Magnetic Force Microscopy of Superparamagnetic Nanoparticles for Biomedical Applications Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Tanya M. Nocera, M.S. Graduate Program in Biomedical Engineering The Ohio State University 2013 Dissertation Committee: Gunjan Agarwal, PhD, Advisor Stephen Lee, PhD Jessica Winter, PhD Anil Pradhan, PhD Copyright by Tanya M. Nocera 2013 Abstract In recent years, both synthetic as well as naturally occurring superparamagnetic nanoparticles (SPNs) have become increasingly important in biomedicine. For instance, iron deposits in many pathological tissues are known to contain an accumulation of the superparamagnetic protein, ferritin. Additionally, man-made SPNs have found biomedical applications ranging from cell-tagging in vitro to contrast agents for in vivo diagnostic imaging. Despite the widespread use and occurrence of SPNs, detection and characterization of their magnetic properties, especially at the single-particle level and/or in biological samples, remains a challenge. Magnetic signals arising from SPNs can be complicated by factors such as spatial distribution, magnetic anisotropy, particle aggregation and magnetic dipolar interaction, thereby confounding their analysis. Techniques that can detect SPNs at the single particle level are therefore highly desirable. The goal of this thesis was to develop an analytical microscopy technique, namely magnetic force microscopy (MFM), to detect and spatially localize synthetic and natural SPNs for biomedical applications. We aimed to (1) increase ii MFM sensitivity to detect SPNs at the single-particle level and (2) quantify and spatially localize iron-ligated proteins (ferritin) in vitro and in biological samples using MFM. Two approaches were employed to improve MFM sensitivity. First, we showed how exploitation of magnetic anisotropy could produce a higher, more uniform MFM signal from single SPNs. Second, we showed how an increase in probe magnetic moment increased both the magnitude and range up to which the MFM signal could be detected from a single SPN. We further showed how MFM could enable accurate quantitative estimation of ferritin content in ferritin-apoferritin mixtures. Finally, we demonstrated how MFM could be used to detect iron/ferritin in serum and animal tissue with spatial resolution and sensitivity surpassing that obtained using conventional biochemical assays. We envisage these advancements will allow MFM to serve as a novel biosensing technique to spatially localize iron/ferritin in small aliquots of clinical samples (i.e. serum) and in tissue biopsies at the ultra-sensitive and ultra- structural level. We also discuss how future work incorporating our advancements could lead to the development of a novel indirect MFM technique, which could enable high-throughput analysis of SPNs for biomedical applications. iii Dedication To my mother, Joni, who has always encouraged me to follow my heart and reach for my dreams, and to my father, Kevin, who has given me practical guidance and a level head to make those dreams come true. To Jason, for keeping me grounded, for never letting me give up and for “(un)quantifiably” loving me no matter what. To Dr. Doros Petasis, for amplifying my love for science, teaching me to “build character” and giving me confidence to pursue my Ph.D. iv Acknowledgements I would like to thank my advisor, Dr. Gunjan Agarwal, for calling me on April Fool’s Day (and it not be a joke) to give me the opportunity to pursue my doctoral degree. This has been the most incredible and most challenging journey, and my scientific and academic successes would not have been possible without her continuous guidance and encouragement. I would also like to thank the members of my dissertation committee, Dr. Stephen Lee and Dr. Jessica Winter, and my candidacy committee, Dr. Chris Hammel and Dr. Vish Subramaniam. Their support, critiques and advice have truly made me a better scientist, and for that I am grateful. A number of individuals have contributed their time and expertise to make the work in this dissertation possible. I would like to thank my co-authors Dr. Christopher Murray and Dr. Jun Chen from the University of Pennsylvania for providing the synthetic Fe3O4 SPNs and SQUID/ZFC characterizations for my anisotropy and probe comparison studies. I am also grateful to Ed Calomeni and Henk Colijn for the generous amount of time they took to teach me electron microscopy and for their thoughtful insights during discussions of my projects. v Thank you to BME undergraduate Yuzhi (Kevin) Zeng, who helped co-author and share in my enthusiasm for the ferritin MFM project. Kevin optimized ferritin/apoferritin sample preparation and assisted with MFM data