New Developments in Isoelectric Focusing and ... New Developments in Isoelectric Focusing and Dielectrophoresis

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  • 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

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    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

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    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.

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    To the women in my life, my mother for guiding me to this point and my wife for

    ensuring I arrived in success.

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    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.

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    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.

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    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

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    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 .........