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University of Alberta University of Alberta The Development of a Micro-Total Analysis System For Gene Detection and Quantitation Using Cycling Probe Technology with Capillary Zone Electropho reçis. Mohammed Youssouf Bada1 O A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry Edmonton, AI berta Spring 200 1 National Library Bibliothèque nationale ofCanada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Weüington Street 345, nie Wellington Ottawa ON K1A ON4 Oüawa ON K1A ON4 CaMda Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence dowing the exclusive pennethnt a la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or seil reproduire, prêter, disrnier ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d' auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. TO rqbear fatber, Late W. A. BADAL, wbose support, love UM~Iimpiratio~ bas maae tbis Oream corne tme. YOM wilf be miss& ........ Thesis Abstract The goal of the thesis was to develop a p-TAS for DNA analysis using cycling probe technology (CPT) as a DNA amplification reaction and using capilfary zone electrophoresis CZE as separatinn technique. Chapter 2 describes a rnethod to modify the surface of channe1 waIls in the chip and subsequently perform capillary zone electrophoresis. Experiments were done to study the electroosmotic flow (EOF) behavior of the poly(dimethy1siloxane) PDMS coated capillaries in the presence of charged and neutral surfactants. The results showed it is possible to fine tune the EOF by adjusting the surfactant concentration. Subsequently, the use of the coating in the CPT project was not pursued. However, we concluded that the coating could be useful in protein separation. In Chapter 3 and 4 we describe a micro-chip device, named GOCPTTT, that was capable or performing enzyme mediated signal amplification of a target DNA sequence, followed by separation and analysis of the reaction products for quantitation. CPT utilizes signal amplification through the cleavage of a 5' end-labeled fluorescein DNA-RNA-DNA probe when hybridized to a target DNA and subsequent enzyme action (RNase H). This thesis work shows how the technique was developed on microtluiaiç devices. which provide a powerful means of intesrriting sample preparation or reaction steps with separation. The samples used in the study were from methicillin resistant Sraplzylococcus crurulu- (Chüpter 3) and Enritzio lzerbicok~(Chapter 4). These bacteria are representative of a typical clinici11 assay. and a simulant of pathogens in the environment, respectively. The probe. enzyme and target were electrokinericaIly mixed on-chip, incubated under isothennal conditions (60 OC) in a reaction zone (integrated on the device), then mobilized for separation on-chip. The cleaved and intact probes were separated using gel-free, open tubular CZE with the heIp of a 3' end-labeled biotin moiety on the proht.. The ratio of accurnulated cieaved probe to intact probe identifies the amount of target present. Amplification of five orders of magnitude was achieved. with a detection limit of about 0.1 attomoles of target DNA and a quantitative response across a wide range of target DNA concentrations. This work shows that each of these steps rnay be integrated together on a single chip, and it establishi-s quantitative performance for the on-chip devices. Special gratitude to my research supervisor, Prof D. Jed Harrison. He introduced me to this exciting field of microfluidics and provided me with valuable guidance and support throughout the course of my research. 1 am very thankful for his patience in reading my thesis and making it in its present form. Also, not to forget the scientific knowledge 1 have gathered from al1 the conferences 1 have attended thanks to Prof D. Jed Harrison. My sincere thanks go to the Harrison's group who reaIly made rny stay as a graduate student very enjoyable in the chemistry department. Special thanks to my friend Thompson Tang for introducing me to the field of cycling probe technology. Thank you to Dr. Hossein SaIimi-Mossavi for his technical and personal advice when 1 started my graduate work. Thank you to Dr. Fahima Ouchen for teaching me microfabrication at the student microfab. 