TECHNOLOGY PLATFORMS FOR TRANSFORMING COMPLEX BIOLOGICAL STUDIES By

251 Pages·2012·12.06 MB·English

by  Chrissy Marasco

TECHNOLOGY PLATFORMS FOR TRANSFORMING COMPLEX BIOLOGICAL STUDIES
By
Christina C. Marasco

Dissertation
Submitted to the Faculty of the
Graduate School of Vanderbilt University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
in
Biomedical Engineering
August, 2012
Nashville, Tennessee

Approved:
Professor John P. Wikswo
Professor Kevin T. Seale
Professor John A. McLean
Professor David E. Cliffel
Professor Hak-Joon Sung
Professor Danny G. Winder
In memory of Ben Liesch and Dean Paras
Without loss, we would lack the drive to save.

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ACKNOWLEDGEMENTS

This work has been financially supported by the Defense Threat Reduction Agency
(DTRA), the National Institute on Drug Abuse (NIDA), the Searle Systems Biology and
Bioengineering Undergraduate Research Experience (SyBBURE-Searle), and the Vanderbilt
Institute for Integrative Biosystems Research and Education (VIIBRE).
The work herein represents a highly collaborative multidisciplinary, inter-institutional
research effort that spans the work of undergraduate students, graduate students, staff
scientists and engineers, research associates, and faculty from Vanderbilt University, Duke
University, and Cornell University. An exhaustive list of contributors and their affiliations can be
found in Appendix A. I am overwhelmingly appreciative for all of their efforts, though this is not
a sufficient display of gratitude for all of their indispensible work. I particularly thank Jeff Enders
and Cody Goodwin for their sizeable contributions to this body of work, for their friendship, for
their commiseration, and for their humor. To the many SyBBURE students who have endured
working alongside me, burdened by my lofty expectations; I thank you for your hard work, for
talking through problems, and for quitting on me or graduating when I needed you, forcing me
to come to terms with my lack of knowledge. These projects also could not have been
completed without the support and expertise of the VIIBRE scientists, engineers, and support
staff. The opportunity to participate in this research was afforded to me at critical time in my
graduate career and without it I would not have achieved this goal. I sincerely thank Kevin Seale,
John Wikswo and John McLean for allowing me to work with SyBBURE and on these projects.
Your continued support has and will continue to be appreciated on many levels.
Finally, I wish to acknowledge those who have supported this eight-year journey. I will
show you my gratitude through my renewed presence in your lives.
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TABLE OF CONTENTS

