DEVELOPMENT OF ENZYME-BASED BIOSENSORS FOR THE DETECTION OF ORGANOPHOSPHATE NEUROTOXINS Except where reference is made to the work of others, the work described in this dissertation is my own or was done in collaboration with my advisory committee. This dissertation does not include proprietary, restricted or classified information. ______________________________ Sheetal Paliwal Certificate of Approval: _________________________ _________________________ Jeffrey Fergus Aleksandr Simonian, Chair Associate Professor Professor Materials Engineering Materials Engineering _________________________ _________________________ Barton Prorok William Moar Associate Professor Professor Materials Engineering Entomology _________________________ _________________________ Vitaly Vodyanoy George T. Flowers Professor Dean Anatomy, Physiology and Graduate School Pharmacology DEVELOPMENT OF ENZYME-BASED BIOSENSORS FOR THE DETECTION OF ORGANOPHOSPHATE NEUROTOXINS Sheetal Paliwal A Dissertation Submitted to the Graduate Faculty of Auburn University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Auburn, Alabama December 19, 2008 DEVELOPMENT OF ENZYME-BASED BIOSENSORS FOR THE DETECTION OF ORGANOPHOSPHATE NEUROTOXINS Sheetal Paliwal Permission is granted to Auburn University to make copies of this dissertation at its discretion, upon the request of individuals or institutions and at their expense. The author reserves all publication rights. _________________________ Signature of Author _________________________ Date of Graduation iii VITA Sheetal Paliwal, daughter of Sri. Hansraj Paliwal and Smt. Savita Paliwal, was born on March 8, 1980, in Hyderabad, India. She graduated from Ratna Junior College, Hyderabad, India in May 1997. She joined MJCET, Osmania University, Hyderabad, India in November 1997 and graduated with a Bachelor of Engineering in Mechanical Engineering in May 2001. She graduated with a Masters in Industrial Engineering from Arizona State University in May 2003. She joined the doctoral program in Materials Engineering at Auburn University in August 2003 under the guidance of Dr. Simonian. She is married to Sai Vamshidhar Nudurupati, son of Sri. Saibaba Nudurupati and Smt. Sarada Nudurupati. iv DISSERTATION ABSTRACT DEVELOPMENT OF ENZYME-BASED BIOSENSORS FOR THE DETECTION OF ORGANOPHOSPHATE NEUROTOXINS Sheetal Paliwal Doctor of Philosophy, December 19, 2008 (M.S, Arizona State University, 2003) (B.E, MJCET, Osmania University, 2001) 183 Typed pages Directed by Aleksandr Simonian Elevated concerns of ecological safety and environmental pollution have facilitated massive research in the area of chemical and biological agent detection. Large numbers of anthropogenic chemicals were introduced in the environment within last 50 years, which posed a serious threat to the natural ecological balance. The series of unfortunate events in the past could have been avoided or minimized by the availability of adequately selective and sensitive detectors, capable of discriminating between common use pesticides and chemical warfare agents. Organophosphates (OPs) in particular are more of a concern because of their toxicity and inhibition of esterase enzymes which are involved in nerve transmission. They have been an integral part of agricultural industry for past decades owing to their specificity. In addition, to their wide usage, they have also been exploited for the development of chemical warfare agents. Consequently, there is an emergent need for rapid and sensitive detection methods. v Enzyme-based studies have been investigated in an effort to develop simple, rapid, user-friendly and sensitive detection of OPs. The methods described have been developed using OPH, an enzyme capable of hydrolyzing OPs. A rapid detection method using a fiber-optic waveguide and a portable fluorimeter Analyte 2000 was developed. Immobilization of OPH was accomplished using avidin-biotin chemistry. Change in fluorescence intensity was observed upon hydrolysis of substrate by OPH, concentration as low as 0.05µM paraoxon (PX) was qualitatively measured. A novel detection method for p-nitrophenol and p-nitrophenyl substituent OPs based on fluorescence quenching of coumarin1, a dye similar in structure to some OPs have also been developed. Decrease in fluorescence intensity of coumarin1 was proportional to paraoxon concentration in the range of 0.7–170 µM. To preserve OPH activity and stability, encapsulation using lysozyme-mediated silica nanoparticles was accomplished and detection limit for PX was found to be 20µM. To improve site accessibility and sensor sensitivity, orientation-specific immobilization of OPH was achieved using site-specific antibodies and different variants of OPH. Finally, the potential use of carbon nanotubes for the electrochemical