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Lipid Bilayers As Surface Functionalizations for Planar and Nanoparticle Biosensors

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Lipid Bilayers As Surface Functionalizations for Planar and Nanoparticle Biosensors Lipid Bilayers as Surface Functionalizations for Planar and Nanoparticle Biosensors by Shell Y. Ip A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Chemistry University of Toronto © Copyright by Shell Y. Ip 2010 Lipid Bilayers as Surface Functionalizations for Planar and Nanoparticle Biosensors Shell Y. Ip Doctor of Philosophy Department of Chemistry University of Toronto 2010 Abstract Many biological processes, pathogens, and pharmaceuticals act upon, cellular membranes. Accordingly, cell membrane mimics are attractive targets for biosensing, with research, pathology, and pharmacology applications. Lipid bilayers represent a versatile sensor functionalization platform providing antifouling properties, and many receptor integration options, uniquely including transmembrane proteins. Bilayer-coated sensors enable the kinetic characterization of membrane/analyte interactions. Addressed theoretically and experimentally is the self-assembly of model membranes on plasmonic sensors. Two categories of plasmonic sensors are studied in two parts. Part I aims to deposit raft-forming bilayers on planar nanoaperture arrays suitable for multiplexing and device integration. By vesicle fusion, planar bilayers are self-assembled on thiol-acid modified flame-annealed gold without the need for specific lipid head-group requirements. Identification of coexisting lipid phases is accomplished by AFM imaging and force spectroscopy mapping. These methods are successfully extended to metallic, plasmon-active nanohole arrays, nanoslit arrays and annular aperture arrays, with coexisting phases observed among the holes. Vis-NIR transmission spectra of the arrays are measured before and after deposition, indicating bilayer detection. Finally, the extraction of membrane proteins from cell cultures and incorporation into model supported bilayers is demonstrated. These natural membrane proteins potentially act as lipid-bound surface receptors. Part II aims to encapsulate in model lipid bilayers, metallic nanoparticles, which are used as probes in surface enhanced Raman spectroscopy. Three strategies of encapsulating particles, and incorporating Raman-active dyes are demonstrated, each using a different dye: malachite green, rhodamine-PE, and Tryptophan. Dye incorporation is verified by SERS and the bilayer is ii visualized and measured by TEM, with support from DLS and UV-Vis spectroscopy. In both parts, lipid-coated sensors are successfully fabricated and characterized. These results represent important and novel solutions to the functionalization of plasmonic surfaces with biologically relevant cell membrane mimics. iii Dedicated to my Mother and Father, Mimi and Ken Ip iv Acknowledgments It is with profound pleasure, humility, and sincerity that I thank those whose help and support have made this endeavour possible. First and foremost, I would like to thank my supervisor, Prof. Gilbert C. Walker for his mentorship, patience, and unvarying support. Though he has undoubtedly provided invaluable scholarly direction throughout the years, it is his good nature, and social acumen that have been most appreciated. I also sincerely thank the members of my committee, Prof. Ulrich J. Krull, and Prof. J. Stewart Aitchison, who, with their enthusiasm, wisdom and expertise, have both contributed significantly to our discussions. Also, it has been my privilege to work with friends and collaborators Matthew J. Kofke, and Prof. David H. Waldeck. Mr. Kofke has skillfully, and reliably provided many FIB samples in timely fashion. Our visits and cooperative efforts in experiments have been grueling at times, but inspired and stimulating. I am grateful to my friend and labmate, Christina MacLaughlin, for her skill and marathon-honed determination, but most of all her spirit, which have made the nanoparticle experiments possible. I also sincerely thank Dr. Nikhil Gunari, not only for his contributions to the TEM imaging of the nanoparticles, but also for years of mentorship, and encouragement. Also to my friend and colleague James K. Li, I extend my gratitude, for sharing with me, his coding expertise and force map analysis code, which forms the framework for the code used within. Several others have made instrumental contributions to this work including my friend and colleague Ruby May A. Sullan, who has been an invaluable partner in many helpful and thoughtful discussions of lipid bilayers. Always willing to share her knowledge and experience, she has had a tremendous influence on the lipid techniques employed. I extend my gratitude also to Natalie Tam in from Gang Zheng’s group at the University Health Network, for the preparation and culturing of cells used in these experiments, not to mention her friendly, generous support and expert advice on cells. I also gratefully acknowledge the helpful discussions of surface plasmons and near-field optics I’ve shared with my friend and colleague Daniel Lamont. For their mentorship and guidance I thank the Post-Docs in our group past and present; specifically, Dr. Weiqing Shi, Dr. Zhara Fakhraai, and Dr. Shan Zou. In particular, I am grateful to Dr. Zou, now with the NRC, not only for her keen insight and pragmatic experimental v sagacity, but also for her support, encouragement, and continued friendship. As a mentor and friend I also thank Dr. Emanuel Istrate of the Institute of Optical Sciences. I also thank the members of the Walker group past and present, who I’ve had the privilege to call my friends and colleagues. Additionally I extend my warm gratitude to Mandy Koroniak and Anna Liza Villavelez for their resourcefulness and friendly handling of all things group and department related respectively. For their use of facilities and equipment, I thank the administrators and staff at University of Pittsburgh Nanoscale Fabrication and Characterization Facility, the Nanofabrication facility at the University of Western Ontario, the Cornel Nanofabrication Facility, The Department of Chemistry’s Center for Nanoscale Imaging, and the labs of Eugenia Kumacheva and Gregory Scholes. Financial support has come from a variety of sources over the years including the Natural Sciences and Research Council of Canada, the Ontario Graduate Scholarship Program, the Department of Chemistry, and the University of Toronto. For their unwavering love, support, and patience, with my love and respect I thank my family: My parents Mimi and Ken; My sister June and her partner Ferhan Last but not least, I genuinely thank Lisa and her family for their support; and especially Lisa for the innumerable ways she has buoyed me, and for her tremendous patience, strength, and perseverance. vi Table of Contents ACKNOWLEDGMENTS ........................................................................................................................................ V TABLE OF CONTENTS ...................................................................................................................................... VII LIST OF TABLES...................................................................................................................................................XI LIST OF FIGURES ............................................................................................................................................... XII LIST OF ABBREVIATIONS........................................................................................................................... XXIII PART I. LIPID BILAYER FUNCTIONALIZATION OF PLANAR NANOAPERTURE ARRAYS ........... 1 CHAPTER 1: INTRODUCTION ....................................................................................................................... 2 1.1 GENERAL INTRODUCTION ............................................................................................................................................2 1.2 PROJECT DESCRIPTION..................................................................................................................................................7 1.3 FUTURE APPLICATIONS.............................................................................................................................................. 10 1.4 CONCLUSIONS .............................................................................................................................................................. 13 1.5 REFERENCES................................................................................................................................................................ 13 CHAPTER 2: BACKGROUND ........................................................................................................................15 2.1 INTRODUCTION ........................................................................................................................................................... 15 2.2 BRIEF REVIEW OF LITERATURE............................................................................................................................... 15 2.2.1 Surface plasmons and SPR sensing..............................................................................................................16 2.2.2 Surface plasmons on nanophotonic structures......................................................................................20 2.2.3 Exploiting cellular interactions.....................................................................................................................26 2.2.4 Phospholipid chemistry ....................................................................................................................................28
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