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 in an Acoustic Levitator: Drop Generation, Reactant Introduction, Maintenance and Fluid Removal...... 25 2.6 Summary...... 26 2.7 References...... 27 2.8 Chapter 2 Figures……...... 54

CHAPTER 3: SAMPLE HANDLING AND CHEMICAL KINETICS: AN ACOUSTICALLY LEVITATED DROP AS A MICROREACTOR...... 62 3.1 Introduction...... 62 3.2 Materials and Methods...... …...... 64 3.3 Results and Discussion...... 69 3.4 Conclusions...... 78 3.5 References...... 80 3.6 Chapter 3 Figures...... 85

CHAPTER 4: STABILITY OF MYELOPEROXIDASE AT THE GAS/LIQUID INTERFACE……………………………………………...………...... 92 4.1 Introduction...... 92 4.2 Materials and Methods...... …...... 93 4.3 Results and Discussion...... 95 4.4 Conclusion...... 100 4.5 References...... 101 4.6 Chapter 4 Figures...... 102

ix CHAPTER 5: CONCLUSIONS AND RECOMMENDATIONS FOR LDR DEVELOPMENTS AND APPLICATIONS………...... 110 5.1 Acoustically Levitated Drop Reactor System Optimization...... 110 5.2 Understanding Dynamics of a Levitated Drop...... 115 5.3 LDR Applications...... …...... 115 5.4 Conclusions...... 118 5.5 References...... 119 5.6 Chapter 5 Figures...... 120

APPENDIX A: PROCEDURES FOR PREPARING 3-CAPILLARY BUNDLES…………………………………………....………………………………...123

APPENDIX B: BASIC INSTRUCTIONS FOR OPERATING THE LEVITATED DROP REACTOR...... 126

APPENDIX C: AUTHOR’S CURRICULUM VITAE……………...………………130

x CHAPTER 1

INTRODUCTION, OVERVIEW AND RESEARCH OUTLINE

1.1 Biochemical Context

The human body is an extremely complex system that has been studied for hundreds of years, yet is still incompletely understood. One serious problem is that although each individual’s body is similar in make-up, there are genetic differences that set each apart and make it difficult to rationalize the many variations among individuals. If one model cannot explain how each individual functions, then it is little wonder that one medicine may give quite different responses to the same disease in different individuals. The human body, its organization and its many reactions, are worth studying and understanding to make preventive care more effective, and to improve drug development. Systems biology is the area of research attacking this issue. By developing theories and conducting research, the idea of personalized health care may become a reality. It will also change the way immunology is studied.

1.2 Systems Biology and Enzyme Kinetics

Systems biology is a useful tool for studying biological networks such as the human innate immune system. Systems biology is the study of interactions between biological compounds, such as enzymes.1 Systems biology is not necessarily focused on the components themselves, but the nature of the links that connect them and the functional states of the networks as a result of the link’s assembly.2 The interactions between enzymes and small molecules are often represented as a connected graph or network.1,3,4 Within the network

1 there are layers of information; each layer increasing in detail. The first layer of information is the statement of the existence of an interaction. The second, more detailed, layer of a network is the approximation of enzymes as a combination of switches that are opened and closed in a specific order to describe feed-back, feed-forward, inhibition, and activation interactions and mechanisms. However, the ultimate description of a network contains the kinetic information of every enzyme-catalyzed reaction in the network. This third layer of detail allows for the formation of a timeline of events of each species in the network. In order to fully maximize the understanding of an enzyme interaction network, the kinetic information of the enzyme-catalyzed reactions must be obtained.

Unfortunately, obtaining kinetic information for every enzyme in a reaction network can be difficult. Reaction kinetics, which are not limited to enzyme-catalyzed reactions, are affected by a number of parameters beyond reactant, product, and enzyme concentrations. The conditions of the environment, as well as other species present in the system, must be understood and controlled to obtain accurate, reliable, and reproducible kinetics data. The sheer number of parameters and species involved in measuring enzyme-catalyzed reaction kinetics as a network of interactions requires the development of novel instrumentation.

Complicating the measurement of kinetics is the difference in environments between in vivo and in vitro measurements. Ideally, all of biology and bioanalytical chemistry would be conducted in vivo. But for many reasons, including but not limited to subject safety, complex heterogeneous systems, uncontrolled environment, ambiguous results, and lack of specificity,

2 not all measurements can be made in vivo, so instead model environments are widely used to mimic environments seen in nature.

One example is a continuously-stirred reactor (CSTR). Traditionally, a CSTR is a quartz, glass, or polymer vial with injection ports and removal ports, and a stirring mechanism, to accurately control the environment. Unfortunately, such systems prove to be problematic when studying enzyme kinetics. Interactions with the reactor walls cause adsorption of reactants and products to the walls can of the vessel. These wall interactions add an additional layer of complexity to the already complex system, changing concentrations and adding surface-catalyzed steps to bulk mechanisms. Developing a reaction system that offers all of the benefits of a CSTR, with minimal wall interactions, is critical to studying biochemical kinetics.

1.3 Research Summary

This thesis recaps the five-year journey to the development of a wall-less instrumentation system to study enzyme reaction kinetics. It begins with the specific motivations behind this research, the specific biochemical reaction system on which we chose to focus, and reasons for choosing the particular system.

We chose to study the peroxidase-oxidase reactions involving the enzyme myeloperoxidase

(MPO) (Figure 1.1). Although understanding the entire immune response system would be profitable, it is too large to study in its entirety with existing measurement technology.

However, with MPOs centrality to the function of neutrophils, we are confident that the

3 system chosen will have a significant impact in immunology research. We have chosen to study MPO because it is arguably one of the most important enzymes involved in fighting foreign microorganisms that invade the human body. This argument is based on the enzyme's ability to generate toxic oxidants such as hypochlorite (HOCl), which has been thought to be responsible for killing foreign invaders (although the role of MPO is more complicated than simply being an HOCl source).4-7 Along with MPOs positive aspects, there are also negative ones. The expression of MPO has been linked to the onset of atherosclerosis and several autoimmune diseases such as osteoarthritis and cystic fibrosis.

Understanding MPO enzyme kinetics can further elucidate its positive and negative effects, as well as provide insight on ways to regulate the enzyme expression and activity so that it fights foreign invaders but not against the host. A review of the published literature concerning this research can be found in Chapter 2.

Next this thesis discusses briefly the design and, in greater detail, the development and employment of an acoustically levitated drop reactor as an instrument for studying enzyme- catalyzed reactions. Much of the design of this instrument was discussed in Christopher

Field’s thesis,8 and is summarized in Chapter 2. Employment of the levitator system to study enzyme kinetics is described in Chapter 3. The development of the levitator’s sample handling system along with proof-of-concept experiments and enzyme kinetics measurements are also reported in Chapter 3. Chapter 4 discusses experiments supportive of work on behavior of MPO at the gas-liquid interface. Chapter 5 suggests next steps for study of MPO kinetics and levitator development now that initial steps have succeeded.

4 Throughout this thesis, examples for potential use of a microliter-sized drop as a microreactor, to study enzyme catalyzed reactions in a manner similar to a CSTR are presented. Also shown are sample sizes that can be reduced by a factor of 1000 compared to macroscale systems. The most important finding is that enzyme catalyzed reactions performed on the microscale, for reactions that do not include free radicals as reactants, intermediates, or products, produce results similar to those generated in macroscale systems.

One thing this research set out to accomplish, but will need to be pursued by the next researcher, is to note the differences in the kinetics data between walled- and wall-free systems for reactions that do involve free radicals. The hypothesis is that there will be significant differences, primarily in the rate constants, due to the lack of wall interactions.

Data are presented to support our theories and are discussed in great detail in the following chapters.

There are several directions research can go in the light of results reported here. Projects may include further developments on the levitated drop reactor and sample handling systems, and experiments that prove a wall-less, microliter-sized drop is useful in studying not only

MPO but an array of biochemical and enzyme-catalyzed reactions. In addition, work on integrating additional detection systems into the levitator to study other changes (structure, activity, pH, side-reactions, etc.) that occur within biochemical reactions would prove useful.

Understanding the drop dynamics for this particular levitator system will also be important for future work. Detailed suggestions are provided in Chapter 5 on recommended future directions.

5 1.4 References

1 A. M. Lesk, Introduction to Bioinformatics. 2nd ed. New York, NY: Oxford University

Press Inc., 2005.

2 B. O. Palsson, System Biology: Properties of Reconstructed Networks. 1st ed. New

York, NY: Cambridge University Press, 2006.

3 K. C. Chen, A. Csikasz-Nagy, B. Gyorffy, J. Val, B. Novak and J. J. Tyson. “Kinetics

Analysis of a Molecular Model of the Budding Yeast Cell Cycle,” Molecular Cellular

Biology, 11, 369-91 (2000).

4 S. J. Klebanoff, “Myeloperoxidase: Contribution to the Microbicidal Activity of Intact

Leukocytes,” Science, 167, 195-196 (1970).

5 E. W. Odell and A. W. Segal, “The Bactericidal Effects of the Respiratory Burst and

the Myeloperoxidase System Isolated in Neutrophil Cystoplasts,” Biochimica et

Biophysica Acta-Molecular Cell Research, 971, 266-274 (1988).

6 T. F. Meyer, “Pathogenic Neisseriae: Complexity of Pathogen-Host Cell Interplay,”

Clinical Infectious Disease, 28, 433-441 (1999).

7 W. M. Nauseef, “How Human Neutrophils Kill and Degrade Microbes: An Integrated

View,” Immunological Reviews, 219, 88-102 (2007).

8 C. R. Field, Developments in Analytical Chemistry: Acoustically Levitated Drop

Reactors for Enzyme Reaction Kinetics nad Single-walled Carbon Nanotube-based

Sensors for Detection of Toxic Organic Phosphonates, Ph.D. thesis, University of

Illinois at Urbana-Champaign (2009).

6 1.5 Chapter 1 Figures

Figure 1.1 Hypothesized Myeloperoxidase Reaction Network. Diagram by A. Scheeline, University of Illinois Urbana Champaign. Used with permission.

7

CHAPTER 2 RESEARCH BACKGROUND

2.1 Immunology: The Innate Immune Response

One of the most complex biological systems is the immune system. The immune system is a collection of mechanisms that protect against infection by identifying and killing pathogens using layered defenses of increasing specificity.1 It detects pathogens and distinguish them from the body’s normal cells and tissues. There are innate and adaptive components to the immune system. The innate subsystem is the first responder. A pathogen is recognized and the innate system responds immediately, but in a generic way. If a pathogen evades the innate response, the adaptive immune response is employed to adapt its response during infection to improve its recognition of the pathogen. The innate immune system is the dominant system of host defense. This thesis focuses entirely on the innate immune response and more specifically the roles of neutrophils and macrophages.

The innate immune response is the first line of defense but lacks the ability to recognize or remember pathogens, or to provide specific protection against re-infection. Adaptive immunity on the other hand is very specific. It is based on a clonal selection from a repertoire of lymphocytes bearing highly diverse antigen-specific receptors that enable the immune system to recognize foreign antigens.2 Both adaptive and innate immunity depend on the activities of the white blood cells, or leukocytes. Innate immunity largely employs granulocytes or macrophages while the adaptive immunity relies on lymphocytes.

Granulocytes, which are also referred to as polymorphonuclear leukocytes, are a diverse

8 collection of white blood cells whose granules provide them with their characteristic staining patterns.

In order to protect the host, the innate and adaptive immune systems must work together.

Most infections are handled successfully by the innate immune system and cause no disease.

Other infections that cannot be resolved by the innate immunity trigger an adaptive immune response. Because there is a delay of between 4 to 7 days before initial adaptive response takes effect, the innate immune response plays a critical role in controlling infections during this period.

The cells of the immune system originate in the bone marrow, where they mature. Once mature, the cells migrate to guard the peripheral tissues, circulating in the blood and in specialized vessel systems called the lymphatic system. The myeloid progenitor is the precursors of the granulocytes, macrophages, dendritic cells and mast cells specific to the innate immune response. Neutrophils, eosinophils, and basophils, are collectively known as granulocytes. Neutrophils, the granulocyte discussed in this remainder of this chapter, are short-lived cells that are abundant in the blood. These phagocytic cells’ key role in the innate immunity is to recognize, ingest and destroy pathogens without the aid of the adaptive immune response.

9 2.2 Myeloperoxidase: Its Role in Immunology, Reaction Mechanisms and Positive/Negative Implications

Of all its host defense mechanisms, the mechanism that involves the enzyme myeloperoxidase (MPO) is the most heavily studied and debated concerning its role, impact, and reaction network dynamics.3-6

The heme-containing enzyme, MPO, is one of the major players in the first line of non- specific immune defense system, responsible for the microbial activity within human neutrophils (Figure 2.1).7 MPO is the product of a single 11kb gene, composed of 11 introns and 12 exons8 located in the long arm of chromosome 17 in segment q12-24.8-12 Mature

MPO (Figure 2.2)13 is approximately 150 kDa and consists of a pair of heavy-light promoters14-16 whose heavy subunits are linked by a disulfide bond along their long axis.14

MPO is a strongly basic protein with an isoelectric point > 10,17 and therefore binds avidly to negatively charged surfaces. The enzyme is packed inside the cytoplasmic azurophilic granules. The concentration of MPO in the granules is relatively high and accounts for up to

5% of the dry weight of the cell.18

Upon invasion of the body by a microorganism, neutrophils are first activated, then they leave the blood stream and migrate towards the infected site. Then the process of bacterial killing, phagocytosis, takes place after the bacterium is completely surrounded by the phagosome, an organelle specialized for this purpose (Figure 2.3). Once the microbe is inside the phagosome, granules containing the biological machinery needed to kill the microbe attach themselves to the membrane of the phagosome and dump their machinery into the extracellular medium between the phagosome membrane and the microbe. This

10 process is known as degranulation. During the degranulation process, the concentration of

MPO within the phagosome equates to roughly 2 mM.19

Within the phagosome (Figure 2.3),20 a bacterium is killed by cytotoxic agents.6 One of those toxic agents is hydrogen peroxide (H2O2). The H2O2 required for the MPO-mediated antimicrobial system may come from a variety of sources; one being NADPH oxidase.21

NADPH oxidase is an enzyme complex that assembles in the phagosomal membrane where -• it converts oxygen into the superoxide radical ion (O2 ), then undergoes dismutation to

1 produce H2O2. MPO exhibits both peroxidase and oxidizing activity, and when released inside the phagosome catalyzes several reactions (Figure 2.4A), referred to as the MPO- mediated antimicrobial system, one being the reaction of hydrogen peroxide (H2O2) with chloride (Cl-) to form hypochlorous acid (HOCl), which has antibacterial activity (Figure 2.4

B).22-24 Figure 2.4 A suggests that MPO-induced killing involves Compound I (CpI) catalyzing a variety of compounds into free radicals and forming Compound II (CpII). The free radicals produced from this reaction then interact with the engulfed microbe. CpII can react with superoxide or additional reducing agents to return to the native state (MP3+) and form oxygen or more radicals, respectively. Native MPO (MP3+) can also react with superoxide to form Compound III (CpIII or MPO2 in the table of Figure 2.5), but CpIII's utility within the phagosome is still in question.25 This simplified reaction network suggest that MPO has a direct involvement in the killing function of neutrophils.

It is highly likely that HOCl is involved in the killing of microorganisms. As HOCl has a pKa of 7.53, it exists as a mixture of the undissociated acid and the hypochlorite ion at physiologic pH levels. When the pH is lowered, as may occur in the phagosome, HOCl

11 26-28 predominates, and it can react with excess chloride to form molecular chlorine (Cl2).

These products, which are the reactive components of standard household bleach, are highly reactive and thus, short-lived, oxidizing agents that can attack the microorganisms at a variety of chemical sites.29 Essentially, any oxidizable group on the organism can be oxidized, and as a consequence, there may be a loss of microbial membrane transport,30 an interruption of the membrane electron transport chain,31 dissipation of adenylate energy reserves,32 and suppression of DNA synthesis with associated disruption of the interaction of the microbial cell membrane with the chromosomal origin of replication.33 HOCl also reacts with nitrogen-containing compounds to form nitrogen-chlorine derivatives such as monochloramines and dichloramines, which can degrade to the corresponding aldehyde.34,35

Some of these compounds retain oxidizing activity. For example, taurine, which is present at a high concentration in neutrophil cytoplasm,36,37 reacts with HOCl to form taurine chloramine, which is less toxic than HOCl, and this reaction has thus been implicated as a mechanism by which neutrophils are protected from HOCl released into the cytoplasm. The chloramines are long-lived, thus providing a mechanism for the prolongation of the oxidant activity of the peroxidase system. This allows for the penetration of MPO-derived oxidants into complex biological fluids, facilitating transport of toxicity to regions sufficiently distant that more reactive products are readily scavenged.

Oscillations in the reactive oxygen species and NADPH concentrations as a result of neutrophil activation and migration have been shown.38,39 Any time there is third or higher order chemical reaction involving autocatalysis and feedback, oscillatory reactions are possible. Neutrophils exhibit intrinsic sinusoidal metabolite oscillations when both resting

12 and migrating.40 Although the mechanisms for such oscillations have not been resolved, it has been proposed that various biochemical oscillators such as glycolysis and cytostolic calcium/inositol and calcium/AMP are possible sources of oscillations within cells and that myeloperoxidase, independent of exogenous oscillators, may initiate oscillations.38 In order to understand neutrophil activation and phagocytosis, it is pertinent to understand the oscillatory behavior and how the cellular composition affects these oscillations.