Page
ACKNOWLEDGEMENTS .............................................................................................................. III
LIST OF TABLES ......................................................................................................................... VII
LIST OF FIGURES....................................................................................................................... VIII
CHAPTER I ................................................................................................................................... 1
1 INTRODUCTION .............................................................................................................. 1
1.1 Objective ........................................................................................................... 1
1.2 Specific Aims ...................................................................................................... 2
1.2.1 Specific Aim 1 ........................................................................................ 2
1.2.2 Specific Aim 2 ........................................................................................ 3
1.3 Technology Innovation, Background and Significance......................................... 3
1.3.1 Microfluidics for the Study of Cellular Response .................................... 3
1.3.2 Mass Spectrometry for Cell State Assessment ..................................... 11
1.3.3 Combining Microfluidics with Mass Spectrometry ............................... 14
1.3.4 Computational Modeling of Complex Biological Systems ..................... 15
1.3.5 Systems Biology Approach for Automated Science .............................. 17
1.3.6 Summary ............................................................................................. 18
1.4 Biological Background and Significance ............................................................ 19
1.4.1 Cocaine Effects .................................................................................... 19
1.5 References ....................................................................................................... 41
CHAPTER II ................................................................................................................................ 60
2 REAL-TIME CELLULAR EXOMETABOLOME ANALYSIS WITH A ......................................... 60
MICROFLUIDIC-MASS SPECTROMETRY PLATFORM ................................................................ 60
2.1 Abstract ........................................................................................................... 61
2.2 Introduction ..................................................................................................... 62
2.3 Methods .......................................................................................................... 69
2.3.1 Microfluidic Bioreactor Design and Fabrication .................................... 69
2.3.2 PDMS Surface Modification ................................................................. 69
2.3.3 Cell Culture and “In Culture” Cocaine Exposure ................................... 72
2.3.4 Metabolomics Sample Preparation and UPLC-ESI-IM-MS Analysis ....... 73
2.3.5 Online Cell Loading and Experimentation ............................................ 74
2.3.6 Solid Phase Extraction Desalter............................................................ 75
2.3.7 Online Cell Effluent Desalting and Mass Spectrometry Analysis ........... 77
2.3.8 Data Processing and Multivariate Statistical Analysis ........................... 78
2.4 Results and Discussion ..................................................................................... 78
2.4.1 Platform Integration and Evaluation .................................................... 78
2.4.2 Cocaine Metabolism in Naïve and Experienced T cells ......................... 84
2.5 Conclusions...................................................................................................... 90
2.5.1 Platform Capabilities and Shortcomings .............................................. 90
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2.5.2 Cellular Memory of Cocaine Experience............................................... 92
2.6 References ....................................................................................................... 94
CHAPTER III ............................................................................................................................... 97
3 PROGRESS TOWARDS A HETERODYNE FRAMEWORK FOR THE GENERATION AND
DETECTION OF OSCILLATORY CHEMICAL SIGNALS IN NONLINEAR LIGHT-PRODUCING SYSTEMS
97
3.1 Abstract ........................................................................................................... 98
3.2 Introduction ..................................................................................................... 99
3.3 Heterodyne Chemistry Theory ....................................................................... 100
3.3.1 Heterodyne Models of Nonlinear Reactions....................................... 103
3.4 Development of Hardware and Data Analysis Methods for Heterodyne
Chemistry ................................................................................................................... 110
3.4.1 Sinusoidal Concentration Generation ................................................ 111
3.4.2 Chemical Signal mixing with a Microfluidic Reactor............................ 123
3.4.3 Detection of Light as Reaction Products ............................................. 123
3.4.4 Fourier Transform Analysis ................................................................ 128
3.5 Nonlinear Chemical Reaction Exploration ....................................................... 129
3.5.1 Fluorescence Quenching.................................................................... 129
3.5.2 Peroxyoxalate Chemiluminescence.................................................... 137
3.6 Summary, Pitfalls, and Problems .................................................................... 146
3.7 Future Solutions............................................................................................. 150
3.8 References ..................................................................................................... 152
CHAPTER IV ............................................................................................................................. 156
4 SUMMARY, FUTURE WORK AND ETHICAL IMPACT ...................................................... 156
4.1 MF-SPE-nESI-IM-MS ....................................................................................... 157
4.1.1 Microfluidic Device Design and Control ............................................. 158
4.1.2 Online Desalting ................................................................................ 158
4.1.3 Data Analysis ..................................................................................... 160
4.1.4 MTNP-SPE-nESI-IM-MS ...................................................................... 161
4.2 Heterodyne Framework ................................................................................. 162
4.2.1 Sinusoidal Concentration Generation ................................................ 163
4.2.2 Upstream Chemical Signal Mixing ...................................................... 165
4.2.3 Downstream Chemical Signal Detection ............................................ 166
4.2.4 Data Analysis Techniques .................................................................. 166
4.2.5 Modeling Heterodyne Chemistry ....................................................... 167
4.2.6 Probing Biochemical Reactions .......................................................... 167
APPENDIX A ALPHABETICAL LIST OF CONTRIBUTORS .............................................................. 168
APPENDIX B MTNP-SPED-NESI-IM-MS USE AND TROUBLESHOOTING GUIDE .......................... 169
APPENDIX C MEDI HEAT MAPS ............................................................................................... 197
APPENDIX D CHEMICAL REACTION MODELS ........................................................................... 200
APPENDIX E RPPM CODE ........................................................................................................ 219
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APPENDIX F HETERODYNE DATA ANALYSIS CODE ................................................................... 227
APPENDIX G PUMP CALIBRATION ANOVA .............................................................................. 238
APPENDIX H HETERODYNE FREQUENCY TABLES ..................................................................... 241

