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Lanthanide-Encoded Polystyrene Microspheres for Mass Cytometry-Based Bioassays

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Lanthanide-Encoded Polystyrene Microspheres for Mass Cytometry-Based Bioassays LANTHANIDE-ENCODED POLYSTYRENE MICROSPHERES FOR MASS CYTOMETRY-BASED BIOASSAYS By Ahmed I. Abdelrahman A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Chemistry University of Toronto © Copyright by Ahmed I. Abdelrahman, 2011 Abstract Lanthanide-Encoded Polystyrene Microspheres for Mass Cytometry-Based Bioassays Ahmed I. Abdelrahman, Ph.D. Thesis (2011) Department of Chemistry, University of Toronto This thesis describes the synthesis and characterization of metal-encoded polystyrene microspheres with a narrow size distribution designed for mass cytometry-based immuno- and oligonucleotide-assays. These particles were prepared by multiple stage dispersion polymerization techniques using polyvinylpyrrolidone (PVP) as a steric stabilizer. As a cytometeric technique, mass cytometry necessitated metal-encoded microspheres to perform the same roles of fluorescent microspheres used in conventional flow cytometry. The first role of the microsphere was to be able to act as a platform (classifier microspheres) for bioassays. Secondly, the microspheres should be suitable for mass cytometry machine calibration as standards. To perform these roles, metal-encoded microspheres were required to have certain size, functionality and metal content criteria. Lanthanide elements were chosen as the metals for encoding the microspheres for their low natural abundance in biological systems and for their similar chemistry. My goal was to employ two-stage dispersion polymerization, of styrene in ethanol, to introduce the lanthanide salts along with excess acrylic acid in the second stage, one hour after the initiation. Acrylic acid deemed to serve as a ligand for the lanthanide ions, through its carbonyl group, so the lanthanide ions get incorporated into the microsphere while acrylic acid is copolymerizing with styrene. Using two-stage dispersion polymerization, I could synthesize ii lanthanide encoded microspheres with narrow size distribution and high lanthanide content. However the lanthanide content distributions were unexpectedly much broader than the size distribution obtained. In addition, I could not attach biomolecules to the surface of such particles. In an attempt to improve the characteristics of these microspheres, I employed modified versions of multiple stage dispersion polymerization and seeded emulsion polymerization to grow functional polymer shell on the surface of the particles prepared by dispersion polymerization. Moreover, I coated the lanthanide encoded microspheres with silica shell which enabled me to grow another layer of functional-silica. Consequently, I could use these particles as classifier microspheres for mass cytometry-based immunoassays as well as fluorescence- based oligonucleotide-assays. iii ACKNOWLEDGEMENTS I would like to express my gratitude and appreciation to my supervisor Professor Mitchell A. Winnik for his support, encouragement and guidance throughout this research work. I am also grateful for the opportunity he gave me to be a part of the exciting lanthanide tags project. I gratefully acknowledge and thank Professor Vladimir Baranov, Dr. Dmitry Bandura, Dr. Olga Ornatsky and Professor Scott Tanner for the valuable suggestions, discussions and collaborations. I would like to thank my friends and colleagues in Winnik group. Particularly, I want to thank Dr. Sheng Dai for giving me a lot of help when I started my research project in Toronto, and Dr. Gerald Guerin and Dr. Conrad Siegers for the very helpful discussions. I owe a debt of gratitude to Dr. Stuart Thickett, Dr. Dirk Weinrich, Yi (Sally) Liang and Wanjuan (Betty) Lin for the fruitful collaborations. It was very enjoyable working with them. I especially thank Stuart for his time and valuable critique of my thesis. Many thanks go to Sally for her important contribution to the success of our project. To my desk-neighbors, Daniel Majonis, Peng Liu and Dr. Yi Hou, thanks for interesting discussions on science. I feel a deep appreciation and gratefulness for my family members in Egypt (my mother, my brother Hany and two sisters Amel and Sara) for their encouragement and unconditional love. This thesis was made possible by their full support and encouragement and is dedicated to them. To my beloved mother: without your prayers, I would not be able to get this thesis done. I would like to express my love and gratitude to my wife Noha, who has always lovingly been by my side through times of frustration and joy. Heartfelt thanks to my lovely kids Ziad, Rofaida and Jana. Before and after all, To Allah Almighty, who endowed me with the strength, enthusiasm and perseverance necessary to see this project through, and to whose glory this work is dedicated. iv Not even the weight of an atom or less than that or greater, escapes from His Knowledge in the heavens or in the earth. ………………………………Holy