Transforming Microtubules into Microshuttles for Advanced Immunoassay Applications by Jenna Marie Campbell A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Mechanical Engineering) in the University of Michigan 2014 Doctoral Committee: Professor Edgar Meyhöfer, Chair Assistant Professor Barry Grant Assistant Professor Ajit P. Joglekar Professor Katsuo Kurabayashi © Jenna Campbell 2014 All Rights Reserved For Lisa Orr and Vivek Tomer— For pushing me forward every time I wanted to take a step back. Your love, support, and guidance have been invaluable. ii ACKNOWLEDGEMENTS This dissertation is the culmination of many years of work, which would not have been possible without the guidance of my advisor, Edgar Meyhöfer. He challenged me, taught me how to ask questions, and how to “do science.” Always having an open door, your support and expertise has been invaluable academically, professionally, and personally. I would also like to acknowledge Katsuo Kurabayashi for your expertise and support. Our meetings gave me a broader perspective on our projects, which played a large role in shaping my academic and professional goals. Committee members Ajit Joglekar and Barry Grant have always been generous with their time, and I am thankful for the advice and expertise they have given throughout my graduate career. My co-workers Charles Jiang and Neha Kaul played a critical role in this work. Thank you for bringing laughter along with great insight into my work. I would like to acknowledge Professor James Gole for encouraging me to continue my education, which brought me to the University of Michigan. I would not have discovered my love of science and research if it were not for your passion to inspire undergraduates and educate young scientists. Finally, my friends and family who have supported me have been critical to my accomplishments over the last five years. Vivek Tomer has given me guidance, advice, and support that was so helpful in overcoming every hurdle I encountered at Michigan. Megan Roberts and Marilyn Gatewood are two brilliant female engineers that I am fortunate to have as role models and friends. My greatest thanks goes to Lisa, Casey, Claire, and Hannah whose patience, love, and support have carried me through my whole life and will take me through many more years. iii TABLE OF CONTENTS DEDICATION .................................................................................................................. ii ACKNOWLEDGEMENTS ............................................................................................. iii LIST OF FIGURES .......................................................................................................... vii LIST OF TABLES ........................................................................................................... viii LIST OF ABBREVIATIONS ........................................................................................... ix CHAPTER 1: MOTIVATION ..........................................................................................1 CHAPTER 2: BACKGROUND ........................................................................................3 2.1 Microtubule-Kinesin Systems: From In Vivo Motility to Lab on a Chip 3 Microtubules as Nanoshuttles .................................................................5 Highly Engineered Cargos ......................................................................7 Kinesin Motor Control .............................................................................8 Mechanical Control of Microtubules ....................................................10 Electrophoretic and Magnetic Control of Microtubules ....................12 2.2 Proteomic Biosensors ....................................................................................13 2.3 Kinesin-Driven Immunoassay Platform ....................................................17 Microtubule Concentrator .....................................................................17 Motivation and Potential for Immunoassay Applications ................19 Hurdles in Combining Kinesin and Microtubules in Proteomic Sensors ......................................................................................................20 CHAPTER 3: ANTIBODY-FUNCTIONALIZED NANOSHUTTLES DRIVEN BY BIOMOLECULAR MOTORS FOR SENSITIVE IMMUNOASSAY APPLICATIONS .............................................................................................................24 3.1 Introduction ...................................................................................................25 3.2 Approach ..........................................................................................................2 iv 3.3 Results .........................................................................................................................28 Specificity and Binding Capacity of Antibody-Functionalized Microshuttles ...........................................................................................28 Motility Properties of Antibody-Functionalized Microshuttles .......36 Limitations for Sandwich Assay Construct .........................................37 3.4 Conclusions ....................................................................................................41 3.3 Experimental Section ....................................................................................42 Microtubule Preparation ........................................................................42 Kinesin Purification ................................................................................43 Microtubule Antibody Conjugation .....................................................43 Ellman’s Reagent Assays .......................................................................44 Binding Capacity Assays .......................................................................45 Motility Assays ........................................................................................46 CHAPTER 4: KINESIN AND MICROTUBULE COMPATABILITY IN HUMAN URINE, SALIVA, AND BLOOD PLASMA .................................................................47 4.1 Introduction ...................................................................................................47 4.2 Biotinylated Microtubules and Their Ability to Bind Streptavidin Cargos in Human Urine, Saliva, and Blood Plasma ......................................49 4.3 Microtubules and Their Motility Properties in Human Urine, Saliva, and Blood Plasma ................................................................................................55 4.4 Motility Characteristics Required for Steering Microtubules in Kinesin- Driven Technologies ...........................................................................................57 4.5 Motility Properties of Kinesin and Microtubules in Human Blood Plasma ...................................................................................................................64 Unfiltered Blood Plasma ........................................................................65 Filtered (10 kDa) Blood Plasma .............................................................69 4.6 Conclusions ....................................................................................................71 4.7 Experimental Section ....................................................................................72 Human Sample Preparation: Blood Plasma ........................................72 Human Sample Preparation: Saliva .....................................................73 Human Sample Preparation: Urine ......................................................73 v Microtubule Preparation ........................................................................74 Binding Capacity Assays .......................................................................75 Quality of Motility ..................................................................................76 CHAPTER 5: CONCLUSIONS AND FUTURE WORK ............................................77 5.1 Conclusions ....................................................................................................77 5.2 Future Work: Fluorescent Detection Via Labeled Detection Antibodies ............................................................................................................79 Modifying Assay Geometry to Support the Transport of Large Cargos .......................................................................................................80 Modifying MT-Antibody Conjugation Procedure to Support the Transport of Large Cargos .....................................................................81 Transport Fewer, Larger Cargos to Sustain Motility .........................84 5.3 Future Work: Kinesin-Driven Immunassay Platforms in Blood Plasma ...................................................................................................................84 Separate Sample Collection from Gliding Assays ..............................85 Engineer Technologies Accommodating for Motility in Blood Plasma .......................................................................................................86 5.4 Future Outlook for Kinesin-Driven Technologies ...................................88 WORKS CITED ................................................................................................................90 vi LIST OF FIGURES Figure 2.1 Geometry of gliding assay…...……………………..………….…..……. 5 Figure