In previous studies, it has been shown that peroxidase-oxidase (PO) reaction (2.1):

Enzyme + 2NADH + O2 2NAD + 2 H2O (2.1)

is activated by melatonin, a substrate found in neutrophils. Such studies have employed horseradish peroxidase (HRP) or lactoperoxidase as catalysts, as these are more readily obtained than MPO. In 2003, Petty and co-workers showed that at pHs between 4 and 6, melatonin induces oscillations in this reaction.41 Unfortunately the experimental data were limited to only one set of conditions (100-125 µM NADH, 200 µM melatonin at a pH of 5.1).

Olsen et al., in 2004, then proposed a mechanism consistent with Petty's observations and obtained transient oscillations using MPO.42

13 Since MPO is similar to lactoperoxidase and melatonin has been shown to be a substrate for the classical peroxidase reaction (2.2),

Enzyme YH2 + O2 Y + 2 H2O (2.2)

which is also catalyzed by MPO, it is suggested that a MPO-catalyzed PO reaction could be involved in neutrophil oscillations. Much of the interest in the peroxidase-oxidase system came from the idea that it is a model system for many aspects of nonlinear dynamics in biology. Through modeling of these complex chemical perturbations, along with performing experiments to confirm results of simulation, will come increased ability to identify important interactions, pathways and compounds that influence biological networks where

MPO is involved.

MPO is arguably the most important enzyme involved in host defense, due to its ability to generate toxic oxidants such as hypochlorous acid (HOCl), which is thought to kill foreign invaders. Unfortunately, the description of MPO’s role in host defense is relatively vague considering the complex network in which MPO is involved. It is clear that although there are many things known or that have been studied in reference to the free radical-radical reactions involving MPO, there are many more details that are not yet well understood by the peroxidase community, which has led to great debate.4, 43-46

Despite its positive role in microbial killing, MPO and it products, have been implicated in a number of diseases like atherosclerosis, rheumatoid arthritis, lung cancer, cardiovascular

14 disease, multiple sclerosis, and Alzheimer’s disease.47-51 While MPO has been implicated in the pathologies of these diseases, MPO’s mechanistic involvement is also largely unknown.

The MPO reaction network is vast, with a large number of reactions that have not been extensively studied. A hypothesized network was shown in Figure 1.1. The vast majority of reactions depicted in Figure 1.1 have no experimentally measured kinetic information.43, 45, 52-

70 The kinetics surrounding MPO function inside and out of the phagosome are prime candidates for investigation both experimentally (rate constant measurements) and theoretically (reaction network simulations).71 Theoretical investigations into the reaction network of MPO have recently been published by Winterbourn, et al. and Olsen, et al. 16,42,53

Both groups used a simplified reaction network of MPO (similar to Figure 2.4), along with rate constants and initial conditions that were either measured experimentally or determined from a best guess when not readily available in the literature. Another issue of concern is that many of the rate constants found in the literature were not measured under biological conditions. This would mean that simulations are only as good as the approximated rate constants and would require further investigation as the quality and quantity of kinetic data for MPO improves.

Since the investigations of Petty, Olsen and Winterborn, much effort has been made to better understand the MPO reaction mechanism and its impact on the human immune response primarily because oscillatory reactions of MPO may be important in immunochemistry.

Unfortunately, because large-volume reactors (0.2 mL and above) require inordinate amounts of enzyme for reactions at physiologically relevant concentrations, and because ordinary microfluidic systems suffer from wall scavenging of free radicals, to our knowledge, the only

15 experimental demonstration of in vitro MPO-catalyzed oscillations is the cited Olsen experiment.42 As a result of Olsen’s work, questions arose as to whether MPO redox dynamics drove these oscillations or whether Ca2+/AMP-driven oscillations72,73 were masters and redox waves phase-locked slaves to cell membrane-controlled transport-driven oscillations. Obtaining definitive answers from discussion of interactions between calcium waves and MPO/redox oscillations is difficult due to the lack of reliable experimental systems where ion transport can be varied at will while the concentrations of Ca2+, inositol,

MPO, oxidants, and reductants are varied. Work in vivo is only suggestive, while theoretical work based on kinetics data involving only small subsets of the full set of species may miss synergistic effects in the reaction network. Numerous rate constants, nominally for homogeneous reaction, have become available from the Obinger,74-84 Winterbourn,75, 85-105 and Davies106 -134 groups, but many measurements may suffer from systematic error due to radical/wall interactions.

Herein lies the importance in studying and understanding biochemical networks and enzyme kinetics. It is clear that MPO is important to life’s cycle, but MPO and other single enzymes do not work alone or in a closed system. What is important is to understand the kinetics of the networks in which the small molecules and MPO are involved. As described above, much of the small-molecule chemistry, critical to both the intra- and extra-cellular MPO function, is sufficiently understood to allow for preliminary ex vivo understanding, but to date, much about MPO in vivo has yet to be discovered. Current research is aimed at gaining sufficient knowledge of these specific enzyme kinetics to learn how best to regulate these biochemical reactions through manipulation of endogenous species concentrations rather

16 than through development of novel compounds. Knowledge of these kinetics will also allow for the development of models that may indicate how to initiate in vivo biochemical oscillations, suppress them, or control their frequency and amplitude. Since oscillatory generation of reactive oxygen species has been correlated with killing of cancer cells,1, 5, 135 such control could prove important for fighting disease. One very important improvement will need to be the development and the utilization of clean and robust instrumentation to obtain experimental data for calculating accurate rate constants.

2.3 Studying Enzyme Kinetics: Macro vs. Micro Reaction Methods

Typical Reaction Methods

Accurate measurement of enzyme kinetics is an essential part of understanding the mechanisms of biochemical reactions. The typical means of studying such systems is using milliliter sized reactors, a microfluidic system on a chip, a stopped-flow system, or other small sample holder. With these setups, typically between 0.1 and 1 mL of reactant solution is needed per experiment. When studying oscillatory reactions, the use of milliliter volumes of enzyme-containing solution per experiment has proven to be expensive, except for the particularly widely-available, plant-derived HRP.42 A shift to microfluidics is an approach that is being explored by many to overcome this problem.

Especially with microfluidic systems, typical reaction vessels have walls that interact with molecules of systems under study and thus potentially alter their reaction kinetics. Major concerns include adsorption of reactants and products to the walls of the reaction vessel,136-

144 wall catalyzed recombination,145 difficulty in maintaining homogeneity,146 and difficulty

17 in maintaining good gas-liquid interface mass transfer (for reactions that produce or consume such species as O2, CO2, or CH4). Using such systems would prove problematic for enzyme systems such as MPO which is cationic at neutral pH (and so can adsorb on glass or quartz, which is anionic above pH 3), consumes O2, and generates free radicals.

In our lab, Lewis and Ruane showed that oscillatory chemistry in a macroscopic system is complicated by wall reactions.147 The study revealed that biofilms form on the quartz reactor surface in less than 48 hours after cleaning with NoChromex under conditions used for the

HRP-catalyzed peroxidase-oxidase reaction. As a result of the biofilms present, the nominally homogeneous peroxidase system exhibited dynamical changes over time that differed from that obtained when using different cleaning procedures in our laboratory and from that observed in other laboratories in, e.g., Indianapolis, IN, Palo Alto, CA, and Odense,

Denmark. The study also suggested that bare quartz also interacts with reaction species.

These studies make clear that wall interaction and surface area-to-volume ratio are important parameters on which the observed dynamics depend. (Figure 2.6) As the dimensions of reactor vessels become smaller (go from macro to micro), surface area-to-volume ratio increases, the effect of wall interactions become more important, and the assumption of homogeneity in a system becomes less assured.

147 The quartz reactor turned out to be a receptive environment for the formation of biofilms.

Biofilms are structures built by and containing microorganisms that adhere to living and inert surfaces. Biofilms can and will interact with a variety of substrates and enzymes and are a highly dynamic system. Biofilms interact with enzymes in an unknown fashion, resulting in

18 147 inconsistent results and poor correlation to biological environments. Scanning Electron

Microscopy (SEM) was used to collect images of the biofilms (Figure 2.7) found by Lewis,

Ruane and Scheeline when using their 5 mL quartz cuvette system, equipped with inlets and outlets for reactants and a stirring rod, to study the Peroxidase-Oxidase oscillatory reaction.

As mentioned above, surface treatments can be used to minimize the effect of biofilms in flow reactors, but they do not eliminate all effects. In many cases, they may further complicate the system.

MPO’s reaction network, as well as those of many enzymes, involves a large number of reactions in which radicals are either reactants or products. This is a problem for quartz cuvettes, which have been known to scavenge free radicals from solution. Since quartz is not present in vivo, it is important to develop an in vitro measurement system that will closely represent in vivo conditions. A wall-less reaction vessel would work best. Both biofilm formation and radical scavenging would be eliminated in a wall-less system.

Levitation: What is it, and why are we interested?

As a way to circumvent the problems exhibited by other reactor types, our lab has developed an alternative approach to studying enzyme-catalyzed kinetics related to free radical and oxidative-stress chemistry. Christopher Field and I have designed, built and characterized an

Acoustically Levitated Drop Reactor (LDR) to use as a microreactor.71 With the LDR, an acoustic wave, produced by a piezoelectric transducer, is reflected by a metallic reflector or quartz window, creating a standing wave. Drops levitate just below the nodes in this field.

Details regarding optimal levitator design are debatable, but the theory is largely

19 understood.148, 149

Levitation is not a new concept. Over the past 15 years, several experiments have been performed in levitated drops including fluorescence measurements, supercritical fluid studies and droplet dynamics. It was not until 2003-2005 that levitators were considered for use in studying biochemical reactions.150 There are four main types of levitation; magnetic, electrostatic,151-154 optical,155,156 and acoustic.157,158

Electrostatic and magnetic levitation generally used in, and are most suitable for, non-contact handling applications.152,153 Magnetic and electrostatic levitation have been used in a variety of applications including bearings, transportation systems and precision actuators.

Optical levitation159 is the leviation of an object within two standing-wave optical modes of a

Fabry-Perot cavity. One large trapping beam provides an optical potential that traps the object near an antinode. Alternatively, optical pressure from an upwardly-directed laser may support a particle absent a resonant cavity. A second more weakly driven cavity mode with a nonvanishing intensity gradient at the trap center is used to cool the motion of the object.

Optical Levitation is most often used for studying microparticles and to achieve enhanced micromanipulation of those particles.160 Optical levitation has also been considered for use in droplet science and studies of aerosols.161,162

20 Acoustic levitation, as mentioned before, uses a standing acoustic wave. Of the various types of levitation, acoustic levitation offers several advantages for studying enzyme kinetics. The lack of magnetic force, electrical charge, or light pressure ensures that there is no added interference or complexity from additional interactions to the already complex enzyme catalyzed reactions. Nanoliter to microliter drops levitate just below the nodes in this field.

The sample volume (and mass) required for experiments is reduced by more than 1000 times compared to that of an experiment with a macroscale reactor. Additional advantages of acoustic levitation are the prevention of chemical contamination between drops and external objects, and potentially increased analytical sensitivity, since no walls disturb detection.163,164

There is also the added advantage of easy access for a wide range of optical, electrochemical, and (in some laboratories) mass-spectral diagnostics.165.166 This technique, first described in

1933,167 has been used to study materials properties, the shape and stability of liquid drops,168 single cell analysis, protein crystallization, and to perform sample preparation (acid-base titrations, liquid/liquid extractions, and solvent exchange). Other examples are cited in

Santesson et al.169 Other than of the static chemical systems that have been studied, there is only one example of use of levitated drops to study reaction kinetics prior to the work described in this thesis. That is a study conducted by David Weis and coworkers, measuring the kinetics of alkaline phosphatase in supercooled water (T< 0˚C).150

21 2.4 An Acoustically Levitated Drop Reactor (LDR)

The acoustic levitator designed and characterized in our lab, although not the first acoustic levitator built, is the most efficient of which we are aware and is completely computer controlled. (Figure 2.8) The design was inspired by Weis’s levitator, but is more user- friendly and versatile, making it useful for studying a variety of reaction systems. Our levitator requires six major components to make it functional; 1) an ultrasound transducer, 2) an ultrasonic horn, 3) a radiating plate, 4) a reflector, 5) the electronics and 6) computer automation.

1) The Ultrasound Transducer. The sound wave necessary for acoustic levitation is

generated by the use of a Langevin, or sandwich, ultrasound transducer. Two

piezoelectric ceramics (5.08 cm diameter, 0.318 cm thickness) are compressed

between two metal blocks and a torque on the four compression nuts of 11.25 inch·lb

is applied to prevent fatigue failure and to ensure good acoustic coupling with the

metal blocks. The pressure bias is achieved using a pair of flanges secured with four

bolts on the ends of the blocks. When a potential is applied longitudinally across the

piezoelectrics, the ceramic crystals convert the electrical energy into mechanical

energy, which results in mechanical motion or sound.

2) The Ultrasound Horn. A horn is used to focus and shape the ultrasound emitted from

the transducer. In our design, a stepped horn is used. The stepped horn is designed in

two quarter-wave blocks with horn radius smaller than transducer radius. This design

maximizes the standing wave amplitude at the tip if the horn. The horn, the upper

22 transducer quarter-wave block, and the radiating plate are made from a single block

of metal to ensure good coupling. The horn is attached to the transducer using a pair

of flanges.

3) The Radiating Plate. A metal radiating plate (2.54 cm diameter, 0.318 cm thickness)

is attached to the end of the stepped horn. This provides proper coupling of the

ultrasound to the medium and provides a nodal structure for acoustic levitation.

4) The Reflector. A concave reflector, with a radius of curvature of 33 mm, is used to

establish a standing wave between it and the radiating plate at 20.73 kHz in a

medium, with in our case is typically ambient air. The distance between the

resonating plate and the reflector determines the reflector’s effectiveness, and the

levitation positions (nodes) within the cavity. Between the resonating plate and

reflector, when a potential has been applied across the piezoelectrics, standing waves

are generated and it is just below the nodes that microliter or smaller volumes of

sample can be acoustically levitated. In order to maintain levitation, the reflector

must maintain a specific distance from the resonating plate. Equations are used to

determine the optimal distance based on the temperature, pressure, and relative

humidity in the room. All equations and specifics of the characterization of this

instrument are discussed in detail in Christopher Field’s thesis.170

23

5) The Electronics. Prior to the design of our LDR, acoustic levitators commonly used a

function generator, a 200 W amplifier, and an impedance matching transformer to

drive the piezoelectrics. This cost on the order of $5000 just to generate enough

power for acoustic levitation (mainly due to the cost of the adjustable, multi-tap

transformer). With our design, we utilize a function generator (Agilent Technologies,

Inc.) and an in-house designed 15 W ten times gain amplifier based on a commercial

power operational amplifier to achieve acoustic levitation. With this design, the

power needed to drive the piezoelectrics, capital cost, and the cost of operation, were

reduced.

6) Computer Automation. The LDR is completely controlled using several

programmed modules in LabView 8.0 (National Instruments, Inc). All of the

electronics mentioned above, in addition to all of the data acquisition hardware, are

controlled using this computer software.

The LDR designed in our lab has been functional and in use since late 2006 to explore opportunities for use of this instrument as a microreactor. As long as the pressure on the piezoelectric/transducer sandwich is stable so that the resonance frequency of the assembly is constant, reliable operation has been achieved.

24 2.5 Sample Handling in an Acoustic Levitator: Drop Generation, Reactant Introduction, Maintenance and Fluid Removal

The development of various sample handling, mixing, and detection techniques for use in levitation would potentially allow for the use of drops as microreactors and specifically the study of biochemical/enzyme kinetics. Microliter-scale droplet generation, reactant introduction, maintenance and fluid removal are all important aspects in conducting reactions in a levitated drop. With enzyme reactions, many reactants involved are costly, in limited supply, or have limited solubility. Therefore small sample volumes, typically on the nanoliter scale, are required.

Typically, gas chromatography (GC) syringes are used to form an initial drop in an acoustic levitation cavity.171,172 From there, drop-on-demand (DOD) dispensers are used to add additional reactants. DOD technology is extremely valuable for the addition of high concentration species in small volumes. Drops are typically removed from the cavity by cutting the power, allowing the drop to fall to the resonating plate and wiping the plate clean.

Alternatively, a capillary can be plunged into the drop, followed by removal by aspiration.

Although the aforementioned sample handling techniques are simple and achieve the goal of levitating drops that contain various reactants, there is room for improvement. There is definitely room for innovation in the areas of drop introduction and drop removal. With this in mind, our lab has explored a unique technique that could be used alone or in addition to the current methods to provide fluid handling to and from an acoustically levitated drop that operates as a microreactor. The development of this system and implementation into the

25 LDR as a drop formation system, using surface-coated capillary bundles, is the central instrument development reported in this thesis; see Chapter 3.

2.6 Summary

Understanding how the immune system operates, normally and when under attack, is very important in biology and medicine. Current research is aimed at understanding of specific enzyme-catalyzed oscillating reactions that generate reactive oxygen species, in order to regulate them.173 This can potentially lead to more enhanced and personalized drug therapy.

