New Developments in Isoelectric Focusing and Dielectrophoresis for Bioanalysis by Noah Graham Weiss A Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Approved November 2011 by the Graduate Supervisory Committee: Mark Hayes, Chair Antonio Garcia Alexandra Ros ARIZONA STATE UNIVERSITY December 2011 ABSTRACT Bioanalytes such as protein, cells, and viruses provide vital information but are inherently challenging to measure with selective and sensitive detection. Gradient separation technologies can provide solutions to these challenges by enabling the selective isolation and pre-concentration of bioanalytes for improved detection and monitoring. Some fundamental aspects of two of these techniques, isoelectric focusing and dielectrophoresis, are examined and novel developments are presented. A reproducible and automatable method for coupling capillary isoelectric focusing (cIEF) and matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) based on syringe pump mobilization is found. Results show high resolution is maintained during mobilization and β-lactoglobulin protein isoforms differing by two amino acids are resolved. Subsequently, the instrumental advantages of this approach are utilized to clarify the microheterogeneity of serum amyloid P component. Comprehensive, quantitative results support a relatively uniform glycoprotein model, contrary to inconsistent and equivocal observations in several gel isoelectric focusing studies. Fundamental studies of MALDI-MS on novel superhydrophobic substrates yield unique insights towards an optimal interface between cIEF and MALDI-MS. Finally, the fundamentals of isoelectric focusing in an open drop are explored. Findings suggest this could be a robust sample preparation technique for droplet-based microfluidic systems. Fundamental advancements in dielectrophoresis are also presented. Microfluidic channels for dielectrophoretic mobility characterization are designed i which enable particle standardization, new insights to be deduced, and future devices to be intelligently designed. Dielectrophoretic mobilities are obtained for 1 µm polystyrene particles and red blood cells under select conditions. Employing velocimetry techniques allows models of particle motion to be improved which in turn improves the experimental methodology. Together this work contributes a quantitative framework which improves dielectrophoretic particle separation and analysis. ii To the women in my life, my mother for guiding me to this point and my wife for ensuring I arrived in success. iii ACKNOWLEDGMENTS I begin with a special thanks to my advisor Dr. Mark Hayes. The experiences, challenges, and guidance you have given me during my time at ASU have transformed me into a new person. I thank you for your time, support, and belief in me. Next I give thanks to my family. First and foremost I thank my beautiful wife and best friend Julie who has helped me tremendously through my entire graduate school journey. Thank you for keeping me grounded and for making every day special. Thanks to my mother who makes me aware of my strengths, challenges me on my weaknesses, and has provided me with so many great opportunities. Thanks to my father who is my inspirational scientist and must have planted scientific seeds in my brain many years ago. Thanks to my big brothers Seth and Jefferson who have taught me so much about the world and have been wonderful supporters in my life. I could not have succeeded in graduate school without my peers. Thank you to Dr. Michelle Meighan for your friendship and for taking me under your wing in the early years of graduate school. Thank you to Dr. Josie Castillo for being a role model and for paving the way one step ahead of me. Thank you to Stacy Kenyon for being a good office mate and even better friend. Thank you to Paul Jones for late afternoon discussions (or was it venting?) over the origins of dielectrophoresis. Thank you to Dr. Prasun Mahanti for helping me understand things outside of my comfort zone. I am very fortunate to have been surrounded by such hard working and fun people. iv There are many other people who deserve special recognition. Thank you to my committee members Dr. Alexandra Ros and Dr. Antonio Gartcia for your contributions toward my graduate education and for challenging me on my work. Thank you to those who contributed to my research projects: Nicole Zwick for guidance in isoelectric focusing, Dr. Randall Nelson for providing MALDI-MS facilities, Jason Jarvis for assistance in MSIA and ESI-MS, Dr. Tom Picraux for nanowire synthesis, Dr. Timo Park for help in superhydrophobic surfaces, Dr. Ana Egatz-Gomez for help in droplet isoelectric focusing, Dr. Rafat Ansari and Jim King for providing NASA facilities and mentorship, Dr. Kang P. Chen for and Dr. Tom Taylor for assistance in dielectrophoretic mobility framework, Saleh Gani for help in particle tracking, and finally the CSSER cleanroom staff for assistance in fabrication and for keeping my research projects alive. Finally, I would like to acknowledge funding from the Jump Start Research Grant sponsored by the Graduate and Professional Students Association (GPSA). In addition, I am especially grateful to the GPSA for conference travel grant sponsorships which tremendously enriched my graduate school experience. I would not be where I am today without help from all of these people. Thank you all dearly for your guidance and support. v TABLE OF CONTENTS Page LIST OF TABLES ..................................................................................................... xii LIST OF FIGURES .................................................................................................. xiii CHAPTER 1 INTRODUCTION .................................................................................. 1 Role of Separation Science in Analytical Chemistry ........................ 1 Linear vs. Gradient Separations ......................................................... 2 The Importance of Bioanalytes .......................................................... 4 Nucleic Acids ......................................................................... 5 Proteins ................................................................................... 6 Cells and Viruses .................................................................... 6 Other Bioanalytes ................................................................... 6 Overview of Isoelectric Focusing and Dielectrophoresis .................. 7 Overview of MALDI-MS ................................................................... 7 Superhydrophobic Surfaces ................................................................ 8 Dissertation Objectives ..................................................................... 10 References ......................................................................................... 11 2 OVERVIEW OF ELECTROKINETIC TECHNIQUES .................... 14 Principle of Charge, Electric Fields, and Electrokinetic Separation Techniques ........................................................................................ 14 Engineering Electric Fields for Separations ..................................... 15 Electrokinetic Separation Techniques .............................................. 17 vi CHAPTER Page Electrophoresis ..................................................................... 17 Isoelectric Focusing .............................................................. 19 Gel Isoelectric Focusing .......................................... 22 Capillary Isoelectric Focusing ................................. 23 Droplet-Based Isoelectric Focusing ........................ 24 Dielectrophoresis .................................................................. 25 References ......................................................................................... 26 3 CAPILLARY ISOELECTRIC FOCUSING COUPLED OFFLINE TO MALDI-MS WITH SYRINGE PUMP MOBILIZATION .......... 29 Introduction ....................................................................................... 29 Materials and Methods ..................................................................... 31 Chemicals and Materials ...................................................... 31 Capillary Isoelectric Focusing ............................................. 31 Hardward Components for Instrumentation ........................ 32 MALDI-MS .......................................................................... 33 Results and Discussion ..................................................................... 34 Concluding Remarks ........................................................................ 39 References ......................................................................................... 39 4 EXAMING SERUM AMYLOID P COMPONENT MICROHETEROGENEITY USING CAPILLARY ISOELECTRIC FOCUSING AND MALDI-MS ......................... 41 Introduction ....................................................................................... 41 vii CHAPTER Page Materials and Methods ..................................................................... 45 Chemicals and Materials ...................................................... 45 Mass Spectrometric Immunoassay ...................................... 46 Capillary Isoelectric Focusing ............................................. 47 Desialylation of SAP ............................................................ 49 Mass Spectrometry ..............................................................