collection. I would also like to thank Dr. Dana McTigue and Dr. Andrew Sauerbeck for their contributions to the work involving rat spinal cord tissue and serum. They generously provided the tissue/serum samples, performed Perls and immunohistochemistry studies and conducted serum ferritin experiments. I am also grateful to Dr. John Moreland from NIST for providing microfluidic silicon nitride membranes for our indirect MFM pilot studies. To my past labmate, Dr. Angela Blissett and my current labmates, Jeff Tonniges and David Yeung: your unending support, advice and breaks have kept me sane and helped me to push through. Thank you for your willingness to help me whenever you could and for making my experience much more enjoyable. I am especially grateful to my softball and Hamptons crew, broomball team and fellow graduate school friends; you have helped me to find many outlets for stress and the balance I desperately needed to face the challenges of grad school one week at a time. Thank you to my furry best friend, Tesla, for appearing on my doorstep two years ago and changing my life. Lastly, I would like to thank Panera Bread for providing bottomless coffee and tea, sometimes two or three meals in a day, free WIFI and kindness. I very well may have never finished writing my papers, proposals and this dissertation without your help. vi Vita August 1987 .................................................................................................. Born, Butler, PA June 2005 .................................................................... Butler Senior High School, Butler, PA May 2009 ........................................................................................... B.S. Physics, Allegheny College, Meadville, PA December 2011 ...................................................... M.S. Biomedical Engineering, The Ohio State University, Columbus, OH August 2009 to August 2012…………………...….AFM Core Laboratory Manager, Davis Heart and Lung Research Institute, Columbus, OH August 2012 to May 2013………………………..…..Graduate Teaching Associate, Department of Biomedical Engineering, The Ohio State University, Columbus, OH June 2009 to Present………..……………………….Graduate Research Associate, Department of Biomedical Engineering, The Ohio State University, Columbus, OH vii Publications Included in Dissertation Chapter 3: Nocera, T.M., Chen, J., Murray, C.B., Agarwal, G., “Magnetic Anisotropy Considerations in Magnetic Force Microscopy Studies of Single Superparamagnetic Nanoparticles,” Nanotechnology 2012; 23 (49): 495704. Included in Dissertation Chapter 4: Nocera, T.M., Sauerbeck, A., Zeng Y., McTigue, D. M., Agarwal, G., “Quantification and Spatial Localization of Ferritin Using Magnetic Force Microscopy,” Revised and resubmitted to Nanomedicine: Nanotechnology, Biology and Medicine. Additional Publications: Stevenson, M.D., Pristine, H., Hogrebe, N.J., Nocera, T. M., Boehm, M., Reen, R., Koelling, K., Agarwal, G., Saraang-Sieminski, A.L., Gooch, K.J., “Self- assembling Peptide Matrix that Independently Controls Stiffness and Binding Site Density Supports the Formation of Microvascular Networks in 3D,” Acta Biomaterialia 2013; 9(8): 7651-7661. viii Agarwal, G., and Nocera, T.M., "Atomic Force Microscopy (AFM)." In The Nanobiotechnology Handbook. Ed.Yubing Xie. Boca Raton: CRC Press, 2012; 369-91. Hilfiger, M.G., Chen, M., Brinzari,T.V., Nocera, T.M., Shatruk, M., Petasis, D.T., Musfeldt, J.L., Achim, C. Dunbar, K.R., “An Unprecedented Charge Transfer Induced Spin Transition in an Fe–Os Cluster,” Angewandte Chemie International Edition 2010; 49(8): 1410–1413. Fields of Study Major Field: Biomedical Engineering Concentrations: Biomedical Micro/Nanotechnology, Biomedical Imaging ix Table of Contents Abstract .................................................................................................................ii Dedication ............................................................................................................iv Acknowledgements ..............................................................................................v Vita ..................................................................................................................... vii List of Tables ...................................................................................................... xii List of Figures .................................................................................................... .xiii List of Abbreviations .......................................................................................... .xv List of Symbols .................................................................................................xviii Chapter 1: Superparamagnetic