Studying enzyme-catalyzed reactions using standard reaction systems requires large sample sizes. Conventional micro-scale reactors have high surface-to-volume ratios, and have been found to alter kinetics. Our lab designed and characterized the most efficient and computer controlled, acoustically levitated drop system for use as a microreactor. In addition, we have developed a unique sample handling system for proper fluid handing in the LDR. The LDR has been discussed in my predecessor’s thesis. This thesis describes the sample handling system and its use in studying a variety of chemical kinetics, including enzyme-catalyzed kinetics. Throughout the chapters, the advantages and challenges of using this system will also be discussed along with future directions for development of the LDR, the sample handling system, and kinetic studies.

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Human Myeloperoxidase," FEBS Letters, 484, 139-143 (2000).

37 77 P. G. Furtmüller, U. Burner, W. Jantschko, G. Regelsberger and C. Obinger, "The

Reactivity of Myeloperoxidase Compound I with Hypochlorous Acid," Redox Report,

5, 173-178 (2000).

78 P. G. Furtmüller, C. Obinger, Y. Hsuanyu and H. B. Dunford, "Mechanism of

Reaction of Myeloperoxidase with Hydrogen Peroxide and Chloride Ion," European

Journal of Biochemistry, 267, 5858-5864 (2000).

79 M. Allegra, P. G. Furtmüller, G. Regelsberger, M. L. Turco-Liveri, L. Tesoriere, M.

Perretti, R. A. Livrea and C. Obinger, "Mechanism of Reaction of Melatonin with

Human Myeloperoxidase," Biochemical and Biophysical Research Communications,

282, 380-386 (2001).

80 J. Arnhold, P. G. Furtmüller, G. Regelsberger and C. Obinger, "Redox Properties of

the Couple Compound I/Native Enzyme of Myeloperoxidase and Eosinophil

Peroxidase," European Journal of Biochemistry, 268, 5142-5148 (2001).

81 W. Jantschko, P. G. Furtmüller, M. Zederbauer, M. Lanz, C. Jakopitsch and C.

Obinger, "Direct Conversion of Ferrous Myeloperoxidase to Compound II by

Hydrogen Peroxide: An Anaerobic Stopped-Flow Study," Biochemical and

Biophysical Research Communications, 312, 292-298 (2003).

82 W. Jantschko, P. G. Furtmüller, M. Zederbauer, C. Jakopitsch and C. Obinger,

"Kinetics of Oxygen Binding to Ferrous Myeloperoxidase," Archives of Biochemistry

and Biophysics, 426, 91-97 (2004).

38 83 D. R. Ramos, M. V. Garcia, L. M. Canle, S. J. Arturo, P. G. Furtmuller and C.

Obinger, "Myeloperoxidase-catalyzed Taurine Chlorination: Initial Versus

Equilibrium Rate," Archives of Biochemistry and Biophysics, 466, 221-233 (2007).

84 D. R. Ramos, M. V. Garcia, L. M. Canle, J. A. Santaballa, P. G. Furtmüller and C.

Obinger, "Myeloperoxidase-catalyzed Chlorination: The Quest for the Active

Species," Journal of Inorganic Biochemistry, 102, 1300-1311 (2008).

85 C. C. Winterbourn, M. B. Hampton, J. H. Livesey and A. J. Kettle, "Modeling the

Reactions of Superoxide and Myeloperoxidase in the Neutrophil Phagosome.

Implications for Microbial Killing," Journal of Biological Chemistry, 281, 39860-

39869 (2006).

86 A. J. Kettle, T. Chan, I. Osberg, R. Senthilmohan, A. L. P. Chapman, T. J. Mocatta

and J. S. Wagener, "Myeloperoxidase and Protein Oxidation in the Airways of Young

Children with Cystic Fibrosis," American Journal of respiratory and Critical Care

Medicine, 170, 1317-1323 (2004).

87 A. J. Kettle, D. F. Sangster, J. M. Gebicki and C. C. Winterbourn, "A Pulse

Radiolysis Investigation of the Reactions of Myeloperoxidase with Superoxide and

Hydrogen Peroxide," Acta Biochimica et Biophysica, 956, 58-62 (1988).

88 A. J. Kettle and C. C. Winterbourn, "Superoxide Modulates the Activity of

Myeloperoxidase and Optimizes the Production of Hypochlorous Acid," Biochemical

Journal, 252, 529-536 (1988).

39 89 C. C. Winterbourn and A. J. Kettle, "Reactions of Myeloperoxidase with Superoxide

and Hydrogen Peroxide: Significance for Its Function in the Neutrophil," Basic Life

Sci. 49 (Oxygen Radicals Biol.Med.), (1988).

90 A. J. Kettle and C. C. Winterbourn, "Influence of Superoxide on Myeloperoxidase

Kinetics Measured with a Hydrogen Peroxide Electrode," Biochemical Journal, 263,

823-828 (1991).

91 A. J. Kettle, and C. C. Winterbourn, "Superoxide-dependent Hydroxylation by

Myeloperoxidase," Journal of Biological Chemistry, 269, 17146-17151 (1994).

92 N. M. Domigan, T. S. Charlton, M. W. Duncan, C. C. Winterbourn, and A. J. Kettle,

"Chlorination of Tyrosyl Residues in Peptides by Myeloperoxidase and Human

Neutrophils," Journal of Biological Chemistry, 270, 16542-16548 (1995).

93 M. B. Hampton, A. J. Kettle and C. C. Winterbourn, "Involvement of Superoxide and

Myeloperoxidase in Oxygen-Dependent Killing of Staphylococcus aureus by

Neutrophils," Infection Immunity 64, 3512-3517 (1996).

94 A. J. Kettle, and C. C. Winterbourn, "Reaction Mechanisms of Myeloperoxidase," in

Plant Peroxidases: Biochemistry and Physiology, C. Obinger, U. Burner, R.

Ebermann, C. Penel, and H. Greppin, eds. (University of Geneva, Geneva, 1996), pp.

134-139.

95 A. J. Kettle, and C. C. Winterbourn, "Myeloperoxidase -- a Key Regulator of

Neutrophil Oxidant Production," Redox Report, 3, 3-15 (1997).

40 96 C. J. van Dalen, M. W. Whitehouse, C. C. Winterbourn, and A. J. Kettle,

"Thiocyanate and Chloride as Competing Substrates for Myeloperoxidase,"

Biochemical Journal, 327, 487-492 (1997).

97 C. C. Winterbourn, H. Pichorner and A. J. Kettle, "Myeloperoxidase-dependent

Generation of Tyrosine Peroxide by Neutrophils," Archives of Biochemistry and

Biophysics, 338, 15-21 (1997).

98 M. B. Hampton, A. J. Kettle and C. C. Winterbourn, "Inside the Neutrophil

Phagosome: Oxidants, Myeloperoxidase, and Bacterial Killing," Blood, 92, 3007-

3017 (1998).

99 A. J. Kettle and L. P. Canadaeis, "Oxidation of Tryptophan by Redox Intermediates

of Myeloperoxidase and Inhibition of Hypochlorous Acid Production," Redox Report,

5, 179-184 (2000).

100 C. C. Winterbourn and A. J. Kettle, "Biomarkers of Myeloperoxidase-derived

Hypochlorous Acid," Free Radical Biology and Medicine, 29, 403-409 (2000).

101 C. C. Winterbourn M. C. M. Vissers, and A. J. Kettle, "Myeloperoxidase," Current

Opinions in Hematology, 7, 53-58 (2000).

102 A. J. Kettle and C. C. Winterbourn, "A Kinetic Analysis of the Catalase Activity of

Myeloperoxidase," Biochemistry, 40, 10204-10212 (2001).

41 103 V. F. Ximenes, S. D. Silva, M. R. Rodrigues, L. H. Catalani, G. J. Maghzal, A. J.

Kettle and A. Campa, "Superoxide-Dependent Oxidation of Melatonin by

Myeloperoxidase," Journal of Biological Chemistry, 280, 38160-38169 (2005).

104 A. J. Kettle, R. F. Anderson, M. B. Hampton and C. C. Winterbourn, "Reactions of

Superoxide with Myeloperoxidase," Biochemistry, 46, 4888 - 4897 (2007).

105 V. F. Ximenes, G. J. Maghzal, R. Turner, Y. Kato, C. C. Winterbourn and A. J.

Kettle, "Serotonin as a Physiological Substrate for Myeloperoxidase and its

Superoxide-dependent Oxidation to Cytotoxic Tryptamine-4,5-dione " Biochemical

Journal, 425, 285-293 (2009).

106 S. Fu, H. Wang, M. J. Davies and R. T. Dean, "Reactions of Hypochlorous Acid with

Tyrosine and Peptidyl-tyrosyl Residues Give Dichlorinated and Aldehydic Products

in Addition to 3-chlorotyrosine," Journal of Biological Chemistry, 275, 10851-10858

(2000).

107 A. Wright, C. L. Hawkins and M. J. Davies, "Single Oxygen-mediated Protein

Oxidation: Evidence for the Formation of Reactive Peroxides," Redox Report, 5, 159-

161 (2000).

108 C. L. Hawkins, B. E. Brown and M. J. Davies, "Hypochlorite- and Hypobromite-

mediated Radical Formation and Its Role in Cell Lysis," Archives of Biochemistry

and Biophysics, 395, 137-145 (2001).

42 109 D. L. Pattison and M. J. Davies, "Absolute Rate Constants for the Reaction of

Hypochlorous Acid with Protein Side Chains and Peptide Bonds," Chemical

Research and Toxicology, 14, 1453-1464 (2001).

110 B. S. van der Veen, M. P. J. de Winther, P. Heeringa, O. Augusto, J. W. Chen, M. J.

Davies, X.-L. Ma, E. Malle, P. Pignatelli and T. Rudolph, "Myeloperoxidase:

Molecular Mechanisms of Action and Their Relevance to Human Health and

Disease," Antioxidant Redox Signaling, 11, 2899-2937 (2009).

111 H. A. Headlam, and M. J. Davies, "Cell-mediated Reduction of Protein and Peptide

Hydroperoxides to Reactive Free Radicals," Free Radical Biology and Medicine, 34,

44-55 (2002).

112 H. Ostdal, M. J. Davies, and H. J. Andersen, "Reaction between Protein Radicals and

Other Biomolecules," Free Radical Biology and Medicine, 33, 201-209 (2002).

113 D. L. Pattison, M. J. Davies and K.-D. Asmus, "Absolute Rate Constants for the

Formation of Nitrogen-centred Radicals from Chloramines/Amides and Their

Reactions with Antioxidants," Journal of the Chemical Society-Perkin Transactions

2- Physical Organic Chemistry, 8, 1461-1467 (2002).

114 A. Wright, W. A. Bubb, C. L. Hawkins and M. J. Davies, "Singlet Oxygen-mediated

Protein Oxidation: Evidence for the Formation of Reactive Side Chain Peroxides on

Tyrosine Residues," Photochemistry and Photobiology, 76, 35-46 (2002).

43 115 M. J. Davies, "Singlet Oxygen-Mediated Damage to Proteins and Its Consequences,"

Biochemical and Biophysical Research Communications, 305, 761-770 (2003).

116 C. L. Hawkins, D. L. Pattison and M. J. Davies, "Hypochlorite-induced Oxidation of

Amino Acids, Peptides, and Proteins," Amino Acids, 25, 259-274 (2003).

117 D. L. Pattison, C. L. Hawkins and M. J. Davies, "Hypochlorous Acid-Mediated

Oxidation of Lipid Components and Antioxidants Present in Low-Density

Lipoproteins: Absolute Rate Constants, Product Analysis, and Computational

Modeling," Chemical Research in Toxicology, 16, 439-449 (2003).

118 M. D. Rees, C. L. Hawkins and M. J. Davies, "Hypochlorite-mediated Fragmentation

of Hyaluronan, Chondroitin Sulfates, and Related N-acetyl Glycosamines: Evidence

for Chloramide Intermediates, Free Radical Transfer Reactions, and Site-specific

Fragmentation," Journal of the American Chemical Society, 125, 13719-13733

(2003).

119 D. L. Pattison and M. J. Davies, "Kinetic Analysis of the Reactions of Hypobromous

Acid with Protein Components: Implications for Cellular Damage and the Use of 3-

Bromotyrosine as a Marker of Oxidative Stress," Biochemistry, 43, 4799-4809

(2004).

120 M. D. Rees, C. L. Hawkins and M. J. Davies, "Hypochlorite and Superoxide Radicals

Can Act Synergistically to Induce Framentation of Hyaluronan and Chondroitin

Sulfates," Biochemical Journal, 381, 175-184 (2004).

44 121 M. J. Davies, "The Oxidative Environment and Protein Damage," Biochimica et

Biophysica Acta, 1703, 93-109 (2005).

122 D. L. Pattison and M. J. Davies, "Kinetic Analysis of the Role of Histidine

Chloramines in Hypochlorous Acid Mediated Protein Oxidation," Biochemistry, 55,

7378-7387 (2005).

123 E. Malle, G. Marsche, J. Arnhold and M. J. Davies, "Modification of Low-Density

Lipoprotein by Myeloperoxidase-Derived Oxidants and Reagent Hypochlorous

Acid," Biochimica et Biophysica Acta, 1761, 392-415 (2006).

124 D. I. Pattison, C. L. Hawkins and M. J. Davies, "Hypochlorous Acid-Mediated

Protein Oxidation: How Important Are Chloramine Transfer Reactions and Protein

Tertiary Structure?," Biochemistry, 46, 9853-9864 (2007).

125 O. Skaff, D. I. Pattison and M. J. Davies, "Kinetics of Hypobromous Acid-Mediated

Oxidation of Lipid Components and Antioxidants," Chemical Research in

Toxicology, 20, 1980-1988 (2007).

126 A. E. Lane, J. T. M. Tan, C. L. Hawkins, A. K. Heather and M. J. Davies, "The

Myeloperoxidase-derived Oxidant HOSCN Inhibits Protein Tyrosine Phosphatases

and Modulates Cell Signalling Via the Mitogen-Activated Protein Kinase (MAPK)

Pathway in Macrophages," Biochemical Journal, 430, 161-169 (2010).

45 127 A. J. Szuchman-Sapir, D. I. Pattison, M. J. Davies and P. K. Witting, "Site-specific

Hypochlorous Acid-induced Oxidation of Recombinant Human Myoglobin Affects

Specific Amino Acid Residues and the Rate of Cytochrome b5-mediated Heme

Reduction," Free Radical Biology and Medicine, 48, 35-46 (2010).

128 C. L. Hawkins, and M. J. Davies, "Hypochlorite-induced Oxidation of Plasma

Proteins: Formation of Nitrogen-centered Radicals and Their Role in Protein

Fragmentation," Current Topics in Biophysics, 22 (Suppl. B), 89-98 (1998).

129 C. L. Hawkins, and M. J. Davies, "Reaction of HOCl with Amino Acids and

Peptides: EPR Evidence for Rapid Rearrangement and Fragmentation Reactions of

Nitrogen-centered Radicals," Journal of the Chemical Society-Perkin Transactions 2-

Physical Organic Chemistry, 9, 1937-1946 (1998).

130 C. L. Hawkins, and M. J. Davies, "Hypochlorite-induced Damage to Proteins:

Formation of Nitrogen-centered Radicals from Lysine Residues and Their Role in

Protein Fragmentation," Biochemical Journal, 332, 617-625 (1998).

131 M. J. Davies, S. Fu, H. Wang, and R. T. Dean, "Stable Markers of Oxidant Damage

to Proteins and Their Application in the Study of Human Disease," Free Radical

Biololgy and Medicine, 27, 1151-1163 (1999).

132 C. L. Hawkins and M. J. Davies, "Hypochlorite-induced Oxidation of Proteins in

Plasma: Formation of Chloramines and Nitrogen-centered Radicals and Their Role in

Protein Fragmentation," Biochemical Journal, 340, 539-548 (1999).

46 133 M. J. Davies and C. L. Hawkins, "Hypochlorite-induced Oxidation of Thiols:

Formation of Thiyl Radicals and the Role of Sulfenyl Chlorides as Intermediates,"

Free Radical Research, 33, 719-729 (2000).

134 C. L. Hawkins, D. L. Pattison and M. J. Davies, "Reaction of Protein Chloramines

with DNA and Nucleosides: Evidence for the Formation of Radicals, Protein-DNA

Cross-links and DNA Fragmentation," Biochemical Journal, 365, 605-615 (2002).

135 J. B. Huang, A. L.Kindzelskii, A. J. Clark and H. R. Petty, “Identification of

Channels Promoting Calcium Spikes and Waves in HT1080 Tumor Cells: Their

Apparent Roles in Cell Motility and Invasion,” Cancer Research, 64, 2482-2489

(2004).

136 E. S. Kirkor and A. Scheeline, "Horseradish Peroxidase Adsorption on Silica Surfaces

as an Oscillatory Dynamical Behavior," Journal of Physical Chemistry B, 105, 6278-

6280 (2001).

137 E. S. Kirkor and A. Scheeline, “Catalyst Adsorption as a Dynamic Phenomenon in

the Peroxidase-Oxidase Oscillator,” in ESPCI, Paris, France (2001).

138 R. Miller, V. B. Fainerman, A. V. Makievski, J. Krägel, D. O. Grigoriev, V. N.

Kazakov and O. V. Sinyachenko, “Dynamics of Protein and Mixed Protein/Surfactant

Adsorption Layers at the Water/Fluid Interface,” Advances in Colloid and Interface

Science, 86, 39-82 (2000).

