ACOUSTICALLY-LEVITATED DROP REACTOR (LDR) EMPLOYABLE FOR KINETICS MEASUREMENTS OF BIOCHEMICAL NETWORKS BY ZAKIAH NACHÈ PIERRE DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry in the Graduate College of the University of Illinois at Urbana-Champaign, 2011 Urbana, Illinois Doctoral Committee: Professor Alexander Scheeline, Chair Professor Jonathan Sweedler Professor Andrzej Wieckowski Professor Arne J. Pearlstein ABSTRACT Accurate measurement of enzyme kinetics is an essential part of understanding the mechanisms of biochemical reactions. The typical means of studying such systems use stirred cuvettes, stopped-flow apparatus, microfluidic systems, or other small sample containers. These reaction-kinetics measurements reactors are usually made of quartz, glass, or an easily-moldable polymer such as polydimethylsiloxane. For studying enzyme kinetics or free radical chemistry, using small volumes with high concentrations is desirable, but interactions of biochemical moieties with these materials can be problematic. The main obstacle is that, as the system volume shrinks, the ratio of surface area to volume increases. As a result, the importance of adsorption can increase, and proteins sticking to walls, biofilms grow on walls, and radicals annihilating on walls can become significant. Thus, under these circumstances, typical microfluidic systems are incompatible with the demands of accurate study of many biochemical systems. Over the past five years, acoustic levitation has become a technique uniquely suited to studying biochemical reactions. Acoustic levitation offers several advantages over typical reaction systems, including small sample volume (and mass), the prevention of chemical contamination between drops and external objects, rapid mixing within the drop as a result of circulation driven by the levitating ultrasound, and freedom from wall interactions. We have developed an Acoustically-Levitated Drop Reactor (LDR) to study enzyme-catalyzed reaction kinetics related to free-radical and oxidative stress chemistry. ii Microliter-scale droplet generation, reactant introduction, maintenance, and fluid removal are all important aspects in conducting reactions in a levitated drop. Therefore, we developed a three-capillary bundle system to address these needs. Since the capillary system is used to introduce drops to the levitation cavity, our LDR system is not 100% wall-less, but the ratio of solid surface area to drops surface area is less than 20%, which is low compared to all the other reaction systems. The largest interface seen by reactants is a liquid/gas interface. Herein, I report kinetic measurements for both luminol chemiluminescence and the reaction of pyruvate with nicotinamide adenine dinucleotide, catalyzed by lactate dehydrogenase in a levitated drop. Observations of the chemiluminescence experiment showed second-order kinetics were detected within experimental error prior to deviations seen after 10 s. For the enzyme-catalyzed reaction kinetic measurements, it was found that the KM for lactate dehydrogenase matched literature values to better than one standard deviation of the literature experiment. Also studied were the effects of laser exposure on the stability of Myeloperoxidase at the liquid/gas interface. It was found that although myeloperoxidase degrades over several hours in buffer, there is no indication that the degradation is due to either denaturation at the free surface or to two-photon photolysis. Based on our accomplishments, we demonstrate the feasibility of using a levitated drop as a microreactor. The reactions discussed utilized in-house developed fiber optic and laser-based optical detection systems to monitor reaction kinetics inside a levitated drop via chemiluminescence and fluorescence. Outgrowths of these accomplishments will hopefully lead to the use of additional detection systems (including electrochemical and mass iii spectrometry) with the LDR to study a wide variety of biochemical reaction kinetics. iv This thesis is dedicated to my husband, Sober Pierre, for standing firm with me through it all. v ACKNOWLEDGEMENTS What a journey this has been. I thank God for the energy and foresight to see this through to completion and I thank all of you for your support along the way. Without you, I am not sure I would have arrived. But I did and I owe much of it to you. To those who know me the best and believed in me the most, my family, I love and thank you. To Mom who always said, “Be the best Z you can be!” and Dad who said, “This thing is yours, so go get what belongs to you.”, those words of encouragement meant the world to me. Sober Pierre. My husband. My rock. The person who refused to let me give up. This PhD is probably more than 