biosensing of OPs has been exploited OPH was covalently immobilized on the surface of these nanotubes and a detection limit of 2.3 µM was obtained for PX. Results demonstrated that the immobilized enzyme retained a significant degree of enzymatic activity, and displayed remarkable stability with only 8% decrease in signal over a period of 10 days. vi ACKOWLEDGEMENTS I would like to express my earnest gratitude to Dr. Aleksandr Simonian for his continuous support and guidance throughout the course of my degree. I would like to acknowledge my committee members, Dr. William Moar, Dr. Jeffrey Fergus, Dr. Vitaly Vodyanoy, and Dr. Bart Prorok for their valuable time and support. My sincere gratitude to Dr. Holly Ellis for acting as an external reader. I would like to extend my special thanks to Maj. Leamon Viveros, who spent effortless hours helping me understand concepts underlying this work and his continued support for all these years. I sincerely acknowledge Dr. James Wild, Dr. Melinda Wales and Dr. Janet Grimsley for providing me the enzyme without which this work was not possible. My nanotube work would not be possible without gracious help from Dr. Dhriti Nepal. Appreciation goes to my entire research group for their help and support. I am deeply indebted to my family, who went far beyond their extremities to provide me with all comforts. I thank them for all the sacrifices they have made and their devotion. The unconditional love and support of my siblings, Richa and Mitrank helped me accomplish this degree. My parents-in law, deserve a special mention for their immense support and affection. Words fail to express my appreciation to my husband, Vamshi whose commitment and persistence have helped me realize my aspirations. His endless love and patience kept me focused and always steered me in right direction. I consider myself fortunate to have him as my life partner. Thanks to one and all. Finally, thanks to Lord Sri Sairam for his grace and blessings. vii Style manual or journal used Sensors and Actuators B Computer software used Microsoft Office XP, Endnote X and OriginPro 8.0 viii TABLE OF CONTENTS LIST OF TABLES ...................................................................................................... xvi LIST OF FIGURES .................................................................................................... xvii 1. INTRODUCTION AND LITERATURE REVIEW .................................................. 1 1.1. INTRODUCTION TO BIOSENSORS ............................................................... 1 1.2. CLASSIFICATION OF BIOSENSORS .............................................................. 2 1.2.1. Biocatalysis-based biosensors ................................................................... 3 1.2.2. Bioaffinity-based biosensors ..................................................................... 4 1.2.3. Microbe-based biosensors ......................................................................... 4 1.2.4. Electrochemical transducers ..................................................................... 4 1.2.4.1. Potentiometric ............................................................................... 4 1.2.4.2. Amperometric .............................................................................. 5 1.2.4.3. Conductometric ............................................................................ 5 1.2.5. Optical transducers ................................................................................... 5 1.2.5.1. Absorption .................................................................................... 5 1.2.5.2. Fluorescence ................................................................................. 6 1.2.5.3. Luminescence ............................................................................... 6 1.2.5.4. Surface plasmon resonance (SPR) ................................................ 6 1.2.6. Piezo-electric transducers ......................................................................... 7 1.2.7. Enthalpymetric transducers ....................................................................... 7 ix 1.3. ENZYME .......................................................................................................... 8 1.4. IMMOBILIZATION OF ENZYMES.................................................................. 9 1.4.1. Physical adsorption ................................................................................. 10 1.4.2. Cross linking ......................................................................................... 11 1.4.2.1. Avidin-biotin system ................................................................... 11 1.4.2.2. Crosslinking using gluteraldehyde..............................................