47 139 J. Deere, E. Magner, J. G. Wall and B. K. Hodnett, “Mechanistic and Structural

Features of Protein Adsorption onto Mesoporous Silicates,” Journal of Physical

Chemistry B, 106, 7340-7347 (2002).

140 K. Lenghaus, J. W. Dale, J. C. Henderson, D. C. Henry, E. R. Loghin and J. J.

Hickman, “Enzymes as Ultrasensitive Probes for Protein Adsorption in Flow

Systems,” Langmuir, 19, 5971-5974 (2003).

141 C. Czeslik, “Factors Ruling Protein Adsorption,” Zeitschrift Für Physikalische

Chemie-International Journal of Research in Physical Chemistry and Chemical

Physics (Muenchen), 218, 771-801 (2004).

142 J. Jenkins, B. Prabhakarpandian, K. Lenghaus, J.J. Hickman and S. Sundaram,

“Fluidics-resolved Estimation of Protein Adsorption Kinetics in a Biomicrofluidic

System,” Analytical Biochemistry, 331, 207-215 (2004).

143 C. A. Lucy, N. E. Baryla and K. K.-C. Yeung, “Surfactant-based Methods for

Prevention of Protein Adsorption in Capillary Electrophoresis,” in Capillary

Electrophoresis of Proteins and Peptides, 276 (Methods in Molecular Biology) 1-14

(Totowa, NJ, 2004).

144 L. S. Roach, H. Song and R. F. Ismagilov, “Controlling Nonspecific Protein

Adsorption in a Plug-Based Microfluidic System by Controlling Interfacial Chemistry

Using Fluorous-Phase Surfactants.” Analytical Chemistry, 77, 785-796 (2005).

48 145 R. M. Noyes, “Wall Effects in Photochemically Induced Chain Reactions,” Journal

of the American Chemical Society, 73, 3039-3043 (1951).

146 T. J. Johnson, D. Ross and L. E. Locascio, “Rapid Microfluidic Mixing,” Analytical

Chemistry, 74, 45-51 (2002).

147 D. D. Lewis, M. L. Ruane and A. Scheeline, “Biofilm Effects on the Peroxidase-

oxidase Reaction,” Journal of Physical Chemistry B, 110, 8100-8104 (2006).

148 W. A. Oran, L. H. Berge and H. W. Parker, “Parametric Study of an Acoustic

Levitation System,” Review of Scientific Instruments, 51, 626-631 (1980).

149 R. Hill and S. M. El-Dardiry, “A Theory for Optimization in the Use of Acoustic

Emission Transducers,” Journal of the Acoustical Society of America, 67, 673-682

(1980).

150 D. D. Weis and J. D. Nardozzi, "Enzyme Kinetics in Acoustically Levitated Droplets

of Supercooled Water: A Novel Approach to Cryoenzymology," Analytical

Chemistry, 77, 2558-2563 (2005).

151 N. D. Draper, S. F. Bakhourn, A. E. Haddrell and G. R. Agnes, “Ion-Induced

Nucleation in Solution: Promotion of Solute Nucleation in Charged Levitated

Droplets,” Journal of the American Chemical Society, 129, 11364-11377 (2007).

49 152 S. F. Bakhourn and G. R. Agnes, “Study of Chemistry in Droplets with Net Charge

Before and After Coulomb Explosion: Ion-Induced Nucleation in Solution and

Implications for Ion Production in an Electrospray,” Analytical Chemistry, 77, 3189-

3197 (2005).

153 M. J. Bogan and G. R. Agnes, “Preliminary Investigation of the Electrodynamic

Charged Droplet Processing to Couple Capillary Liquid Chromoatography with

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry,” Rapid

Communications in Mass Spectrometry, 18, 2673-2681 (2004).

154 X. Feng, M. J. Bogan, E. Chuah and G. R. Agnes, “Chemical Probe Using Field-

Induced Droplet Ionization Mass Spectrometry,” Journal of Aerosol Medicine-

Desposition Clearance and Effects in the Lung, 32, 1147-1159 (2001).

155 M. Trunk, J. Popp, M. Lankers and W. Kiefer, “Microchemistry: Time Dependence

of Acid-Base Reaction in a Single Optically Levitated Microdroplet,” Chemical

Physical Letters, 264, 233-237 (1997).

156 I. Hartmann, J. Popp, M. Lankers, M. Trunk and W. Kiefer, “Theory for Morphology

Dependent Resonances in the Raman Spectra of Optically Levitated Microspheres,”

Berichte Der Bunsen-Gesellschaft-Physical Chemistry Chemical Physics, 101, 809-

813 (1997).

157 G. Kaduchak, D. N. Sinha and D. C. Lizon, “Novel Cylindrical, Air-Coupled

Acoustic Levitation/Concentration Devices,” Review of Scientific Instruments, 73,

1332-1336 (2002).

50 158 T. L. Stephens and R. S. Budwig, “Three-Axis Acoustic Device for Levitation of

Droplets in an Open Gas Stream and Its Application to Examine Sulfur Dioxide

Adsorption by Water Molecules,” Review of Scientific Instruments, 78, 014901

014901-014908 (2007).

159 D. E. Chang, "Cavity Opto-Mechanics Using an Optically Levitated Nanosphere."

Proceedings of the National Academy of Sciences USA, 107 1005 (2010).

160 G. G. C. Veneranda , D. Kishan and G. C. Spalding, “Extended-area optically

induced organization of microparticles on a surface,” Applied Physical Letters. 86,

(PA: 90188) (2005)

161 E. J. Davis, “A history of single aerosol particle levitation,” Aerosol Science and

Technology, 26, 212 (1997).

162 C. Y. Yung, M. D. Barnes, N. Lermer, W. B. Whitten, and J. M. Ramsey, Single

Molecule Analysis of Ultradilute Solutions Using Guided Streams of 1-mm Water

Droplets,” Applied Optics 38, 1481-1487 (1999).

163 V. Vandaele, P. Lambert and A. Delchambre, “Non-Contact Handling in

Microassembly: Acoustic Levitation,” Precision Engineering, 29, 491-505 (2005).

164 F. Priego-Capote and L. de Castro, “Ultrasound-Assisted Levitation: Lab-on-a Drop,”

Trends in Analytical Chemistry, 25, 856-867 (2006).

165 R. T. Hilger, M. S. Westphall and L. M. Smith, "Controlling Charge on Levitated

Drops," Analytical Chemistry, 79, 6027-6030 (2007).

51 166 M. S. Westphall, K. Jorabchi and L. M. Smith, "Mass Spectrometry of Acoustically

Levitated Drops," Analytical Chemistry, 80, 5847-5853 (2008).

167 W. Meissner and R. Ochsenfeld, “Ein Neuer Effekt Bei Eintritt der

Supraleitfahigkeit,” Naturwissenschaften, 21, 787-788 (1933).

168 H. Liu, and P.K Dasgupta, “A Liquid Drop: A Windowless Optical Cell and a

Reactor Without Walls for Flow Injection Analysis,” Analytica Chimica Acta, 326,

13-22, (1996).

169 S. Santesson, E. S. Cedergren-Zeppezauer, T. Johansson, T. Laurell, J. Nilsson, and

S. Nilsson, “Screening of Nucleation Conditions Using Levitated Drops for Protein

Crystallization,” Analytical Chemistry, 75, 1733-1740 (2003).

170 C. R. Field, Developments in Analytical Chemistry: Acoustically Levitated Drop

Reactors for Enzyme Reaction Kinetics nad Single-walled Carbon Nanotube-based

Sensors for Detection of Toxic Organic Phosphonates, Ph.D. thesis, University of

Illinois at Urbana-Champaign (2009).

171 S. Santesson, and S. Nilsson, "Airborne Chemistry: Acoustic Levitation in Chemical

Analysis," Bioanalytical Chemistry, 378, 1704-1709 (2004).

172 E. H. Trinh, "Compact Acoustic Levitation Device for Studies in Fluid Dynamics and

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Instruments, 56, 2059-2065 (1985).

52 173 R. A. Siegel, Y. Gu, A. Baldi and B. Ziaie, “Novel Swelling/Shrinking Behaviors of

Glucose-Binding Hydrogels and Their Potential Use in a Microfluidic Drug Delivery

System,” Macromolecular Symposia, 207, 249-256 (2004).

53 2.8 Chapter 2 Figures

Figure 2.1 A) A cartoon representation of the reactions occurring within an activated neutrophil which will generate toxic oxidants to destroy foreign microorganisms. Reprinted from Current Opinion in Microbiology, 7, Mayer-Scholl, A. et al., “How Do Neutrophils and Pathogens Interact?,” 62, 2004 with permission from Elsevier publishing.

54 Figure 2.2 A) Crystal structure of MPO emphasizing B) the non-polar porphyrin ring in MPO and its covalent attachments to the preotein via two ester bonds. Reprinted from the Annual Review of Immunology, 23, Segal, A., “How Neutrophils Kill Microbes,” 197, 2005, with permission of Annual Reviews.

55 Figure 2.3 A cartoon representation of the steps involved in a macrophage ingesting and killing a pathogen. Reproduced from New Scientist, 1605, “The Human Immune System: The Lymphocyte Story,” 1, 1988 with permission of Elsevier publishing.

56 A.

B.

Figure 2.4 A) Products of the MPO-mediated antimicrobial system and the B) MPO complex formation. Reproduced from the Journal of Leukocyte Biology, 77, Klebanoff, S.J., “Myeloperoxidase: Friend or Foe,” 598, 2005, with permission of the Federation of American Societies for Experimental Biology.

57 Figure 2.5 List of reactions in the simplified reaction network of MPO. Units on rate constants are s-1 for first-order reactions and M-1s-1 for second- order reactions. Reprinted from the Journal of Biological Chemistry, 281, Winterborn, C. C. et al., “Modeling the Reactions of Superoxide and Myeloperoxidase in the Neutrophil Phagosome,” 39860, 2006, with permission from ASBMB publications.

58

Figure 2.6. Fluctuations in [O2] in the PO oscillator for various reactor compositions. Reprinted, with permission, from Dean Olson’s thesis (personal communication).

59 Figure 2.7 A pair of scanning electron microscopy images of biofilm formation on quartz slides. Reproduced with permission from Christopher Ryan Field’s PhD dissertation, 2009, University of Illinois Urbana-Champaign. Original data obtained by D. D. Lewis and M. L. Ruane.

60 Concave Reflector

Linear Actuator Resonating Plate

Transducer Stepped Horn

Copper Electrodes

Piezoelectrics

Bolt

Figure 2.8 Details of the Acoustically-Levitated Drop Reactor with labeled components. Alibre drawing generated by Christopher Field in Scheeline Research Group. Reprinted with permission via personal communication.

61 CHAPTER 3

SAMPLE HANDLING AND CHEMICAL KINETICS: AN ACOUSTICALLY LEVITATED DROP AS A MICROREACTOR

Much of this chapter, coauthored by Christopher R. Field and Alexander Scheeline, has been previously published in Analytical Chemistry, 81(20), 8496-8502 (2009) DOI:

10.1021/ac901400y. While Field and Scheeline contributed helpful suggestions to the work, all fluidics apparatus are of my construction and I obtained all kinetics data. Additional figures and material have been added to note changes and provide additional clarity.

Figures from the above publication have been noted and reprinted with permission of the publisher.

3.1 Introduction

In an effort to thoroughly understand the mechanisms of biochemical reactions, accurate measurement of enzyme kinetics is essential. Common methods used to study reaction systems employ stirred cuvettes, stopped-flow systems, lab-on-a chip/microfluidic flow channels, or some other sample container together with an appropriate monitoring system.1-3

With many of the standard reaction rate methods, adsorption of reactants and products to the walls of the reaction vessel proves problematic. Reaction rates may be altered by biofilm buildup on reactor walls.4 In an effort to avoid such problems, we report the use of an acoustically levitated drop reactor (LDR).5 Precedent measurements of kinetics in levitated drops include work by David Weis and others.12-14

62 In order to achieve such reactions in a levitated drop, droplet generation and sample delivery with minimal sample loss, is important. Most research employing levitators uses syringes, pipettes or picoliter (pL) drop-on demand ballistic injection. In the present study, we investigated the efficiency of using an Acoustically-Levitated Drop Reactor (LDR) (Figure

3.1) to study reaction kinetics coupled with a pressure driven capillary bundle system to achieve sample introduction, mixing, drop maintenance, and fluid removal.

Kinetics measurements for both luminol chemiluminescence and lactate dehydrogenase

(LDH) catalyzed reaction of NADH with pyruvate were conducted. These reactions have been studied previously and are well understood.25-27 By studying these reactions, we were able to evaluate both the advantages and challenges of running reactions in an LDR. We address questions such as the degree of mixing in the drop, correspondence of luminescence in a 5 mL capillary-fed drop to that expected in larger reactors, and how long a drop can be maintained.

63 3.2 Materials and Methods

Materials. All reagents were purchased from either Sigma-Aldrich (St. Louis, MO) or Fisher

Scientific (Fair Lawn, NJ), unless otherwise noted.

For chemiluminescence experiments, solutions of 0.07 M luminol (CAS 521-31-3), 3.0 M sodium bicarbonate (NaHCO3, CAS 144-55-8), 0.5 M sodium carbonate (Na2CO3, CAS 497-

19-8), 0.05 M ammonium carbonate ((NH4)2CO3, CAS 506-87-6), 0.015 M copper II sulfate

(CuSO4, CAS 7758-98-7) and 0.10% hydrogen peroxide (H2O2, CAS 144-55-8) were prepared using 18-MΩ ultrapure water purified using a Q-POD ultrapure water filtration system (ZMQSP0D02, Millipore, Billerica, MA). Reagents were both prepared and stored at room temperature. Literature concentrations and procedures were modified so that reaction would occur at a measurable rate in a 5 µL drop.28 When running experiments, 0.2 mL of luminol, NaHCO3, Na2CO3, (NH4)2CO3, and CuSO4 solutions were each added to one sample injection vial, while 1 mL of H2O2 solution was added to a second. For each run, 2.5 µL of the luminol mixture was injected into the LDR followed by 2.5 µL of H2O2.

For the enzyme-catalyzed fluorescence experiments, solutions of 1 mM sodium pyruvate

(CAS 113-24-06), 0.15 mM nicotinamide adenine dinucleotide, reduced form (NADH, CAS

606-68-8), 0.25units/mL of L-lactate dehydrogenase (LDH, CAS 9001-60-9), 0.1% w/v poly-L-lysine, MW 150,000 – 300,000 (CAS 25988-63-0), and 0.25 mM bovine serum albumin (BSA, CAS 9048-46-8) were prepared, separately, in 0.1 M 1X-phosphate buffer solution (PBS, CAS 7558-79-4) (HyClone, Logan, UT). All reagents, except LDH, were refrigerated (10 ˚C) when not in use. LDH was frozen (-20 ˚C). Solution preparation and reaction procedures were detailed previously.29 When running experiments, 0.75 mL each of

64 sodium pyruvate and NADH solutions were each added to one injection vial and 1.5 mL

LDH to the second. For each run, a 4 µL drop was generated for reaction; 3.5 µL of the

NADH mixture was injected into the LDR followed by 0.5 µL of LDH solution. To prevent

LDH from adhering to the walls of the pipette tips, when transferring it from stock solution to LDR sample vials, transfer pipette tips were coated with BSA before each transfer.

CYTOP® (ASAHI Glass Company. Maiden, MA), a fluorinated polymer, with a contact angle of greater than 110°, was applied to the outer surface of the capillaries to provide a super-hydrophobic outer coating so that the drops would not wick to the sides of the capillaries. Walls of capillaries used to convey LDH were coated with poly-L-lysine30 by pumping a 0.01% solution through capillaries following cleaning with piranha solution (3 parts concentrated H2SO4, 1 part H2O2). Warning: piranha solution is a strong oxidant, must be kept away from significant quantities of oxidizable organic compounds, and must be disposed of by careful neutralization.

LDR Setup and Capillary Positioning. Before drops are introduced into the LDR via the capillary system, the capillaries must be positioned within one millimeter of the LDR pressure node. This is achieved by first manipulating the levitator so that it is optimized for levitation. Optimized levitation means the drop is as positionally stable as possible in the levitation cavity, close to a spherical shape, and with shape fluctuations. Using a LabView

8.0 module (see Appendix), the levitator is activated. Next, temperature, pressure, and humidity are recorded, and reflector height computed as previously described.5 A syringe is used to introduce a 5 µL drop into the LDR. This drop is used as a guide for the tip of the

65 capillary system to indicate the optimal position for delivering reactants. This procedure only has to be completed once daily.

Sample Introduction. Sample introduction was achieved using a Poiseuille flow capillary system developed in-house (Figure 3.2). The setup consists of a polyamide adhered, poly-L- lysine (PLL) flushed, CYTOP®-coated, capillary bundle to deliver the initial drop to the levitator, add reactants, and remove the droplet once reacted. The capillary bundle is made of three 1 m long, 363 µm OD, 180 µm ID, flexible fused silica capillaries from Polymicro

Technologies (Phoenix, AZ). They are adhered to one another using a polyimide resin (CAS

68410-23-1). Each capillary is connected to a 2 mL vial using a series of PEEK® fittings, ferrules, unions and tubing sleeves (Upchurch Scientific, model numbers F33NX, P-702, P-

440, and F-238X), in which reagents can be placed for delivery to the LDR or from which waste can be collected. 0.01 % w/v PLL in 0.1 M Phosphate buffer is flushed through the capillaries to coat the inner walls of the capillaries to eliminate cationic enzyme adsorption or build-up on the walls. The coated capillaries are used for up to 60 reaction runs before flushing with PLL again. The capillary bundle is positioned in the LDR using an XYZ stage

(TSX-1A, Newport, Irvine, CA).