1 wish to express to my gratitude to Mrs. Arlene Figley for reading my thesis and making grammatical corrections. My appreciation also extend to the technical support staff in this chemisti-y department especialty to those of the glass, electronic and machine shops. Thank you to Dr. WiIIiam E. Lee and Douglas. E. Bader from the Defence Research Establishment at Suffield, Terina Dickingson-Laing from C.W. Bios Inc., and Dr. Faouzi Bekkaoui from ID Biomedical for providing DNA samples and also for valuable discussion and suggestions for this research. My thanks also go to the members of my candidacy and defence committee in the persons of Dr. Frederick Cantwell, Dr. Norman Dovichi, Dr. Mark Mcderrnott, Dr. Rik Tykwinski, Dr. Martin Somerville and Dr. Ulrich Krull. Special thanks to my country mates from Mauritius, Soleiman and Asvina for their personal support. 1 thank Dr. Ahmad Khodabocus, Senior Iecturer at the University of Mauritius, for his support and encouragement. Thank you to rny family, My late father. Waheb. my mother, Rosida, and my two brothers Rezah and Nazim for their support, love and inspiration. And most importantly thank you God for giving the courage and patience to persevere during the course of my research. Table of Contents CHAPTER 1: Introduction Page Miniaturization Micro (or Miniaturized) Total Analysis Systems Microfluidics and "iab on a chip" Focus of Chapter One Mode of Capillary Electrophoresis Capillary Zone Electrophoresis Micel tar Elecuokinetic Capiltary Chrornatography Capillary Gel Electrophoresis Capillary Isoelectric Focusing Capillary lsotachophoresis Theory of Capillary Electrophoresis Double Layer Theory Generation of Electroosmotic Flow Fundamentais of CE Eftïciency of Separation Characterization of Electroosmoric FIow Resolution Between Two Peaks Surface Modification in Capillary Electrophoresis Importance of Coating Different Coating Procedures Covalent Bonding Adsorption Methods The Development of Micro-Total Analysis Systems pTAS for Gene Analysis Mixing Basic DNA Concepts Deoxyribonucteic Acid (DNA) Structure Properties of RNA Denaturation and Renaturation of DNA Renaturation Identification of Microorganisms or Bacteria by Conventional Methods Polymerase Chain Reaction Cycling Prohe Technology DNA-RNA Hybrid Recoznition by Rnase H Application of CPT PubIished to Date PhysicaI Artifacts in DNA and RNA Hairpin Loop Formarion Primer-Dimer Scope of the Thesis References CHAITER 2: Electroosmotic Flow Study on Poly(dimethylsi1oxane) Coated Capillaries Page Introduction Experimental Instrumentation Rertgents Coating of Capillaries Protein Separation EOF Measurement Results and Discussion Coating Evaluation Effect of pH on the EOF in Coated Capillaries Effect of SDS on the EOF Effect of CT.4B on the EOF Effect of pH on the study in the Presence of Charged Surfactants 2.4 Conclusion 2.5 References CHAPTER 3: Methicilin Resistant Staplrylococcus aureus DNA Assay Using Cycling Probe Technology with Capillary Zone Electrophoresis on a Chip Introduction Experimental CPT Components and Conditions Device Design and Fabrication Method of Fabricating the Drilled Cover Plates Method of Fabricating the Etched Plates Cold Bonding the Substrate with the Cover Plate to Make the Microtluidic Devicr Thermal Bonding of the Substrates Device Layouts Channel Derivatization Instrumentation Set-up and Optirnization CO do CE on Chip Signal Optimization Instrumentation Set-up and Optirnization for On-chip Cycling and Separation LiF Detection Using an lnverted Confocal Microscope Instrumentation Alignment for Optimum Fluorescent Signal On-chip Cycling and Separation Results and Discussion Cycling Probe TechnoIogy and Capillary Electrophoresis On-Chip Mixing, Reaction, Separation and LE Detection Using the GOCPTTT Device Temperature Control On-chip On-chip CPT process: Mixing, Reaction and Separation Diffusional Mixing CZE Separation Calibration with On-chip CPT Reaction Conclusion References CHAPTER 4: A p-TAS for Detection of Pathogens in the Environment Ushg Cycling Probe ~tkhnolo~~for DNA Amplification Page Introduction 1 24 Experimental 1 26 Materials and Methods 126 Target DNA 126 CPT Reagents 126 Chernical Reagents 1 27 Devices 1 27 Channel Derivatization 128 Instrumentation 128 On-chip Cycling and Separation 128 Results and Discussion 131 Earlier Work 13 1 Development of CPT Probes to Detect for Envinia herbicoln Target DNA 131 Assessrnent of CPT Probes 133 CPT Assay for Envinia herbicola On-chip Format 13.5 Calibration 138 On-chip Reaction Using 15 min Incubation Time 141 Conclusion 141 References 142 CHAPTER 5: Conclusion And Future Suggestions Conclusion from thesis work 146 Surface Modification
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