Quraan 34:3 Dedicated to the soul of my dad v TABLE OF CONTENT ABSTRACT ii ACKNOWLEDGMENTS iv TABLE OF CONTENT vi LIST OF TABLES x LIST OF FIGURES AND SCHEMES xii LIST OF APPENDICES xix ABBREVIATIONS xx 1 GENERAL INTRODUCTION 1 1.1 Polymer Particles by Dispersion Polymerization 4 1.1.1 Dispersion Polymerization: Mechanism and Kinetics 5 1.1.2 Different Processes for Dispersion Polymerization 8 1.1.2.1 Dispersion Polymerization by The Batch Process 8 1.1.2.2 Semi-Continuous, Continuous and Seeded Dispersion Polymerizations 9 1.1.2.4 Two-Stage Dispersion Polymerization 11 1.1.3 Functional Microspheres by Dispersion Polymerization 12 1.1.3.1 Microspheres with Functional Groups 13 1.1.3.2 Bioconjugation Reactions as Post-Functionalization for Microspheres 15 1.1.4 Fluorescent Dye- and Metal-Containing Microspheres by Dispersion Polymerization 18 1.1.4.1 Fluorescent Dye-Labeled Microspheres 18 1.1.4.2 Precious Metal-Containing Microspheres 19 1.1.4.3 Magnetic Polymer Microspheres 20 1.1.4.4 Polymer Microspheres Functionalized with Silica 20 1.2 Flow Cytometry 21 1.3 Lanthanides: Physical and Chemical properties 23 1.4 Research objectives 25 1.5 Thesis outline 25 References: 27 2 INSTRUMENTAL METHODS AND EXPERIMENTAL DETAILS 38 2.1 Instrumental Methods 38 2.1.1 Inductively Coupled Plasma Mass Spectrometer 38 2.1.1.1 Introduction 38 2.1.1.2 Plasma Source 39 vi 2.1.1.3 Mass Analyzers 40 2.1.1.4 Sample Preparation for ICP-MS 40 2.1.2 Mass Cytometry 41 2.1.2.1 Introduction 41 2.1.2.2 Instrument Design 42 2.1.2.2 Sample Preparation for Mass Cytometry 44 2.2 Materials 45 2.2.1 Solvents 45 2.2.2 Reagents for Particles’ Synthesis and Characterization 45 2.2.2 Reagents for Bioconjugation 46 2.3 Synthetic Procedures 47 2.3.1 Microspheres’ Synthesis by Dispersion Polymerization 47 2.3.2 PVP pyrrolidone ring opening (PVP activation). 51 2.3.3 Dispersion polymerization using modified PVP. 52 2.3.4 Seeded Emulsion Polymerization with Methacrylic Acid (MAA). 53 2.3.5 Seeded Emulsion Polymerization with Glycidyl Methacrylate (GMA). 53 2.4 Characterization Methods 54 2.4.1 Scanning Electron Microscopy (SEM) 54 2.4.2 Dynamic Light Scattering (DLS) 54 2.4.4 Gel Permeation Chromatography 54 2.4.5 Gravimetrical measurements 55 2.4.3 Titration of Acid Groups 55 2.4.6 Bioconjugation 56 2.4.7 Fluorescence Emission 57 References: 58 3 MICROSPHERES BY TWO STAGE DISPERSION POLYMERIZATION 60 3.1 Introduction 60 3.2 Designing the Synthesis of the Microspheres 62 3.2.1 Techniques to Fulfill Size Requirements 62 3.2.2 Metal-Content Requirements 65 3.3 Results and Discussion 66 3.3.1 Synthesis of the Microspheres 66 3.3.2 Determining Lanthanide Content by Mass Cytometry 69 3.3.3 Microsphere Encoding Protocols: Variability and Dimensionality 79 3.3.4. Lanthanide Incorporation, Particle-to-Particle Variability and Surface Functionality 84 3.4 Covalent Attachment of Proteins to the Surface of the Microspheres 87 3.5 Summary 91 vii References 92 4 MICROSPHERES BY THREE STAGE DISPERSION POLYMERIZATION 94 4.1 Introduction 94 4.2 Three Stage Dispersion Polymerization 96 4.3 Factorial design 99 4.2.1 Design of the Factorial Experiments 100 4.2.2 Factorial Experiments: Results and Discussions 101 4.3 Testing Ion Leakage 110 4.4 Synthesis of Particles with Higher Variability 111 4.5 Bioconjugation 117 4.6 Summary 119 References: 121 5 SURFACE FUNCTIONALIZATION OF LANTHANIDE-ENCODED MICROSPHERES 123 5.1 Introduction 123 5.2 Results and Discussion 125 5.3 SUMMARY 143 References: 146 6 SILICA-COATED PARTICLES 147 6.1 Surface Functionalization with Silica Shell 147 6.2 Immunoassays Based on the Amino-Functionalized Particles 153 6.2.1 Streptavidin -Coated Lanthanide-Encoded Particles via Biotin- Streptavidin Sandwich (Fluorescence Assay) 153 6.2.2 Streptavidin -Coated Lanthanide-Encoded Particles via Biotin- Streptavidin Sandwich (Mass Cytometry Assay) 157 6.2.3 Streptavidin-Coated Lanthanide-Encoded Particles via covalent attachment of Streptavidin (Mass Cytometry Assay) 160 6.3 Oligonucleotide Assays-Based on the Amino-functionalized Particles 161 viii 6.5 Conclusions 166 References: 168 7 ANALYTICAL ASPECTS 170 7.1 Introduction 170 7.2 Results and Discussion 172 7.2.1. Microsphere Synthesis and Metal Incorporation 175 7.2.2. 3-Stage Dispersion Polymerization 179 7.2.3. Ion-release behavior and mass cytometry calibration standard 181 7.2.4. Lanthanide-Containing Microspheres as Internal Standards for Cell Samples. 183 7.2.5. Reproducibility of the Synthesis of Lanthanide-Containing Microspheres. 188 7.3 Conclusions 193 References 194 APPENDIX 1 198 ix LIST OF TABLES 1 General Introduction 1 Table 1-1: Different functional monomers used in the synthesis of polymer particles (by dispersion polymerization). 13 Table 1-2: Configuration of outer electrons of Ln atoms and Ln3+ ions 24 2 Instrumental Methods and Experimental Details 38 Table 2-1. Recipe for the synthesis of microsphere sample AA0069 as an example for 2-stage dispersion polymerization (2-DisP) of styrene with PVP55 as a dispersant in ethanol.
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