Fluids are fed into the levitator through two of the capillaries in the bundle using pressurized argon (40 psi) and a drop is formed on the tip of the capillaries. The inlet flow rate is

2.3 µL/s. A belt driven, HYVAC vacuum pump (-12 psi, 600 rpm, CENCO, New Brighton,

MN) is connected to the third capillary in the bundle. A 1-mL vial, used to collect the sample waste as drops are removed, is positioned between the capillary and the pump. A

66 series of two-way, 5V solenoid valves (Precision Dynamics, Inc. New Britain, CT) is used on each capillary line to control the fluid flow. This stopped-flow system is controlled using a program written in LabVIEW 8.0. Solenoid drivers are connected directly to the digital outputs on a multifunction analog-to-digital (ADC) card (PCI-DAS6014, Measurement

Computing Inc., Norton, MA). The program allows for the injection of fluids from the sample vials sequentially within seconds with the click of one button on the LabVIEW module screen. Drops are removed from the LDR in the same way. Without using the vacuum to remove the drop, a 5 µL levitated drop will evaporate completely from the tip of the capillaries within 6 minutes in a dry atmosphere close to room temperature, 23°C. For the experiments conducted in this paper, all reactions were complete within 1 min.

Kinetics Measurements. The kinetic measurements for the luminol chemiluminescence and

NADH fluorescence were performed using the LDR detailed previously.5 Since that publication, foot switches (model FTSW-SM, MicroRidge, Sunriver, OR) were added to allow hands-free, fine-tuning adjustments (0.01 mm increments, up and down) of the reflector height for optimum levitation. Initially, a closed-circuit digital camera (CCTV

Camera, 24 V AC 60Hz 3.4 W, UltraLum, Claremont, CA) and television (model WV-BM-

990, Panasonic, Secaucus, NJ) were used to view the levitation cavity. This was later replaced with a higher speed CMOS camera (Stingray IEEE1394 C-Mount Camera, Allied

Vision Technologies GmbH, Stadtroda/Germany), coupled with a 0.63× magnification telecentric lens (Invarigon 59 LGU 042 with 59 LGZ 415 teleconverter, CVI/Melles Griot,

Covina, CA) to better view the levitation cavity during experiments in real time.

Temperature, pressure, humidity, levitation bimorph drive voltage, and reflector height were

67 recorded before each run. Measurements were carried out close to room temperature.

For the chemiluminescence experiments, the time course of the luminol reaction with H2O2 was detected using a 910 µm core diameter fiber optic (BFH22-910, ThorLabs. Newton, NJ) coupled to a photomultiplier tube (PMT) (R928, Hamamatsu. Bridgewater, NJ) with a variable high voltage supply (model 205A-01R, Bertan Associates), set to 375 V (Figure 3.1

A). The output current from the PMT was sensed through a 1V/µA linear amplifier. The signal output from the amplifier was T-ed to an oscilloscope (7603, Tektronix, 7A14 plug-in) through a 1 MΩ signal input and to the same multifunction ADC used for solenoid valve control mentioned earlier. The ADC was configured with 16 analog single-ended inputs. A data acquisition program was written in LabVIEW 8.0 (National Instruments. Austin, TX) using the Measurement Computing Universal Library for LabVIEW to collect, save, and display the PMT signal. The time course of the intensity change was fitted to a second order rate law.

For the NADH fluorescence, either a 200 mW 357 nm argon ion laser (Innova 90, Coherent

Inc., Santa Clara, CA) or a 16 mW, 372 nm wavelength diode laser (1Q micro 1A, Model

LDCU8/8940, Power Technologies Inc. Alexander, AR) was used for excitation (See ‘Note’ at end of Chapter for clarification). Reaction concentrations, sampling rate (1kHz), total sampling time (30s), and the fiber optic detection system were the same for both sets of experiments.

68 3.3 Results and Discussion

Microfluidic Setup. The LDR functions as a stirred batch microreactor with convection induced by the ultrasound.20 In order for this system to function properly, the capillary system explained in the previous section is vital. The 3-capillary bundle system (Figure 3.1

B) provides the means to introduce samples into the LDR, in part to mix reactants, and to remove spent drops. The orientation of the capillaries is very important to the success of experiments. Reactants can be delivered or removed from any capillary, but the capillaries must be positioned so that there are two capillaries with axes parallel to the earth’s surface and at equal height, with the third capillary at the bottom (Figure 3.2). Improper orientation results in wicking of the drop to the side of the capillary bundle. Even with proper orientation, it is essential to coat the capillary tip with CYTOP® to prevent drop adhesion.

Reactants are introduced sequentially from the capillaries. Mixing in the drop appears to be effectively complete in less than one second, as is discussed below concerning the chemiluminescence experiments. Without adequate mixing of H2O2 into a pre-existing luminol/Cu2+ solution drop, homogenous emission would not have been observed. The 2.3

µL/s rate at which reactants are introduced contributes to drop rotation and mixing. Details are shown in Figure 3.3.

69 Chemiluminescence Experiments. Typical chemiluminescence data are shown in Figure 3.3.

The reactions are:

Cu2+ + luminol Cu2+luminol (3.1)

2+ 2+ Cu luminol + H2O2 Cu + 4-aminophthalate* (3.2)

4-aminophthalate* 4-aminophthalate + hν (3.3)

The primary reasons for performing these experiments were to demonstrate that reactions initiated by reactant flow from capillaries could be observed in the drop, and that reactant mixing in the LDR from diffusion, flow-induced convection, and ultrasound-induced circulation was sufficiently rapid to be useful for study on a second or faster time scale. Data seen in Figure 3.3 and the still images extracted from one of the runs (Figure 3.5) show that mixing is essentially complete within 1 s after reactant influx is stopped, as emission appears to be uniform throughout the drop.

Figure 3.3A shows a single raw data transient, one of eight chemiluminescence experiments in a successful data set. The reaction is nearly complete within 15 seconds. Figure 3B is an inset of Figure 3.3A that shows the last second of the luminol/Cu2+ solution feed and the introduction of the H2O2. As the H2O2 is introduced at ~ t = 3 s, the drop begins to luminesce, causing a sharp increase in the photocurrent. Each chemiluminescence transient showed baseline offset (Figure 3.3C) from one run to the next that was trivially corrected

(Figure 3.3D). While valve switching is precise to better than 0.1 s, mixing time varies from

0.5 to 1.5 s. Inset D of Figure 3.3 shows every trace offset to a common baseline, with

70 transients shifted in time so that all decays go through 0.4 µA simultaneously.

Averaging all the traces in D, one obtains Inset E. If the decay were a pure second-order

2+ reaction between luminol and H2O2 with the effect of Cu limited to increasing the pseudo- second order rate constant k2, the fluorescence intensity would follow a time course,

B Fk (B ! A)2 e(B!A)kt I = A (3.4) " B %2 $ e(B!A)kt !1' # A & where A = [luminol]0, B = [H2O2]0, F is an arbitrary scale factor dependent on observation system and reaction quantum yield, and k is an effective rate constant, dependent at least on pH, ionic strength, and [Cu2+]. By design, B>A. As expressed here, (B-A) can either be computed from the initial values of A and B or used as a separate fitting parameter. When it is separately optimized, one obtains the best possible fits. Obviously, an alternative interpretation is that the rate law governing chemiluminescence is more complicated than pure second order. Yu et al. postulate a mechanism that would be at least third order or involve a pre-equilibrium.31 The point here is not to elucidate the mechanistic details but rather to demonstrate that such elucidation is plausible with this apparatus.

In Figure 3.4, one can see that as H2O2 is delivered to the cavity within the first 2.5 seconds, the relative standard deviation (RSD), from averaging the 6 transients, is extremely high; at several times reaching as high as 1000% RSD. After 2.5 s, where the luminol has begun to flow into the drop, the RSD drops to below 20%. Once the luminol delivery was complete,

71 at 4.5 s, the RSD dropped to its lowest of 1%. As reaction continued in the drop for the final

10 seconds, the RSD gradually increased at a rate of 1% s-1, ending at 10%. Figure 3.4 shows that the reproducibility other than baseline and initiation time offsets (Figure 3.3D) can be seen to be less than 10% once mixing is complete.

Figure 3.5 shows individual frames from a video recording of a luminescence transient.

Between 1 and 2.97 s there is heterogeneity within the drop. As the H2O2 is introduced, beginning at 3.05 s, the drop begins to gradually illuminate in a non-uniform manner.

Circulation from solution introduction causes the drop to precess in a circular motion which ultimately results in a well mixed drop that reaches maximum intensity at 3.7 s, and only 0.7 s after completion of H2O2 introduction. At 3.7 s there is a noticeable decrease in intensity that rebounds at both 4.0 and 4.1 s, neither reaching the intensity observed at 3.7 s. After 4.2 s, the drop luminescence intensity steadily decreases. Between 7.34 s and 10.23 s, a great difference in drop intensity can be seen. In addition to the intensity changes, inhomogeneity can be seen over time. At 3.13 s, the ripples in the drop are evidence of mixing. For each time noted on the plot beyond 3.13 s, small drop modulations are observed. Figure 3.3 inset

F shows that some small signal modulation inconsistent with a pure mass-action, monotonic approach to equilibrium is observed. Whether this "hump" is due entirely to drop inhomogeneity or if dust or some other source causes this modulation has not been determined.

72 LDH Experiments: NADH/LDH/Pyruvate kinetic data can be seen in Figures 3.6 and 3.7.

The reaction is LDH NADH + Pyruvate + H+ NAD+ + lactate (3.5) where LDH catalyzes the reduction of pyruvate to lactate using NADH in the LDR.

Attempts to study this well-known reaction presented several difficulties. Although a difference is observed between the transients of reacting samples and these of water or nonreacting NADH, reproducibility between runs was difficult to obtain. When a run was successful, it followed Michaelis-Menten32 kinetics remarkably well, as detailed below.

Figures 3.6 and 3.7 show LDH experiments from two series of drops formed at 1 min intervals without changing levitation parameters (See ‘Note’ at end of chapter for clarification). Figure 3.6A shows fluorescence from a blank, a 4 µL drop of 18 MΩ deionized water. Figure 3.6B shows the transient for a newly formed drop that contains only

NADH. Besides the intensity increase as a result of the NADH fluorescence, the drop is relatively stable and exhibits little change over the 30 second observation period. This demonstrates that NADH fluorescence is steady when laser-excited but when no pyruvate or enzyme is present.

Figures 3.6 A-C and E and Figures 3.7 A-C present our most recent LDH experiments. The

LDR parameters were: reflector height (17.463 mm), transducer amplifier drive (4.6 Vpp), temperature (27.5 ˚C), humidity (15.1%), and pressure (980.8 mbar). For detection, PMT voltage was set to 450 V. Illumination was with the Power Technologies diode laser.

Figures 3.6D and F and 3.7 D-F are representative of LDH experiments conducted a year earlier. The LDR parameters for these reactions were: reflector height (17.463 mm),

73 transducer amplifier drive (6.2 Vpp), temperature (22.2 ˚C), humidity (22.9%), and pressure

(996.1 mbar). The 357 nm, 200 mW argon ion laser was used for NADH excitation. The

PMT voltage was set much lower for this laser, at 325 V. Reaction concentrations, sampling rate (1 kHz), total sampling time (30 s), and fiber optic detection system were the same for both sets of experiments.

Figures 3.6C and D show data from successful LDH runs. Within the first four seconds, the

NADH/Pyruvate mixture and LDH have formed a levitating drop and have begun reacting.

As the NADH and pyruvate are consumed, the signal decays. Each transient reaches a steady state as all of the pyruvate and approximately 10% of the NADH are consumed. If all of the

NADH had been consumed the signal intensity would have eventually dropped to zero; instead the intensity drops by about 20% in each case. Figures 3.6 A-C and E exhibit the same high frequency fluctuations. We attribute the majority of the fluctuations to shot noise.

The Savitzky-Golay33 algorithm was used to filter the data.

While we attempted to derive a rate law and integrated rate expression based on an extensive set of fundamental reactions in this system, the number of fitting parameters exceeded what could be statistically justified from the noisy data. This is hardly a novel problem.34

We thus approximated the reaction model as pure Michaelis-Menten, with pyruvate as the kinetically-limiting substrate,

d[lactate] V [ pyruvate] = max (3.6) dt KM + [ pyruvate]

74 where Km and Vmax have the usual meanings of substrate binding constant and maximum reaction rate, ignoring product inhibition.

d[NAD+ ] d[lactate] d[ pyruvate] = = ! (3.7) dt dt dt

By conservation of matter,

[NADH] = [NADH]0 – [lactate] (3.8)

Integration of equation 6 gives:

[lactate] K & [lactate] # T M ln$1 ! = ' $ ' ! (3.9) Vmax Vmax % [ pyruvate]0 "

This is conveniently rewritten in terms of the fraction of reaction completed, f.

[ pyruvate] f K T = 0 ! M ln(1! f ) (3.10) Vmax Vmax

Vmax is known from the assay of enzyme used in the reaction, so KM is the free fitting parameter. Equations 8 and 10 were used iteratively to fit the data in Figures 3.6C and D.

Data in Figure 3.6C was offset to a zero baseline. The experimental concentration of substrate, enzyme concentration, and time were used as parameters to generate the fitted

35 decay of Figures 3.6C and D, as shown in Figures 3.6E and F. The fitted KM, 0.2 µM, matched literature values under the conditions used, offering validation that the LDR could serve, at least in this case, as a useful microreactor for studying biochemical reactions. The reaction in the 5 µL drop in the LDR, in a volume comparable to other microfluidic systems and orders of magnitude smaller than conventional systems, provides the same data as a

75 macroscopic system. It is important to note that the reactions were conducted without active environmental controls (temperature, pressure, humidity, etc.) other than the approximate regulation provided by building heating systems. Nevertheless, results were reproducible.

The data in Figure 3.6 demonstrates the potential for LDRs to be used as microreactors.

Figure 3.7, on the other hand, presents several non-idealities of the LDR system that must be understood and rectified, if possible, before the system will offer the reproducibility expected in a routinely useful instrumental system. Contributions to these non-idealities in drop signal in addition to the obvious shot noise include rapid spinning of the drop, breathing modes of the drop, bubble formation and dissipation and scattering from debris present in the drop.

Examples of these other phenomena in drops have been previously documented.36, 37 Figure

3.7 shows three examples of non-idealities in the drops, classified using images obtained using the CMOS camera.

In Figures 3.7 A-F, each drop exhibited bubbles forming as the drop was being generated; these eventually dissipated. For droplets that have bubbles present, whether large or small, stationary or in-motion, random increases in intensity and large frequency fluctuations in the samples are observed (Figure 3.7A and D). When a droplet is breathing, expanding up and down or side to side, dips and valleys are present as seen in Figures 3.7B and E. Although the signal is compromised as a result of the breathing motions of the drop, convection is necessary to mix the reactants and burst the bubbles to form a homogeneous drop. Spinning

76 of the drop also aids in the mixing of the drop’s contents; spinning rapidly correlates with noisy signals from the drop. When a drop is spinning, evenly spaced sharp increases in intensity appear as in Figure 3.7B and E. Being able to recognize these nonidealities will allow development of ways to reduce or eliminate them.

Limitations and Improvements. A number of changes to the LDR system are required to make it useful for its design purpose of studying kinetics of enzyme reactions involving reactive oxygen species. Active temperature and humidity controls are being added.

Improvements in sample introduction using ballistic injection of "drops on demand" are also in process. Understanding and optimizing mixing of separate reactant streams (whether continuous from capillaries or discrete from droplet injection38, 39) is critical. While mixing times ~ 0.5 s are useful for some experiments, times below 0.1 s are necessary for study of many free-radical systems, and times below 0.01 s are necessary to be competitive with, e.g., stopped-flow systems.

77 3.4 Conclusions

This chapter demonstrated that a LDR can be used to obtain enzyme and other reaction kinetics data in a system free of solid-liquid interfaces. Success at such measurements has been shown for LDH. Also noted briefly were drop stability, mixing, and instrument limitations, most of which can be remedied with engineering improvements. Some improvements are discussed in the next chapter. Chapter 5 includes more detail on the limitations of the LDR and suggestions for future engineering and experiments that can be used to address those issues.

78 Note (as cited in text)

LDH Experimental Data

The LDH experiment data presented in this paper were collected on two different dates using different lasers; a 200 mW 357 nm argon ion laser (Innova 90, Coherent Inc. Santa Clara,

CA) or a 16 mW, 372 nm wavelength diode laser (1Q micro 1A, Model LDCU8/8940, Power

Technologies Inc. Alexander, AR). Figures 3.4 and 3.5 contain data from both series of data.