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LIST OF TABLES

Table Page
1 Methods of Cellular Communication 8
3.1 Methods of Cellular Communication 99
3.2 Fluorescein Quenching Frequencies 106
3.3 Peroxyoxalate Chemiluminescece Frequencies 109
3.4 Input Speed and Average Resulting Flow Rates for Constant Speed Calibration 121
3.5 Input and Output Parameters for Sinusoidal Calibration 121
3.6 Fluorescein Quenching Experimental Variable Flow Frequencies 133
3.7 Fluorescein Quenching Experimental Corrected Frequencies 135
3.8 Reservoir Contents and Sample Input Parameters for Peroxyoxalate Reaction 138
3.9 Pre- and Post-calibration Results for Peroxyoxalate Reaction 142
3.10 Comparison of Model and Experimental Peroxyoxalate Reaction Frequencies 145
3.11 Heterodyne chemistry hardware performance metrics 147

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LIST OF FIGURES

Figure Page
1.1 Device Fabrication, Design and Cell Loading 6
1.2 Examples of Microfluidic Sinusoidal Concentration Generators 10
1.3 Ion Mobility Mass Spectrometry 12
1.4 Waters Synapt G2 13
1.5 Conceptual Pathway from Experimental Data to Discovery of Conservation Laws 16
1.6 A Comparison of Substances of Abuse 20
1.7 Potential Mechanisms of the σ1 Receptor 26
1.8 Summary of Effects of Cocaine on Immune Function 28
1.9 Brain-immune Bi-directional Connections 37
2.1 Cocaine Metabolic Pathways 67
2.2 Experimental Scheme Showing the Potential Cell Fates 68
2.3 Device Fabrication and Design 70
2.4 PDMS Silanization 71
2.5 Cocaine Exposure Scheme 72
2.6 Solid Phase Extraction Desalter Setup 76
2.7 MTNP-SPE-nESI-IM-MS Platform 79
2.8 Insulin Treatment of Silanized PDMS Channels 83
2.9 Walking Principal Component Analysis of Exometabolomic Profiles 85
2.10 Benzoylecgonine Time Course and Fragmentation Spectrum 86
2.11 Additional Metabolite Time Course Data 88
2.12 Metabolite Expression Dynamics Inspector Heat Maps 89
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2.13 In Culture Principal Component Analysis of Exometabolomic Profiles 91
3.1 Heterodyne Chemistry Basics 101
3.2 Reaction Rate Equations for Fluorescein Quenching 104
3.3 Fluorescein Quenching Model Data 105
3.4 Reaction Rate Equations for Peroxyoxalate Chemiluminescence 107
3.5 Peroxyoxalate Chemiluminescence Model Data 108
3.6 Examples of Microfluidic Sinusoidal Concentration Generators 112
3.7 Multiple Pump Sinusoidal Concentration Profile Generation 113
3.8 Y-offset Contributions to Frequency Sprectrum 114
3.9 Rotary Planar Peristaltic Micropump 116
3.10 Single Pump Sinusoidal Flow Rate 118
3.11 Pump Calibration 120
3.12 Sinusoidal Pump Calibration 122
3.13 Microfabrication Procedure 124
3.14 Continuous Flow Photometer 125
3.15 CFP Evaluation 127
3.16 CFP Detection of Sinusoidal Concentration Profiles 128
3.17 Experimental Setup for Fluorescein Quenching 131
3.18 Fluorescein Quenching Sample Data Set 132
3.19 Corrected Fluorescein Quenching Sample Data Set 134
3.20 3D Plot of Varying FL Drive Frequencies 136
3.21 Experimental Setup for Peroxyoxalate Chemiluminescence 139
3.22 Pre- and Post-calibration Peroxyoxalate Chemiluminescence Experimental Data 141
3.23 3D Plot of Varying H2O2 Drive Frequencies 143
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3.24 Model versus Experimental Peroxyoxalate Chemiluminescence 144

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