50% yours. When I wanted to run for the door, you advised otherwise. You helped me to see the light at the end of the tunnel when all I could see was an oncoming train. This journey has taken me for quite a ride but you always represented consistency. For all of your patience and understanding, I truly thank you. To the best group, or should I say extended family, a girl could ask for, thank you. To Professor Scheeline, I may not have always welcomed ALL (there was so much) of the feedback, remarks, critiques, words-o-advice, S-grams, etc., that came my way but I am so grateful to have had a boss who cared enough to provide such thorough guidance. I also thank you for allowing me to be me, allowing me to explore outside opportunities and not forcing me into a mold common for PhD Chemists. I have grown tremendously as a result. Thank you. To the group members, it was all worth it having you along for the ride. Dr. Christopher Ryan Field, you were one cool side-kick. The LDR and I both missed you when you left. Dr. Rebekah Koch Wilson, my right hand woman, through the laughs and the tears, vi you were always right there. You shared in the frustration like they were your own and always offered suggestions on how to move research forward. What a friend I found in you. Edward Chainani, the man with a heart of gold. If I needed anything you were always there with a helping hand leaving me to wonder, "Does he ever say No?" Your kindness and generosity were so greatly appreciated. To Haylee Thomas who, although long gone, still found the time to send words of encouragement. For being your amazing selves and allowing me to share in it, I thank you all. To those who made achieving this goal possible, thank you. For your time and support, throughout the years, I thank my other committee members, Professor Andrzej Wieckowski, Professor Jonathan Sweedler, Professor Arne Pearlstein, and Professor Neil Kelleher. For aiding with day-to-day research, I would like to acknowledge a number of people from various units on campus. Thank you, Mike Thompson and Jim Wentz of the School of Chemical Sciences (SCS) Electronics Shop for your hard work and expertise for various electrical needs. Thank you to Bill Knight and the others (Roger Smith, Ed Runyon, Tom Wilson, Mike Harland, and Dave Hire) that run the SCS Machine Shop, for building the levitator and a number of other items that I designed. Dorothy Loudermilk of the SCS Graphic Services, thank you for your hard work seeing to it that I had great graphics to put in my presentations and professional posters to present at conferences. A very special thank you goes to the mother of the Analytical Chemistry Area, Julie Sides, for always checking in on me to make sure that not only was I okay but also that all of my paperwork was in order so that when the time came, my transition out would be smooth. Thanks Julie. vii Last but definitely not least, thank you to all of the funding institutions that made not only the research discussed in this thesis possible but also provided for a roof over my head, food in my mouth and clothes on my back. To the National Institutes of Health (NIH, Ruth L. Kirschstein National Research Service Award 5 R01 GM067193-06) (Thank you again Professor Kelleher for the NIH support), National Science Foundation (NSF, Grant 08-52383 ARRA), Research Corporation (Grant RA0333), The National Consortium for Graduate Degrees for Minorities in Engineering and Science (GEM), and Proctor and Gamble (P&G), thank you so very much. viii TABLE OF CONTENTS CHAPTER 1: INTRODUCTION, OVERVIEW, AND RESEARCH OUTLINE…...1 1.1 Biochemical Context............................................................................................1 1.2 Systems Biology and Enzyme Kinetics................................................................1 1.3 Research Summary...............................................................................................3 1.4 References …........................................................................................................6 1.5 Chapter 1 Figures..................................................................................................7 CHAPTER 2: RESEARCH BACKGROUND................................................................8 2.1 Immunology: The Innate Immune Response .......................................................8 2.2 Myeloperoxidase: Its Role in Immunology, Reaction Mechanisms and Positive/Negative Implications...........................................................................10 2.3 Studying Enzyme Kinetics: Macro vs. Micro Reaction Methods.......................17 2.4 An Acoustically Levitated Drop Reactor (LDR)................................................22 2.5 Sample Handling