Data obtained using the argon laser is presented in Figures 3.4D, 3.4F and 3.5D-F. Levitation parameters were: reflector height (17.46 mm), transducer amplifier drive (6.2 Vpp), temperature (22.2 ºC), humidity (22.9%), and pressure (996.1 mbar). PMT voltage was set to

450 V.

Due to failure of the 200 mW laser, the 16 mW, 372 nm wavelength diode laser was used for the second series of data, which is presented in Figures 3.4A-C, 3.4E and 3.5A-C. Levitation parameters for the diode laser were: reflector height (17.46 mm), transducer amplifier drive

(4.6 Vpp), temperature (27.5 ºC), humidity (15.1%), and pressure (980.8 mbar). The PMT voltage was set reduced to 325 V. Approximately one year transpired between the two sets of experiments.

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Lactate Dehydrogenase X,” Biochemistry, 9, 4372-4380 (1970).

36 A. V. Anilkumar, C. P. Lee, T. G. Wang, “Stability of An Acoustically Levitated and

Flattened Drop: An Experimental Study,” Physics of Fluids, 5, 2763-2774 (1993).

37 A. L. Yarin, D. A. Weiss, G. Brenn and D. Rensink, “Acoustically Levitated Drops:

Drop Oscillation and Break-up Driven by Ultrasound Modulation,” International

Journal of Multiphase Flow, 28, 887-910 (2002).

38 J. C. Yang, W. Chien, M. King and W. L. Grosshandler, “A Simple Piezoelectric

Droplet Generator,” Experiments in Fluids, 23, 445-447 (1997).

39 Sauter, A. D., US Patent 6,149,815, “Precise Electrokinetic Delivery of Minute

Volumes of Liquids,” (2000).

84 3.6 Chapter 3 Figures

A

CCD Camera Fluid B Reservoirs PMT VAC 2 1 Capillaries + CW Laser Ar CW Laser

Figure 3.1 Acoustic Levitation. A. Schematic of the entire acoustic levitation drop reactor (aLDR) sysetm. B. Levitation cavity setup of the aLDR. Sample vials with connections to silica fused capillary bundles system. Continuous wave argon laser, drop and optical detection system also shown. Reprinted from Analytical Chemistry, 81, Pierre, Z., Field, C. and Scheeline, A., “Sample Handling and Chemical Kinetics in an Acoustically Levitated Drop Microreactor,” 8496, 2009, with permission from ACS publications.

85 Inlet Capillaries

CYTOP Coating

Poly-L-Lysine 180180µµmm 180µm Coating 180µm 180µm

Polyamide Resin 180µm Fused Silica Capillaries Vac-Line Capillary

Figure 3.2 3-Capillary Bundle System. Three 1 m, 180 µm ID fused silica capillaries bonded together with polyamide resin. Orientation with two capillaries parallel to earth’s plane on the top and one capillary on the bottom is needed for successful experiments. Reprinted from Analytical Chemistry, 81, Pierre, Z., Field, C. and Scheeline, A., “Sample Handling and Chemical Kinetics in an Acoustically Levitated Drop Microreactor,” 8496, 2009, with permission from ACS publications.

86 3.1 3 3 A B 2.9

2.9 2.8 Luminol

2.8 / 2 2+ O 2.7 2 H 2.7 Cu 2.6 2.6 2.5 2.5

) 2.5 3.5 4.5 5.5 0 5 10 15 A 3.1 µ C 0.6 D 3 0.5 2 3 2.9 0.4 4 2.8 5 0.3 6 2.7 0.2 8 2.6 0.1

Photocurrent ( Photocurrent 2.5 0 0 5 10 15 0 5 10 15

0.6 0.2 E "Hump" Inconsistent F 0.5 Mean Run with Simple Rate 0.15 Law; Run 5 Raw Data 0.4 Consistent Fit Consistent Fit Independent Fit Independent Fit 0.3 0.1 0.2 0.05 0.1

0 0 2.5 5 7.5 10 8 10 12 14 Time (s)

Figure 3.3 Chemiluminescence Kinetics in Levitated Drops. A. Single chemiluminescence transient. Introduction of copper/luminol solution and H2O2 solution are noted on the plot. B. An inset of A showing luminescence during reaction initiation. C. Six raw chemiluminescence transients. D. Data from A, baseline and time-offset. E. Average of data in D. Fit to second-order rate law is

"Consistent Fit." Allowing [luminol]/[H2O2] to be fit independently from [luminol] – [H2O2] gives "Independent Fit." F. Scale expansion of fits in Inset E. Nonmonotonic decrease in luminescence data is evidence of dynamics other than simply mass-action kinetics. Reprinted from Analytical Chemistry, 81, Pierre, Z., Field, C. and Scheeline, A., “Sample Handling and Chemical Kinetics in an Acoustically Levitated Drop Microreactor,” 8496, 2009, with permission from ACS publications.

87 Figure 3.4 Relative Standard Deviation of Light Emission During Drop Mixing During Chemiluminescence Experiment. Red trace is ~ 20x scale expansion of blue trace. Property of the Scheeline Reseaech Group.

88 Figure 3.5 Chemiluminescence Kinetics in Levitated Drops. A single chemiluminescence transient with visual representation of the drops from individual frames of a video recording of a luminescence experiment. Photoinsets and photomultiplier traces are from the same experiment. Changes in the drop color intensity, size and shape can be seen over time. Reprinted from Analytical Chemistry, 81, Pierre, Z., Field, C. and Scheeline, A., “Sample Handling and Chemical Kinetics in an Acoustically Levitated Drop Microreactor,” 8496, 2009, with permission from ACS publications.

89 A B

C D

E F

Figure 3.6 Lactate Dehydrogenase Catalyzed Reduction of Pyruvate to Lactate in the LDR. A. Transient of 18MΩ deionized water. B. Transient in the absence of pyruvate. (NADH Only) C. -D. Two 30-second transients from runs in the LDR on different dates. Each transient relaxes to steady state with consumption of all pyruvate and ~ 10% of NADH. E.-F. Represents the Michaelis-Menten fit for C and D, respectively. (Data smoothed using a 21 point Savitzky-Golay algorithm (A and B) and 51 points (C). D-F data was not smoothed.). Reprinted from Analytical Chemistry, 81, Pierre, Z., Field, C. and Scheeline, A., “Sample Handling and Chemical Kinetics in an Acoustically Levitated Drop Microreactor,” 8496, 2009, with permission from ACS publications.

90 A B C

C D F

Figure 3.7 Drop Pathological Phenomena. A-F. Transients of Lactate Dehydrogenase Catalyzed Reduction of Pyruvate to Lactate in the LDR . Drop breathing (A and C), bubble formation and dissipation (B and E) , and spinning (C and F) are all presented here. Bubbles formed during drop generation; most dissipate within 15 s of drop formation. Reprinted from Analytical Chemistry, 81, Pierre, Z., Field, C. and Scheeline, A., “Sample Handling and Chemical Kinetics in an Acoustically Levitated Drop Microreactor,” 8496, 2009, with permission from ACS publications. .

91

CHAPTER 4

STABILITY OF MYELOPEROXIDASE AT THE GAS/LIQUID INTERFACE

Myeloperoxidase has been mentioned repeatedly in this thesis because it is the motivation behind much of the development of the LDR. Although wall interactions are not a concern in the LDR system, there is the possibility for denaturing or instability of enzymes at the gas/liquid interface. This chapter addresses the issue of Myeloperoxidase stability at the liquid/gas interface and in solution.

4.1 Introduction

Three-dimensional, biologically active structures of proteins are maintained by a fragile balance of electrostatic and hydrophobic interactions among the residues.1 For many proteins, the slightest imbalance in temperature, pH, surfactant, etc., causes denaturation and impairs its activity. The primary reason this occurs is because of the way water-soluble proteins are arranged. Their nonpolar, saturated hydrocarbons are buried in the interior while the surface is covered with polar groups. When such a structure comes in contact with an interface, there is a strong tendency for the nonpolar parts to be attracted to the hydrophobic parts of the interface. As a result, the protein undergoes a conformational rearrangement.

This rearrangement, performed in order to maximize the number of favorable interactions, leads to unfolding of the protein; also known as surface denaturation.2 Surface denaturation of proteins has been studied by a variety of methods such as X-ray and neutron reflections,3 sum frequency generation,4 conventional spectroscopic techniques,5 and the stability of proteins at the liquid/gas interface has also been discussed.2

92 If surface denaturation universally occurred, then even a fully-developed LDR to study enzyme-catalyzed reactions would be of little value. Therefore this chapter details the investigation of surface denaturation of the protein Myeloperoxidase using both sum frequency generation (SFG) and UV-Vis spectroscopy. The SFG (and associated surface tension measurement) work was done in collaboration with Professor Elsa Yan of Yale

University. Details of the work have not yet been published, so only results of the study will be presented here based on correspondence with Yan. My contribution to the work was to test whether Yan's results were correlated with laser-induced photochemistry or if they were more likely were due to light-independent of photochemistry.

4.2 Materials and Methods

Materials. All reagents for the buffer were purchased from Fisher Scientific (Fair Lawn,

NJ). A 100 mL stock solution of 0.1 M phosphate buffer (pH 7.0) was prepared from 30.5 mL of 0.2 M sodium phosphate, dibasic, heptahydrate (Na2HPO4•7H2O, CAS 7782-85-6),

19.5 mL of 0.2 M sodium phosphate monobasic monohydrate (NaH2PO4•H2O, CAS 10049-

21-5), and 50 mL of 18 MΩ ultrapure water purified using a Q-POD ultrapure water filtration system (ZMQSP0D02, Millipore, Billerica, MA). The buffer was stored at room temperature.

Myeloperoxidase (MPO) was obtained from Lee Biosolutions Inc. (St. Louis, MO). The source of the MPO was from human neutrophils (CAS 9003-99-0, EC 1.11.1.7, 1 mg

MPO/vial, > 98% purity by SDS-PAGE).

93 MPO was prepared directly in a 1 cm path length, micro volume (580 µL), Spectrosil® quartz spectrophotometer cell purchased from Starna Cells, Inc. (Catalog# 28/9/Q/10;

Atascadero, CA). The laser used for protein exposure was a 500 mW average power at 800 nm, picosecond-pulsed, mode locked, Ti:Sapphire laser made by Spectra Physics (Model:

Millennia Vs). The UV-Vis spectrophotometer used to obtain spectra of MPO after laser exposure was made by Shimadzu (Model # UV-1650PC). Both the laser and UV-Vis spectrometer used in these experiments are in the Martin Gruebele lab.

Methods. Yan observed changes in MPO surface spectra over a 3-4 hour period. In order to investigate the potential changes in MPO on this time scale, independent of the liquid/gas free surface, and to detect plausible photoproducts that may arise to complicate the chemistry when using lasers for MPO kinetic studies, MPO was exposed to the laser described above for 30 minute intervals, after which any changes in the spectra of MPO were sought using

UV-Vis spectroscopy. Before beginning, 0.43 mL of phosphate buffer was added to the micro volume cuvette and a spectrum was obtained using the UV-Vis to determine the I0 for the buffer. 0.075 mL of 35 µM MPO was then added to the buffer in the cuvette, bringing the final concentration to 5.2 µM MPO and the total volume to 0.50 mL in the cuvette. A spectrum of unexposed MPO over the entire range of the spectrophotometer (200 - 850 nm) was then obtained.

The setup was simple (Figure 4.1). A sample holder was used to hold the cuvette containing

MPO in place while being exposed to the laser beam. The cuvette holder was held at a constant temperature by flowing water through the holder from a temperature bath set at

94 27˚C. The sample was exposed to the laser for 30 minutes at a time, then removed to obtain a spectrum. A blank spectrum of phosphate buffer was taken before each MPO spectrum was obtained. The MPO samples were out of the temperature-controlled environment for approximately 10 min for each spectral scan. The experiment was carried out for a little over

2.5 h, providing for 6 absorbance spectra of MPO to be obtained for comparison. MPO sample was capped and inverted several times in order to mix the sample before each spectrum was taken and before placing it back into the path of the laser. A control experiment was also conducted where a 5 µM MPO sample was held at constant temperature, following the same time intervals for analysis, but with no exposure of the sample to lasers.

The SFG work conducted at Yale by Yan observed a 4 µg/mL solution of MPO prepared in phosphate buffer (0.1 M, pH 7). The experiments were carried out in a Teflon beaker. Sub- picosecond pulses from a regeneratively-amplified Ti:sapphire laser (80 mW average power at 800 nm) and broadband infrared radiation as light sources were used to obtain vibrational spectra at the air/liquid interface. Any spectral change was expected to indicate MPO structural changes over time. Spectra were acquired every 5 m over a span of 5.5 h. Surface tension was also measured (without laser exposure) to determine if the protein at the gas/liquid interface was restructuring on this time scale.

4.3 Results and Discussion

Surface tension and SFG measurements. Results of the surface tension measurements showed that surface tension of MPO increased smoothly by 2.5-3 millinewton m-1 over a 3 h period following introduction of a freshly diluted solution to their apparatus. It was not until

95 the stabilization of surface tension after 3 h that a peak at 1653 cm-1, which corresponds to an amide I vibrational transition, appeared. There are at least two possible explanations for these results. One is that even at a nominally motionless interface, an organized monolayer of protein will slowly form. This time scale is promising because it suggests that over the tens of minutes that we plan for reactions in drops, there is a low probability of any molecule spending significant time at the interface, making the likelihood of denaturation at the surface even lower. Due to MPO’s solubility, circulation in the drop should prevent protein island formation at the interface. The other possibility is that no MPO is detectable at the solution/air interface for the first three hours because little is there. This is plausible because

MPO is so highly charged (isoelectric pH ≥10). If this is true, MPO may be a nearly-ideal enzyme for study in levitated drops. How then, would a hydrophobic form of MPO migrate to the surface? A possibility is photochemical degradation by the laser. It was this possibility I sought to examine.

MPO stability at the surface/Laser exposure. One of the questions that the SFG experiment results raised was; ‘What is causing the appearance of amide vibrations after several hours?’

A hypothesis was that since absorbance at 800 nm at low fluence is negligible, perhaps multiphoton absorption (to vibrationally-excited components of the heme Soret band, ~400 nm) leads to photo-oxidation and hydrophobic degradation products of MPO. As a result, it might take several hours for the protein at the interface to form a sufficiently organized monolayer. The laser exposure experiments conducted were to test the surface organization argument.

96 Figures 4.2 to 4.4 are the UV-visible light absorbance spectra for MPO exposed to laser light

(Figures 4.2A and 4.4) and MPO solution that was not exposed (Figures 4.2B and 4.3).

Comparing the spectra in Figure 4.2 A and B, there are visible differences in the absorbance intensity between the laser exposed and non-exposed MPO. In Figure 4.2 A (non-exposed

MPO), as the time increases. the absorbance intensity of MPO across the spectral range decreases by a similar increment for each time interval. The same cannot be said for Figure

4.2 B (laser-exposed MPO). Compared to the initial spectrum of MPO (t=0 min, red line), the spectrum at t = 30 min (yellow line) actually increases in intensity by about 5%, and then decreases similarity to the non-exposed MPO. This phenomenon could probably be explained by the temperature change experienced with solution as a result of initial exposure to the laser. Heme spectroscopic signatures are particularly useful in characterizing the impact of the protein environment on the heme moiety and the perturbation by chemical or temperature stress.7 In this case, stability of the protein was monitored via absorption at 428 nm (Soret band). Both solutions were prepared using the same phosphate buffer, therefore the laser is the only variable that was different.

In Figures 4.3 (no laser exposure) and 4.4 (laser exposure), I have taken the plots shown in

Figure 4.2, normalized at 430 nm, then zoomed in on the X-axis, to get a better view of the possible changes taking place within the spectra. Figure 4.3 shows spectra just incubated at

27 ˚C in phosphate buffer for 2.5 h with spectra taken approximately every 30 minutes. In

Inset B, which is in the UV range of the spectrum (250-310 nm), the trend is reversed from that seen at 428 nm. Instead of the absorbance intensity decreasing at the same rate of change from the lowest time to the highest, here the absorbance intensity at t=155 min has

97 the highest intensity and t=0 min has the lowest intensity (which it shared with t=30 min).

Figure 4.4 B follows the same trend as 4.3 B except there is a clear distinction between the absorbance intensities of t=0 min and t =30 min. In inset C for both Figures 4.3 and 4.4 there is something occurring between the 300-340 nm. In Figure 4.3 C, MPO at t=0 min has a step increase at 325 nm that has completely disappeared for all the other time intervals. Yet in

Figure 4.4 C an absorbance increase is observed at each time interval. This may be explained by the shifts and intensity increases observed in 4.3 D and 4.4 D, respectively.

Inset D for both Figures 4.3 and 4.4 (390-470 nm range), include the 428 nm Soret band for

MPO. Because this wavelength was used to normalize the entire UV-vis spectrum, not many changes can be seen without zooming in. For practical purposes, there is little net change in this region of the spectrum. In Figure 4.3 D a similar trend to what is observed in Figure 4.6 is seen. As MPO is incubated in the buffer, there is a slight shift in the 428 nm peak to lower wavelength. Although the change is small, it is there. So while the absorbance of the spectra in Figure 4.4 D increases in magnitude as a result of the laser exposure and possible changes in temperature from t=0 min, the spectra in Figure 4.3 D remain at a constant intensity and instead the 428 nm peak shifts to shorter wavelengths the longer the solution is incubated. In both cases, there is evidence that, over time, that some denaturation of protein is taking place.

