MAGNETIC ISOLATION OF FECAL INDICATING BACTERIA USING BIO- FUNCTIONALIZED MAGNETIC MICRODISCS FOR WATER QUALITY MONITORING By KEISHA YARIE CASTILLO-TORRES A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2020 © 2020 Keisha Yarie Castillo-Torres To my beloved parents ACKNOWLEDGMENTS I would like to thank the University of Florida (UF) Office of Technology Licensing (OTL) and the US Army Medical Research and Materiel Command under SBIR Contract led by Innovative Space Technologies, LLC for their financial support for this project. I also thank my supervisory committee: Dr. Y.K. Yoon, Dr. Carlos Rinaldi, Dr. Eric McLamore, and Dr. David Arnold for their support, guidance, and ideas throughout this process. Special thanks to Dr. Eric McLamore for his continuous help and guidance on the biological concepts and experiments. Also, my immense gratitude goes to my supervisory committee chair and advisor, Dr. David Arnold for giving me the opportunity to work in his group and for his continuous and valuable support, guidance, advice, mentorship, and patience. I would also like to thank staff from the University of Florida (UF) Nanoscale Research Facility (NRF) for their assistance during the microfabrication process and the staff from the Electron Microscopy core in the UF Interdisciplinary Center for Biotechnology Research (ICBR) for their assistance during confocal imaging. Special thanks go to my lab mates and friends from Dr. Arnold’s group: Dr. Nicolas Garraud, Dr. Camilo Velez, Dr. Alexandra Garraud, Dr. Xiao Wen, Yuzheng Wang, Connor Smith, and Beatriz Jimenez, whose help, support, feedback, friendship, and/or positive vibes have helped throughout this path. Also, special thanks to Dr. Fan and his students, specially Jingling Zhang, Kangfu Chen, Jose Varillas, Pablo Dopico, and Minh-Chau Le for their help and availability to help, teach, and let me use their fluorescence scope, and microfluidic fabrication tools. As well as Dr. Rinaldi and his students, especially Angelie Rivera-Rodríguez and Melissa Cruz-Acuña for their help. Finally, special thanks go to my “cubicle row-mates” and friends from the 4 Interdisciplinary Microsystems Group (IMG), specifically, Brittney, Justin, Ashley, and Brett, whose support, friendship, and/or positive vibes have also helped on a daily basis. Also, I would like to deeply thank professors and mentors that encouraged me to consider, pursue, and/or continue my doctoral degree, specifically Dr. Nayda Santiago, Dr. Domingo Rodríguez, Dr. Néstor Rodríguez, and Dr. María Vera. Special thanks go to my long-distance friends (Mónica, Kathyria, Sharlene, Débora, Lorraine, and Deneiz) for their support, motivation, and positive vibes when I needed them the most (even when miles away). But most specially, I would like to thank my dearest friend Sylmarie Dávila-Montero, for her unconditional and constant support and feedback, and for walking this path with me from the very beginning when we were lab mates until today when we are miles away pursuing our PhD degrees. Immeasurable thanks to my parents, Irma L. Torres-Plaza and Luis A. Castillo- Dávila, whose endless love, understanding, and support have meant the world to me throughout every single step of my personal, academic, and professional paths. Also, I will be forever grateful with my loving husband, and best friend, Adail A. Rivera-Nieves, whose unconditional love, motivation, understanding, and support have made this path easier by just listening, celebrating even the smallest of my victories, and for walking this path right by my side while working towards his own PhD degree. Finally, and most importantly, I would like to thank God for giving me the strength and persistence to start, continue, and complete my PhD degree. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 9 LIST OF FIGURES ........................................................................................................ 10 LIST OF ABBREVIATIONS ........................................................................................... 15 ABSTRACT ................................................................................................................... 16 CHAPTER 1 INTRODUCTION .................................................................................................... 18 1.1 Overview ....................................................................................................... 18 1.2 Motivation ...................................................................................................... 18 1.3 Previous Works on Bacteria Detection .......................................................... 20 1.3.1 Standard Methods ............................................................................... 20 1.3.2 Bio-Nano-Magnetic Methods ............................................................... 22 1.3.3 Microfluidic Magnetic Separation Technologies .................................. 28 1.4 Research Approach ...................................................................................... 31 1.4.1 Objectives ........................................................................................... 32 1.4.2 Methodology ....................................................................................... 32 1.5 Dissertation Overview ................................................................................... 34 2 BACKGROUND ...................................................................................................... 36 2.1 Overview ....................................................................................................... 36 2.2 Biosensors Overview .................................................................................... 36 2.3 Magnetic Materials Overview ........................................................................ 41 2.4 Spin-Vortex Magnetic Behavior ..................................................................... 45 2.5 Magnetic Forces and Torques Overview ....................................................... 49 2.6 Summary ....................................................................................................... 52 3 BIO-FUNCTIONALIZED MAGNETIC MICRODISCS ASSAY TO DETECT PROTEIN-COATED PARTICLE OR BACTERIAL TARGETS ................................ 53 3.1 Overview ....................................................................................................... 53 3.2 Microfabrication of Gold-Coated Magnetic Microdiscs .................................. 55 3.3 Surface Bio-Functionalization of Magnetic Microdiscs .................................. 58 3.3.1 Surface Bio-Functionalization for Avidin-Coated Particles .................. 58 3.3.2 Surface Bio-Functionalization for Bacterial Targets ............................ 59 3.4 Sample Preparation ...................................................................................... 60 3.4.1 Protein-Coated Particle Target Samples ............................................. 60 6 3.4.2 Bacterial Target Samples .................................................................... 61 3.5 Bio-Functionalized Microdiscs Targeting Particles and/or Bacteria ............... 62 3.6 Viability Assay Preparation for Bacterial Targets .......................................... 62 3.6.1 SYTO9 / Propidium Iodide (PI) Viability Labelling ............................... 63 3.6.2 Other Fluorescent Labels .................................................................... 63 3.7 Magnetic Isolation and Detection of Target Bacteria/Particles Overview ...... 64 3.8 Fluorescence Inspection ............................................................................... 64 3.9 Summary ....................................................................................................... 65 4 METHODS FOR THE MAGNETIC SEPARATION OF MICRODISCS.................... 66 4.1 Overview ....................................................................................................... 66 4.2 Magnetic Microdisc Capture Efficiency Quantification ................................... 67 4.3 Multiphysics Simulations ............................................................................... 71 4.3.1 Magnetic Simulations .......................................................................... 72 4.3.2 Microfluidic and Particle Tracing Simulations ...................................... 76 4.4 Magnetic Separation Configurations ............................................................. 78 4.4.1 Single Magnet Configuration Setup..................................................... 78 4.4.2 Dual Magnet (Attracting and Repelling) Configuration Setups ............ 79 4.4.3 Column Array of Magnets with Alternating Polarizations ..................... 80 4.4.4 Microfluidic Magnetic Separation (µFMS) Device ............................... 82 4.4.4.1 µFMS device fabrication .............................................................. 83 4.4.4.2 µFMS device experiments ........................................................... 85 4.4.4.3 Estimation of magnetic capture efficiency of µFMS device .......... 89 4.4.4.4 Magnetic capture efficiency vs. flow rate for µFMS device .......... 91 4.5 Filtering MNPs from Large-Volume