o permittivity of a free space 8.85x10 Fm

Ɛ i Dielectric constant of the material

Material Ɛ i Dielectric constant

Cellulose Acetate 6

Durimid 115° 3.1

Teflon®AF 1.93

A is the electrode area = 11.3 mm2 (using the contact area to the electrode of a 10 l droplet of sample and measured with the software Dynamic Contact Angle System (DCAS) modified and the Droplet Handling System (DHS) development by Herberth 2006 [Herberth, 2006], see chapter 3). d is the distance between electrodes. However, this study is completely agrees with the suggestion from Sally L. Gras et al. [Gras, 2007], who believe that electrode shape (droplets contact area to the bottom electrode) and distance between electrodes have a significant effect on the distribution of the electric field and degrees of electrowetting. For practicability, here d was assumed as the thickness of layers as Jeong-Yeol Yoon et al. [Yoon, 2003].

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Material d (TENCOR P11, Profilometer)

Cellulose Acetate ~ 400-500 nm

Durimid 115A 3800 nm

Teflon®AF (2%) ~100-150 nm

For Cellulose Acetate

(8.85 X10 12 F / m)(6)(11.3X10 6 m2 ) C 1.5nF CA 400 X10 9 m For Durimid 115A

(8.85 X10 12 F / m)(3.1)(11.3X10 6 m2 ) C 0.08nF Durimid 3800 X10 9 m

For Teflon®AF

(8.85 X10 12 F / m)(1.93)(11.3X10 6 m2 ) C 1.93nF TeflonAF1600 100 X10 9 m

For the system Cellulose Acetate and Teflon®AF the total capacitance estimated is: 1 1 1

CCA T 1.5nF 1.93nF

CCA T 0.84nF

For the system Durimid 115A and Teflon®AF the total capacitance estimated is:

1 1 1

CD T 0.08nF 1.93nF

CD T 0.076nF

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The current flows through the capacitor system until it will fully charge. The double- layer charging current I

t V RsCd I

The solution conductivity, for PBS puffer 0.1 M is 23.7 mS/cm then the solution resistance is Rs = 1/ Cs = 42.12 M cm

Cd Double layer capacitance is assumed to be equal to the total Capacitance C estimated above.

The double-layer charging current I for the system: Cellulose Acetate and Teflon®AF by switching time of the electrodes approximately each t = 3ms and 60V.

3X10 3 s 60V 42.12X106 cm)(0.84X10 9 F I 42.12 X10 6 cm

6 I 1.3X10 A

The double-layer charging current I for the system Durimid 115A and Teflon®AF by switching time of the electrodes approximately each t = 3ms and 60V.

3X10 3 s 60V 42.12X 106 cm)(0.076X10 9 F I 42.12 X10 6 cm

7 I 5.58 X10 A

For the homogeneous layers, this small double-layer charging current should be indicate that the system of layers behaved as insulator and only for heterogeneous layer or defect, should be undergo dielectric breakdown. However, see commentary in chapter 4.8.

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