Based on results from a study conducted 1988 by Svensson, there is a possibility that the slight increases in intensity observed at 428 nm for the laser exposed MPO solution could be the induction of MPO complex II. There is also the possibility that due to the lack of metal ions in the buffer, such as Ca2+, protein dissociation is occurring. As seen in Figure 4.8, circled in orange are several of the calcium ions found in the MPO crystal structure. If that

98 calcium were to dissociate, protein unfolding is highly likely. This could change the conformational arrangement of MPO and affect its activity and spectrum. Conducting a similar study to the ones described here, but adding metal ions to the phosphate buffer, would provide further insight as to whether or not this would positively or negatively impact MPO in solution.

Inset E of both Figures 4.3 and Figure 4.4 reveal that not much activity is occurring at 570 nm. The total change in intensity is less than a 0.01 absorbance unit range. The reason for looking closely at this particular wavelength is to determine if there are photoproducts of

MPO that developed over time with exposure to laser light. Insets Figures 4.3 and 4.4 E offer no suggestion that photoproducts of MPO are being generated. The characteristic peaks of MPO are still present at 570 nm, and there are no shifts or major increases in absorbance that would suggest any products of MPO interactions are being formed.

Figure 4.7 offers a look at the trends of absorbance over time at the characteristic wavelengths of MPO (428 nm, 570 nm, and 800 nm). For both the laser-exposed and the non-exposed MPO, the absorbance trends remain consistent; as time increases, the absorbance of MPO decreases. There is a slight increase in the ratio of absorbance at 570 to

430. The striking trends in absorbance changes at 280 nm and 320 nm could be correlated with a number of things. Possible causes for the changes in the UV and deep UV could be a result of tryptophan and tryrosine or even cysteine sites being oxidized. There is also the possibility of localized heating of the laser exposed MPO solution. Another possibility is

99 oxidation by dissolved oxygen, perhaps involving radical chain reactions. All of these are possible, but without further investigation and the generation of more spectra to test reproducibility, all of these hypotheses are unproved. This experiment will need to be redone to test time and concentration dependence of the changes observed.

4.4 Conclusion

The data above suggest that even at low concentrations and with minimal environmental disturbances, for the most part the MPO-typical heme to protein bonds will remain intact and enable (nonspecific) heme coordination with the protein. This is in strong contrast to heme peroxidases with noncovalently linked heme which lose their prosthetic group with the consequence of a significant blue shift of the Soret maximum to ~365 nm. The short time period needed to study MPO reaction kinetics (tens of minutes), the fact that there is not a great concern for conformational changes, oxidation, denaturation or surface organization/restructuring of the protein at the interface, makes MPO promising as a protein to study in the LDR. There is room for additional experiments to be conducted to be certain that this holds true under a wider set of conditions. As mentioned above, it would be useful to repeat the experiment, varying the amount of Ca2+ in solution to see if it prevents MPO from unfolding. Although this research did show that there was some type of chemical changes occurring within the MPO solution, a concern that of instability of the enzyme at the gas/liquid interface would be a dominant source of denaturation has been shown to be unlikely. This is a step towards validating experiments on MPO in the LDR.

100 4.5 References

1 M. Arai and K. Kuwajima, “Role of the Molten Globule State in Protein Folding,”

Advances in Protein Chemistry, 53, 209 (2000).

2 F. Macritchie, ”Protein Adsorption/Desorption at Fluid Interfaces,” Advances in

Protein Chemistry, 32, 283 (1978).

3 D. Gidalevitz, Z. Huang, and S. Rice, “Protein Folding at the Air-Water Interface

Studied by X-ray Reflectivity,” Proceedings of National Academy of Sciences of the

USA, 96, 2608-2611 (1999).

4 J. Wang, S. M. Buck and Z. Chen, “The Effect of Surface Coverage on

Conformation Changes of Bovine Serum Albumin Molecules at the Air–Solution

Interface Detected by Sum Frequency Generation Vibrational Spectroscopy,” Analyst,

128, 773-778 (2003).

5 V. Lechevalier, T. Croguennec, S. Pezennec, C. Guerin-Dubiard, M. Pasco and F.

Nau, “Ovalbumin, Ovotransferrin, Lysozyme: Three Model Proteins for Structural

Modifications at the Air-Water Interface,” Journal of Agricultural and Food

Chemistry, 51, 6354-6361 (2003).

6 B. E. Svensson, “Myeloperoxidase Oxidation States Involved in Myeloperoxidase

Oxidase Oxidation of Thiols,” Biochemical Journal, 256, 751-755 (1988)

7 S. Banerjee, J. Stampler, P. G. Furtmuller and C. Obinger, “Conformational and

Thermal Stability of Mature Dimeric Human Myeloperoxidase and a Recombinant

Monomeric Form from CHO cells,” Biochimica et Biophysica Acta – Proteins and

Proteomics, 1814, 375-387 (2010).

101 4.6 Chapter 4 Figures

Water In

Temperature Bath Cuvette Holder

MPO

Laser

Micro-Volume Cuvette Water Out

Figure 4.1 Setup for investigating the effects of direct laser exposure of MPO at the liquid interface.

102 0.4 2 A. Blank 0.35 t=0min 1.8 t=30min 0.3 t=65min 1.6 t=95min 0.25 1.4 t=125min 0.2 t=155min 1.2 0.15

1 0.1

0.8 0.05

Absorbance(AU) 0.6 0 200 300 400 500 600 700 800 0.4

0.2

0 200 300 400 500 600 700 800 Wavelength (nm)

Figure 4.2 Absorbance spectra of MPO (5 µM in phosphate buffer)over the entire UV-Vis spectral range; 200-850 nm. A) No laser exposure B) Laser exposure using a Ti-Sapphire 800 nm, 500 mW laser. A spectrum of MPO was obtained every thirty minutes for experiments lasting 2.5 hours. Temperature was held at 27˚C in both cases. Both insets are the original spectra, zoomed in on the Y-axis to see the activity between 250-500 nm.

103 Figure 4.3 Control. No laser Exposure. Absorbance spectra of MPO that was exposed to laser light. A) Across the entire spectral range of the UV-vis spectrophotometer, 200-850 nm, normalized at 430 nm. B-E boxed in red in A are sections of the spectra that were blown up to analyze the activity at those particular wavelengths. B) 250-310 nm C) 300-400 nm D) 390-470 nm and E) 540-610 nm. Digitization granularity can be seen in inset E.

104 Figure 4.4 Laser Exposed MPO. Absorbance spectra of MPO that was exposed to laser light. A) Across the entire spectral range of the UV-vis spectrophotometer, 200-850 nm, normalized at 430 nm. B-E boxed in red in A are sections of the spectra that were blown up to analyze the activity at those particular wavelengths. B) 250-310 nm C) 300-400 nm D) 390-470 nm and E) 540-610 nm. Digitization granularity can be seen in inset E.

105 Figure 4.5 The induction of MPO complex II by buffer. MPO (56 nM) was added to 50 mM-potassium phosphate buffer, pH 7.0 (with supplements and substitution as indicated below), and the visible-light-absorption spectrum were scanned after 30 s (solid line). The MPO3+ spectrum (dotted line) is included as reference (see e). (a) No supplement; (b) freshly deionized water with resistivity 16 ΜΩ⋅cm (Milli-Q; Millipore Corp., Bedford, MA) substituted for the buffer; (e)+ 16 µg of catalase/ml + 200 µM-DTPA. These typical results were obtained by Svensson, B.E. Biochemical Journal, 256, “Conformational and Thermal Stability of Mature Dimeric Human Myeloperoxidase and a Recombinant Monomeric form from CHO Cells,” 751, 1988. This figure has been altered (utilized only insets a, b and e) and reproduced with permission of Elsevier Publishing.

106 Figure 4.6 Denaturation of mature MPO by GdnHCl as monitored by UV- Vis spectroscopy. A) Comparison of UV-vis spectra of 1.35 µM native ferric myeloperoxidase in 5mM phosphate buffer, pH 7 (gray line) and 1.35 µM myeloperoxidase incubated for 18hr in 6.0 M GdnHCl (black line). These results were obtained by by Svensson, B.E. Biochemical Journal, 256, “Conformational and Thermal Stability of Mature Dimeric Human Myeloperoxidase and a Recombinant Monomeric form from CHO Cells,” 751, 1988. This figure has been altered (utilized only inset a) and reproduced with permission of Elsevier publishing.

107 0.35 A. Abs430 Abs570 0.3 Abs800 Abs570/Abs430 0.25 Abs800/Abs430

0.2

0.15

Absorbance(AU) 0.1

0.05

0 0 20 40 60 80 100 120 140 160 180 Time (min)

Abs430 B. 0.3 Abs570 Abs800 0.25 Abs570/Abs430 Abs800/Abs430

0.2

0.15

0.1 Absorbance(AU)

0.05

0 0 30 60 90 120 150 180 Time (min)

Figure 4.7 Absorbance of MPO at five different wavelengths at various time intervals as a result of A) exposure to a Ti-Sapphire, 500 mW, 800 nm picosecond pulsed laser and B) no laser exposure. Each experiment was held at a constant temperature of 27˚C.

108

Figure 4.8 Cyrogenic crystal structure of human MPO isoform c. Obtained from the Protein Data Bank. The green dots circled in orange represent several calcium ions that may be associated with maintaining tertiary protein structure.

109 CHAPTER 5

CONCLUSIONS AND RECOMMENDATIONS FOR LDR DEVELOPMENTS AND APPLICATIONS

Chapters 3 and 4 discussed the development of an acoustically levitated drop reactor system for use in to studying chemical kinetics in a single, microliter-sized drop. Over the course of this research the levitation system has evolved from an idea and several drawings into a system that is fully operational. The past few years of research and development have really positioned the next generation of researchers to focus primarily on chemistry, running experiments in the LDR. This chapter discusses recommended future directions as it applies to both enzyme kinetics experiments in the drop and LDR optimization.

5.1 Acoustically Levitated Drop Reactor System Optimization

The levitator system described in the previous chapters, used to conduct various experiments, works. For what it was designed for, which was to study chemical kinetics in a single drop, minimizing wall interactions, the levitator gets the job done. There are areas where further development or optimization would save the next generation of researchers hours of preparation work and minimize frustration. The areas that need to be addressed are in 1) sample-handling, 2) environmental controls and 3) computer controls.

Sample handling:

The 3-capillary, pressure-driven sample handling system described in chapter three has been improved several times. The major issues with using the fused silica capillaries in the LDR system are 1) they are fragile and break easily, 2) they clog often and 3) without

110 pretreatment, they are not hydrophobic, so our generated drops are attracted to the sides of the capillary instead of staying on the end where the best optical measurements are obtained.

To minimize these issues, we have developed a process by which to prepare bundles and coat them (inside to reduce protein adsorption and outside to reduce the amount of drop wicking).

Although not optimal, it works, and as a result the capillary system has been effective in showing the potential for use of a levitated microdrop as a reactor. This is useful, but there is still the limitation of having ~ 20% of the drop in contact with the capillaries in a system that we are referring to as wall-less. The dead volume between the inlet vials and the drop is close to 0.5 mL (100 times the volume needed to form one drop), and it takes hours to prepare the bundles and an entire day for coatings to cure. To reduce difficulties and move in a more efficient direction of sample handling, for batch reactions, replacement of the capillaries with Drop on Demand (DOD) dispensing is desirable.

Drop on Demand dispensing ejects droplets from a syringe tip or nozzle into a desired space

(Figure 5.1). Based on the nodal structure in the levitation cavity, there is a high probability that a small drop aimed towards the levitation cavity will tend to the node and levitate without requiring much precision in its initial velocity. Having tested this theory several times, it is clear that although a precise initial velocity is not necessary, proper position of the drop dispenser at a specific distance from the levitation cavity is necessary. Assuring the same positioning of the drop dispensers each time will also be required to achieve reproducibility. Determining this position may take some time initially, but once achieved will not require further attention. The time saved makes a good case for investing time into the drop dispensers.

111

In addition to the time saved, a 100% wall-less reaction chamber will be achieved. By using multiple drop dispensers, multiple reactants can be mixed together when ejected into the levitation cavity one after another. Convective mixing in the growing, spinning drop and the continued circulation in the drop driven by the no slip boundary condition between gas and liquid aids mixing, as does the violent shaking of the drop when a droplet impacts (Figure

5.2).

We currently own DOD dispensers from two manufacturers. They both can eject drops down to picoliters in size. The dispensers from MicroFab Technologies can be run using computer controls and the dispensers from NanoLiter LLC come with drive electronics that are activated by a foot pedal. Each has the capability to be computer controlled, which would be the most efficient approach.

To remove drops, a single capillary connected to a vacuum line and a collection vial could be used. It is desired in the future to use the levitation system as a continuously stirred tank reactor (CSTR), I would suggest a using a stopped-flow system or syringe pump with flow monitor and control system. Unlike the current system, these systems would help with accuracy and reproducibility in drop formation. The inlet lines would need to be short, with small inner diameters, to reduce the dead volume.

Environmental Controls

While adsorption to a reaction vessel wall is not a concern in a levitated drop, evaporation and condensation are major concerns.1,2 These concerns are addressed by using a controlled environment in which humidity, temperature, ambient pressure, and gas composition

112 surrounding the drop are regulated. Currently there are minimal systems in place to control the aforementioned variables. There is an environmental box surrounding the levitator which can be closed during experiments to shield from drafts, debris, and other interferences. This environmental box can be enhanced with various sensors and systems to control humidity, temperature, composition, and pressure. For example, flowing heated or cooled gases through the environmental box can regulate the temperature around the drop. Humidity control will require controls to avoid high or low humidity levels from adversely affecting the levitated drop. One way to accomplish this would be to saturate the feed gas stream at a low temperature, then reheating the gas to operating temperature before releasing it around the drop.

Considering most of the reactions will involve enzymes, maintaining the pH of analytes will also be very important. Using dye in the substrates to monitor pH visually is one was to approach pH monitoring. This could be used in conjunction with the computer controlled

DOD dispensers to add buffer to the drop in small amounts until the pH is once again at the desired level. There are many ways that environmental controls can be approached and will need to be easily adjustable to suit varying reaction conditions. Installation of environmental controls should be a high priority before moving forward with enzyme kinetic experiments.

Computer Control

Although there is a small levitation community, we pride ourselves on having developed the most power-efficient and completely computer-controlled levitator available. Unfortunately, those same computer controls display their limitations all too often. (See Appendix B:

113 Operating an LDR.) We rely on computer controls to control power to the levitator and gather the necessary information (temperature, pressure, and relative humidity surrounding the levitation cavity) needed to determine the reflector height that matches cavity resonance to transducer resonance frequency. It there is a failure in this process and the reflector height is miscalculated, then the distance between the resonating plate and the reflector is off, no nodes are created, and levitation is difficult to achieve. In addition to having to get the reflector height correct, it is also imperative to get the drive voltage correct; too high and the drop will explode, too low and the drop will fall out of levitation. Both the initial drive voltage and reflector plate height are determined during the initial computer controlled- sequence, and if that system fails to deliver the correct values, they have to be adjusted manually.

An effective feedback loop system needs to be implemented that is always monitoring the levitation cavity temperature, pressure, and humidity and utilizes that information to adjust both the reflector height and drive voltage for optimal levitation. A system that hunts for resonances would also prove useful in immediate levitation. Implementation of these systems will shave time off experiments that are currently spent just trying to levitate the initial drop.

In addition to controlling the levitation using a computer, a computer-controlled sample handling system is also needed. Once environmental controls are in place there will be a need for a 100% hands-free sample handling because the environmental box will be sealed.

These types of computer controls that have quick calibration systems and multiple feedback

114 loops so they are always checking the status of the levitation system will make levitation fast, reliable, wall-less, robust, and aid in reproducibility.

5.2 Understanding Dynamics of a Levitated Drop

The most important piece of the levitated drop system is the drop. What is taking place inside the drop is what we are most interested in. It is our reaction vessel. By understanding the way the drop behaves in the levitation cavity, we will learn how to utilize those behaviors and changes in behavior to our advantage. Studying the drop dynamics is an experiment well worth exploring.

While I was doing the NADH chemiluminescence experiments discussed in chapter 2, I observed some interesting trends in the output spectrum when the drop was behaving abnormally. Figure 5.3 shows examples of time series obtained while collecting data for

Chapter 3. These are less-than-ideal time series, as were obtained in over 80% of experiments. The questions that I ask are 1) what causes these different phenomena to occur,

2) how do the phenomena affect the reactants in the drop and 3) how can I initiate this phenomenon to achieve a desired result within my reaction?

5.3 LDR Applications

As mentioned earlier, aside from a few developments that the LDR could use to make it run more efficiently, the levitator system is useful in its current condition. The intended purpose for this instrument was to study free-radical enzyme-catalyzed reactions and this thesis showed that this is possible. The experiments presented here demonstrated the potential for

115 this wall-less, micro-environment system to be used as a micro-reactor to study a variety of biochemical reaction kinetics. In addition to showing the use of the LDR for various experiments, good next steps would be to examine the feasibility of utilizing the other detection systems with the LDR.

Two experiments that I attempted over the past year that would be good places to extend once the environmental controls and sample handling issues are addressed would be

1) examining the Peroxidase-Oxidase oscillatory reaction within a levitated drop3 and

2) sensing hydrogen peroxide in studying the inhibition effects of tryptophan and its analogs on myeloperoxidase4. Both of these experiments involve reaction mechanisms important to human immune response. They also can both be done on the macro-scale and then compared to the results obtained on the microscale (in the LDR). One experiment deals with

Myeloperoxidase, which in Chapter 4 we demonstrated might be one of the best-suited enzymes for conducting reactions in the LDR. This experiment would be a beginning to the desired MPO research. These experiments, which both employ electrochemistry to monitor either oxygen of H2O2, will also provide insight on how well electrochemical sensing is achieved in the levitation cavity.

