ABSTRACT Botulism, the disease caused by the introduction of botulinum toxin (BT) into the body, is a rare but severe ailment that frequently results in death from respiratory failure. Cholera, the disease caused by the introduction of cholera toxin (CT) into the body, is a much more common problem in third world countries, causing severe diarrhea and dehydration. In this study, the design of previously developed sensitive biosensors for the detection of BT and CT has been scrutinized, with the intention of improving upon the detection limit the BT assay. This biosensor is a test strip assay which utilizes ganglioside-incorporated liposomes, and toxin antibodies which are immobilized on an analytical zone of a plastic-backed nitrocellulose membrane strip to form a sandwich-type detection mechanism. The intensity of the band could be visually estimated or measured by densitometry, using computer software. Previous studies with this design obtained a limit of detection (LoD) of 15 pg/mL and 10 fg/mL in 20 minutes for BT and CT, respectively. Difficulties in obtaining any concentration gradient in the detection of BT, converted this study into a reassessment of this prior design. All attempts to obtain a BT biosensor similar to that developed in the previous study failed to obtain a LoD greater than 10 g/mL, and took at least 35 minutes to complete. A similar attempt to recreate the CT biosensor design yielded a LoD no greater than 0.64 ng/mL, but took only 15-20 minutes to complete. The results from tg/mL, and took at least 35 minutes to complete. A similar attempt to recreate the CT biosensor design yielded a LoD no greater than 0.64 ng/mL, but took only 15-20 minutes to complete. The results from this study suggest that the design of the bioassay developed for BT should be reconsidered, and that further studies shouldBIOGRAPHICAL SKETCH Ryan Urbanowicz was born in the town of New Milford, CT in 1982. Parents Maureen and Gary Urbanowicz raised him in the town of Sherman, CT where he grew up and lived. His father Gary, a former Top Gun pilot, passed away in 1995 from a combination of leukemia and lymphoma. His mother Maureen faithfully serves as a 2nd grade teacher in the family’s hometown of Sherman. Ryan and his brother John became involved in the Boy Scouts of America as youths, in which they are still active as Eagle Scouts and Adult Leaders. In High School, Ryan initiated basic chemical carcinogenesis research performed at the Danbury Hospital Diagnostic Cytogenetics Lab, and Boehringer Ingelheim R&D in Ridgefield CT. After graduating from New Milford High School in 2000, he moved to Ithaca, NY where he began his degree in Biological Engineering (BEE) at Cornell University. At Cornell, Ryan concentrated in courses linked to biomedical engineering, and upper level biology. His work load also satisfied his interests in psychology, music, and design. In 2003, he obtained an internship in environmental engineering and lake management under Larry Marsicano at Candlewood Lake, CT. In 2004, he was awarded a New Horizons research fellowship, which he used to work under Professor Ruth N. Collins in the Molecular Medicine Department at Cornell. This opportunity helped Ryan to redefine his interest in “wet lab” biomedical research, and better prepare himself for a PhD program. Ryan graduated from Cornell in 2004 with a Bachelors of Biological Engineering and a Minor in Biomedical Engineering. He then began a Masters of Engineering at Cornell under advisors Antje J. Baeumner and Richard A. Durst. Outside of his Engineering degree, Ryan dedicated his time at Cornell to environmental interests through the Ecology House program, and to public service, through Cayuga Heights Volunteer Fire and Rescue. He also volunteered at the local ER, and became certified as a EMT-B in 2002, and an EMT-I in 2004. In the Fall of 2005 Ryan will begin his trail to PhD in the Dartmouth Molecular and Cellular Biology Program. He hopes to continue in academia, and spend his days learning, teaching, and researching. To my parents, my brother, my godson, and my brother eagles. ACKNOWLEDGMENTS I would like to sincerely thank Dr. Antje Baeumner for acting as my advisor, and taking me on as an MENG student, and Dr. Richard A. Durst for taking me into his lab group in Geneva. I am lucky to have taken their course in Biosensors, and to have stumbled into this MENG program. Cornell University has been the best place and the best time, and I will never forget it. My truest thanks goes to Thomas DeCory, who was both generous and keen in his technical and moral support. I will miss your playful banter, as well as your perspectives on science and life in general. I would also like to extend special thanks to Dr. Cathy Wen for both her laboratory expertise and her lessons in English, and to Jason Chen for being himself. And to Dr. Wlodzimierz Borejsza-Wysocki, a fellow Pollock, and a very interesting man to talk with. Outside of this lab, I would like to acknowledge and thank some of the key people who have served as mentors in life. First to my parents, who did a great job. To my brother, who is so much cooler than I will ever be. To Phil, Regina, Nora, Mathew, and my godson Tim, whom I love so much and never cease to make me laugh. To my Boy Scout brothers, who have always been there for me. To my undergraduate advisor Katrina Overton, the queen BEE, and a person to which I could always turn for help. To my scientific mentors, Mr. John Halloran of the Sherman Elementary School, Dr. Jacqueline P. Burns of Danbury Hospital, Mr. Larry Marsicano of the Candlewood Lake Authority, and Dr. Ruth N. Collins of Cornell University. TABLE OF CONTENTS Abstract i Biographical Sketch ii Dedication iii Acknowledgments iv Table of Contents v List of Tables and Figures vii List of Abbreviations viii Chapter 1. Literature review of Botulinum and Cholera Toxins: Characterization and Detection Assays. 1 Introduction 1 Botulinum Toxin 2 Detection methods for botulinum toxin 2 Cholera Toxin 9 Detection methods for cholera toxin 10 Chapter 2. Reassessment of a ganglioside-liposome biosensor for the detection of botulinum and cholera toxins. 14 Introduction 14 Materials and Methods 17 Materials 17 Preparation of ganglioside-incorporated liposomes 18 Liposome characterization 19 Preparation of test strips 19 Assay formats 20 Detection and quantification 22 Results and Discussion 22 BT Initial trials 22 Liposome Concentration 24 Preliminary UCSF Medical Center Antibody Trials 25 Increased assay speed 26 Incubation time 26 Fresh toxins and antibodies 26 Test strip preparation method 27 Liposome characterization 28 Toxin Variations 28 Blocking solution 29 Human Error 30 Result Summary for BT assay 30 CT assay 30 Conclusions 34 Future Studies 35 Chapter 3. References 36 LIST OF TABLES AND FIGURES _____________________________________________________________ Table 2.1. Toxin Test Concentrations _____________________________________________________________ Figure 1.1. Structure of Botulinum Toxin and it’s Activation Figure 1.2. Mode of action of Botulinum Toxin Figure 1.3. Structure of Cholera Toxin Figure 2.1. Cross-sectional representation of liposome used in design Figure 2.2. Diagram – Sandwich Assay Figure 2.3. Test Strip Assay Figure 2.4. Antibody Density Trials Figure 2.5. Initial Trial of full BT spectrum Figure 2.6. Liposome Dilution Trial Figure 2.7. Typical BT Trial up to this point Figure 2.8. Toxin type variation Figure 2.9. Initial CT assay trial Figure 2.10. CT trial (membrane / liposome dilution variables) Figure 2.11. CT trial (Test strip fabrication variable) Figure 2.12. Toxin Step-wise Dilution Narrowed. LIST OF ABBREVIATIONS BT: botulinum neurotoxin CLISA: chemiluminescence immunosorbent assay CT: cholera toxin ELSIA: enzyme-linked immunosorbent assay GM1: monosialoganglioside GT1b: trisialoganglioside LoD: limit of detection NC: nitrocellulose