Another experiment that I attempted was to investigate the effects of hydrogen peroxide

- H2O2, Cl , and HOCl on MPO. The idea was to expose MPO to the aforementioned analytes and use various types of mass spectrometry to see if these interactions caused any structural changes to MPO. The idea came from an assertion made recently by Winterbourn and coworkers, based on results of a study they conducted, that modeled oxidant production

116 within phagosomes. They suggested that the oxidation of neutrophil proteins within phagosomes is likely to affect their function.5 The study showed that MPO, treated with

HOCl, killed a variety of bacteria and that amino acids and peptides reacted with HOCl to form dichloroamine groups which were unstable, yet bactericidal. In this reaction, MPO utilizes H2O2, which is generated by NADPH oxidase found in the phagosomal membrane, to oxidize Cl- to generate toxic oxidants, in this case HOCl, to destroy foreign microorganisms found in the body.6 The hypothesis is that the changes in MPO, for example, formation of polypeptide fragments could result in those fragments acting as an antibiotic, instead of relying only on the oxidizing chemistry of HOCl. Unfortunately, we were only able to try one type of top-down mass spectrometry approach and the changes observed were not enough to draw any definite conclusions. To further explore this scenario would also be worth the time. Not only would mass spectrometry be coupled with the LDR, but it would directly address a critical facet of the hypothesized reaction network of MPO.

There is much to be learned from these preliminary studies that will aid in the future studies of MPO dynamics and its involvement in ridding the body of foreign invaders. The MPO network may be complicated, but the idea is to utilize this wall-less, micro-reactor to unravel the mystery.

117 5.4 Conclusions

One goal of the Scheeline research group is to understand the innate immune system, particularly to unravel how neutrophils kill. Our key to determining this is by understanding the myeloperoxidase reaction network, considering it is arguably the most important enzyme involved in host defense. By understanding this reaction network we will potentially be able to determine how MPO is directly involved in host defense, and also how MPO contributes to atherosclerosis and other pathogenesis. In order to effectively study this system, we have developed a wall-less microreactor system. The system utilized ultrasound to levitate microliter-sized drops that are used to conduct reactions using a three-capillary bundle sample handling system. Over the past few years, development of this levitation system has taken place and proof of concept experiments have been conducted. It is clear that with a few further developments of this system, it will be optimal for studying enzyme-catalyzed reactions. This system will aid in unraveling the complex myeloperoxidase reaction network for use in aiding future drug development.

118 5.5 References

1 A. L.Yarin, G. Brenn, O. Kastner, D. Rensink, and C. Tropea, “Evaporation of

Acoustically Levitated Droplets,” Journal of Fluid Mechanics, 399, 151-204 (1999).

2 J. Sloth, S. Kiil, S., A. D. Jensen, S. K. Andersen, K. Jorgensen, H. Schiffter and G.

Lee, “Model Based Analysis of the Drying of a Single Solution Droplet in an

Ultrasonic Levitator,” Chemical Engineering Science, 61, 2701-2709 (2006).

3 C. G. Steinmetz, T. Geest and R. Larter, “Universality in the Peroxidase-Oxidase

Reaction: Period Doublings, Chaos, Period Three, and Unstable Limit Cycles.”

Journal of Physical Chemistry, 97. 5649-5653 (1993).

4 I. Sliskovic, I. Abdulhamid, M. Sharma and H. M. Abu-Soud, “Analysis of the

Mechanism by which Tryptophan Analogs Inhibit Human Myeloperoxidase,” Free

Radical Biology and Medicine, 41, 1005-1013 (2009).

5 A. Kettle, M. Coker, A. Chapman, C. Bishop and C. Winterbourn, “It’s a Gas Man!

Ammonia Chloramines and Bacterial Killing”. Free Radical Research Group. The

5th International Human Peroxidase Meeting. New Zealand. (2007).

6 D. I. Pattison and M. J. Davies, “Reactions of Myeloperoxidase-Derived Oxidants

with Biological Substrates: Gaining Chemical Insight into Human Inflammatory

Diseases,” Current Medicinal Chemistry, 13, 3271-3290 (2006).

119 5.6 Chapter 5 Figures

Figure 5.1 A) Drive waveform required to generate drops using Drop-on-Demand technology. B) Microfab droplet dispenser generating a continuous stream of drops. (+V: 15V, -V: 15V)

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Figure 5.2 Left inset shows 5 µL drop prior to step perturbation of acoustic field. Middle and right insets show two disruptions of the same drop. Drop returns to original shape between (or following) perturbations. Exposure is 7 ms (150 frames s-1). Data obtained by undergraduate Oluwafemi Masha.

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A B C

C D F

Figure 5.3 Drop Pathological Phenomena. A-F. Transients of Lactate Dehydrogenase Catalyzed Reduction of Pyruvate to Lactate in the LDR . Drop breathing (A and C), bubble formation and dissipation (B and E) , and spinning (C and F) are all presented here. Bubbles formed during drop generation; most have dissipated within 15 s of drop formation. Reprinted from Analytical Chemistry, 81, Pierre, Z., Field, C. and Scheeline, A., “Sample Handling and Chemical Kinetics in an Acoustically Levitated Drop Microreactor,” 8496, 2009, with permission from ACS publications.

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APPENDIX A

PROCEDURES FOR PREPARING 3-CAPILLARY BUNDLES

Materials:

You will need the following materials to prepare a capillary bundle for use in the LDR:

3 – 0.30 m, 151 µm ID, 665 µm OD flexible fused silica capillary tubing (TSP10665, PolyMicro Technologies, Phoenix, AZ)

A 1.65 mm ID, 0.1 m long tube (metal or plastic, to hold the bundles together during assembly and in LDR

2 – 1.50 m ID (0.06 in ID) O-rings

Pyrolin® Polyimide Coating (2-817A, Supelco, Bellefonte, PA)

Small-tip paint brush

Any type of hot plate, ring stand and claw clamp

Fine grain Emery polishing paper (AA2, 2/0, 3M, St. Paul, MN)

A BIC lighter

Isopropanol

Kimwipes® (Delicate Task Wipers, Kimberly-Clark, Roswell, GA)

30 mL beaker

Pirhana Solution = 15 mL Sulfuric Acid (H2SO4) + 5 mL of Hydrogen Peroxide (H2O2) (Do not prepare until ready to use. SCS has specific safety rules for Pirhana solution. Use cautiously.)

Storage vial filled with 10 mL MilliQ water

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Procedures:

1) Take the 3 flexible fused silica capillaries and bundle together using the 0.1 m

long tubing and o-rings.

2) Assure that at one end the capillaries align as flush as possible.

3) Using the small-tip paint brush, starting at ~ 2.5 cm from the aligned tip, apply

polyimide coating to the capillaries so that they form a bundle (apply down 0.1 m

of the capillaries).

4) Heat the hot plate to high heat (between 4 and 5 heat setting), set up the ring

stand, and attach the claw clamp. Use claw clamp to hold the capillary bundle ~

0.5 in from the hot plate surface for 20 minutes. (This will cure the polyimide and

secure the capillaries together).

5) Remove capillaries from hotplate. Allow to cool 5 minutes, and then using the

polishing paper, file down the capillary bundle side that was aligned in step #2.

By doing this, you assure that the capillaries are not only aligned but as flush and

smooth as possible for fluids to flow through, and for drops to hang from the tip.

6) Once the tip that has been smoothed, heat it using the lighter until the capillaries

glow red. After cooling, rinse with isopropanol. Wipe off remaining burnt

coating with a Kimwipe. This is done to remove the outer coating from the

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capillaries. This will allow for good adhesion of the CYTOP coating to be

applied just before use of the bundle in the LDR.

7) Prepare the piranha solution (NOTE: Prepare in a glass container. Be sure to add

H2O2 to the acid. It will bubble and the container will become HOT!!). Then

immerse the tip of the capillary bundle, up to where the coating has been

removed, into the solution. Let sit for 20 minutes. This will clean any debris or

silica that may have gotten caught up in the capillary outlet during polishing.

8) Store the capillary bundle in water to keep hydrated until ready for use.

Pictorial Representation:

3 Flexible Fused Silica Capillaries Tubing Front View

3-Capillary Bundle O-rings with exposed tip

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APPENDIX B

BASIC INSTRUCTIONS FOR OPERATING THE LEVITATED DROP REACTOR

LDR Major Components

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1. Turn on the computer. Open the ‘LDR Programs’ folder that can be found on the desktop. Double-click on the LabView ‘LDRStart.vi’ to open it. (This may take a few minutes)

2. Turn ON all Hardware. (see above schematic as a reference) a. the Agilent function generator b. the 20 KHz ×10 amplifier c. the fluid injection control system (be sure that the control switch is set to ‘Remote’) d. the argon valve (it is above the LDR; turn knob so that is parallel with the pipe) e. the video camera (double click on ‘AVT Smartview’ icon on Desktop, click on OHCILynx, Click on O: Stingray F033B (AVT), Camera screen will appear on desktop, Press play on camera to get see LDR cavity.

3. Using the LabView LDRStart.vi, start the levitator by clicking the white arrow. This will open the other LabView Vis useful in operating the levitator.

a. The Agilent function generator will set to 20.73 kHz and 4.5 Vpp b. The argon gas, used to push fluids through the injection system, valve will open and release gas at between 35 and 40 psi. c. The reflector will automatically adjust to a height based on the temperature, pressure, and relative humidity in the room.

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d. The following useful LabView Vis will open on the computer desktop: i. Linear Actuator Control (used to display and adjust the reflector height. This program is linked to the foot-petals so they can be used instead of clicking buttons in this VI.)

ii. Pressure Flow Valve Control (controls the gas flow necessary for fluid injection and removal)

4. Assure that the capillary bundle is removed from the acoustic field before attempting this step. Using a 5µL syringe, levitate a drop of water.

If drop does not levitate on the first attempt, try the following (in this order): a. Increase the voltage on the function generator to 5.8Vpp (or to a voltage just lower than 6Vpp, just before you hear a piercing noise coming from the levitator. If you hear the piercing sound, the voltage you chose is too high.) b. Using the foot pedals, adjust the height of the reflector: i. Up, if the drop is falling to the sides of the radiating plate ii. Down, if the drop levitates but is bouncing up and down, rapidly i. ii.

c. Reduce the voltage if the drop is spinning rapidly

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5. Once a drop is levitating, Record the variables from LDRStart.vi and the reflector height for future reference.

6. Use the micro-manipulators to move the capillary bundle towards the levitated drop until the drop is drawn to the capillary tip. If the drop wicks to the side of the capillary bundle, a. Wipe the capillary clean with a Kimwipe b. Move the capillaries out of the acoustic field c. Repeat steps 4 through 6

7. To inject fluid into the LDR, use the Pressure Flow Valve Control VI. (see 3.d.ii) a. Flush the capillaries with fluid samples before beginning experiments (a typical flush volume is 5µL) b. Before forming droplets, be sure to set the desired injection volumes for the each vial c. You can inject samples individually (using ‘Inject Capillary 1 or 2’ buttons) or select the ‘Form Drop’ button to have the specified drop volumes of each sample injected automatically. d. The ‘New Drop’ button is used to remove a current drop and then form a new drop as soon as the old drop is gone. e. Select ‘Remove Drop’ if you just want to remove a drop and stop there.

8. From here proceed with desired sampling and analysis.

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APPENDIX C: AUTHOR’S CURRICULUM VITAE Zakiah N. Pierre

905 W. Eads St. • Urbana, IL 61801 • (217) 365-9531 • [email protected]

EDUCATION

Ph.D. in Chemistry • University of Illinois at Urbana-Champaign (UIUC) • Urbana, IL . Anticipated graduation date: January 2011 . Dissertation title: “Acoustically-Levitated Drop Reactor (LDR) Employable for Kinetics Measurements of Biochemical Networks” . Dissertation advisor: Alexander Scheeline

Certificate in Business Administration • University of Illinois at Urbana-Champaign • Champaign, IL • 2007

BS in Chemical Engineering • Tuskegee University • Tuskegee, Alabama • 2003 . Magna cum laude

ANALYTICAL AND RESEARCH EXPERIENCE

Consultant/Data Analyst • Education Pioneers (EP) • Washington, DC • June 2010 – Aug 2010 . Worked for Friendship Public Charter Schools (FPCS) charter management organization . Compiled and analyzed student and teacher assessment data to evaluate performance . Assisted in the revision of the Performance Management Plan for FPCS instructional staff

Commercialization Analyst • Office of Technology Management • Urbana, IL • May 2009 – 2010 . Performed competitive analysis and market research on university developed technology/inventions . Made recommendations for commercialization and filing patent applications . Developed industry market reports for several technology portfolios for senior management

Research Assistant • University of Illinois at Urbana-Champaign • Urbana, IL • Aug 2004 – Jan 2011 . Analyze enzyme-catalyzed reaction kinetics related to the human innate immune response . Collaborated on the development of the world’s most efficient Acoustically Levitated Drop Reactor (LDR) with sample handling system for LDR

Research Analyst • The DOW Chemical Company • Freeport, TX • Summer 2004

. Investigated EPA regulations for industry CO2 emission limits for the state of Texas . Monitored CO2 emission levels from ethane production lines to determine if the CO2 met or surpassed EPA limits

Associate Design Engineer • Westinghouse Savannah River Co. • Aiken, SC • 2003-2004 . Developed and designed equipment and transfer paths necessary for nuclear waste removal . Assessed composition/concentration changes of waste samples and forecasted future changes

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COMMUNICATION AND INTERPERSONAL EXPERIENCE

Executive Board Member/Events Chair ▪ Women Chemist Committee (WCC) ▪ 2007 - 2009 . Sponsored events for females of all ages to attract women to a profession in the sciences . Arranged for chemical industry and academic professionals to visit UIUC to give seminars

Graduate Counselor • Office of Minority Student Affairs (OMSA) • 2007 – 2009 . Mentored 19 undergraduate freshman engineering students, annually . Collaborated with OMSA to assist students in a smooth transition into college . Helped students identify and achieve their personal, academic and professional development goals

Chemistry Instructor • University of Illinois at Urbana-Champaign • Urbana, IL • 2004 – 2006 . Facilitated small group and team interactions, encouraging students to bring their strengths to the general discussion . Developed engaging curriculum to effectively conveyed technical information to students from various backgrounds

CONSULTING AND BUSINESS EXPERIENCE

Engagement Manager • Illinois Business Consulting (IBC) • Champaign, IL • Fall 2008, Fall 2009 . Managed 13-student lead Finance related consulting projects for top financial firms . Provided goals and direction to team leaders throughout the engagement . Maintained the commitment to assure a high level of client and participant satisfaction

Project Manager • Illinois Business Consulting (IBC) • Champaign, IL • Summer 2008 . Managed a team of 5 consultants on a project for a not-for-profit start-up . Successfully developed a business strategy for establishing a not-for-profit organization . Established goals and provided direction and coaching for the team . Interacted regularly with the client to ensure our goals aligned with the client’s vision

Event Consultant (Founder/Owner) • Events by Zee, Inc. • Champaign, IL • May 2006 - Present . Create original, cost-effective events for individual and corporate clients . Track and manage budgets throughout the planning process . Manage a production staff which assist with event execution

AWARDS AND RECOGNITIONS

. Education Pioneers Fellowship Recipient - DC Metro Area, 2010 . Proctor & Gamble Co. (P&G) PhD Chemist Fellowship, 2009 . National Institute of Health (NIH) Ruth L. Kirschstein National Research Service Award for Individual Predoctoral Research Assistantship, 2007 . National Consortium for Graduate Degrees in Engineering (GEM) Ph.D. Science Fellowship Recipient (Sponsor: The DOW Chemical Company), 2004-Present . Office of Civilian Radioactive Waste Management HBCU Undergraduate Fellowship Recipient, 2002 . 3M Undergraduate Scholarship/Internship Recipient, 1998-2002

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. Girl Scouts of the USA Gold Award Recipient, 1998

PUBLICATIONS AND PRESENTATIONS

. Pierre, Z., Field, C., and Scheeline, A. Sample Handling and Chemical Kinetics in an Acoustically Levitated Drop Reactor. Analytical Chemistry 81, 8496-8502 (2009) . “Biochemical Kinetics in an Acoustically Levitated Drop Reactor”, Oral Presentation, Federation of Analytical Chemistry and Spectroscopy Societies (FACSS), Louisville, KY, October 18-22, 2009 . “Acoustically Levitated Drop Reactor: Droplet Generation, Maintenance and Control”, Oral Presentation, The Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (PITTCON), New Orleans, LA, March 2009

REFERENCES

Alexander Scheeline, Professor of Chemistry University of Illinois Urbana-Champaign 217-333-2999, [email protected]

Valerie Mitchell, Program Director, DC Metro Area Education Pioneers 202-577-6961, [email protected]

Liezl Bowman, Assistant Director, Illinois Business Consulting (IBC) 217-244-8890, [email protected]

Leslie Millar, Director, Office of Technology Management University of Illinois Urbana-Champaign 217-333-6807, [email protected]

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