Lanthanide-Containing Microgels Intended for Bead-Based Bioassays by Mass Cytometry
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Lanthanide-Containing Microgels Intended for Bead-Based Bioassays by Mass Cytometry By Wanjuan Lin A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Chemistry University of Toronto © Copyright by Wanjuan Lin, 2013 Lanthanide-Containing Microgels Intended for Bead-Based Bioassays by Mass Cytometry Wanjuan Lin Doctor of Philosophy Graduate Department of Chemistry University of Toronto 2013 Abstract This thesis describes the synthesis and characterization of functional polyelectrolyte copolymer microgels intended for bioassays based upon mass cytometry. The microgels are based upon copolymers of N-isopropylacrylamide (NIPAm), N-vinylcaprolactam (VCL) and methacrylic acid (MAA), crosslinked with methlene-bis-acrylamide, poly(NIPAm/VCL/MAA). The microgels were loaded with Ln(III) ions, which were then converted in situ to LnF3 or LnPO4 nanoparticles (NPs). Very specific conditions were required for confining the NPs to the core of the microgels. I used mass cytometry to measure the number and the particle-to-particle variation of Ln ions per microgel. By controlling the amount of LnCl3 added to the neutralized microgels, we could vary the content of individual microgels from ca. 106 to 107 Ln atoms, either in the form of Ln3+ ions or Ln NPs. ii Leaching profiles of Ln ions from the Ln-containing microgels were measured by traditional ICP-MS. Under neutral or basic conditions, the leakage of Ln ions into the aqueous medium was not significant. In acidic buffer solutions, however, the leakage of metal from Ln3+ ion- containing microgels was prominent. On subsequent exposures to buffers, the microgels underwent continuous loss of metals. For LnF3-containing microgels, there was much smaller extent of metal leakage and very small of continuous loss of metal upon subsequent exposures to buffers. For LnPO4-containing microgels, there was very little detectable leakage of metals to the acidic buffers. In order to test the use of Ln nanoparticle as reagents of enhanced sensitivity in mass cytometry, we employed TmF3 encoded microgels as model cells. In one sample, Tm-encoded poly(NIPAm/VCL/MAA) microgels were coated with streptavidin (SAv) and used as model biomarkers, in quantitative mass cytometric bioassays. The sensitivity of different reagents (including a biotinylated metal cheating polymer, containing 50 Tb3+ ions per probe and a biotinylated NaHoF4 NP, containing 15,000 Ho atoms per probe) was examined for detecting and quantifying the number of SAv per microgel by mass cytometry. By using the Bi- 4 PAsp(Tb)50 probes, a biomarker level at ~10 per cell was detected. By using the Bi-NaHoF4 NP probes, a biomarker level as low as ~102 per cell was quantified. iii Acknowledgments 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, Professor Mark Nitz, Dr. Olga Ornatsky and Professor Walter Richtering for the valuable suggestions, discussions and collaborations. I am also grateful to Professor Eugenia Kumacheva and Professor Dwight Seferos for being on my supervisory committee, and for their valuable insights and helpful discussions. I would like to thank my friends and colleagues in the Winnik group. Particularly, I want to thank Professor Andrij Pich, Dr. Sheng Dai and Dr. Xiaomei Ma for giving me a lot of help when I started my research project. I owe a debt of gratitude to Dr. Yi Hou, Dr. Yijie Lu, Dr. Ahmed Abdelrahman, Dr. Jieshu Qian, Dr. Dirk Weinrich, and Ms. Yi (Sally) Liang for the fruitful collaborations. It was very enjoyable working with them. I especially thank Yi and Yijie for their important contribution to the success of our project and Dr. Chun Feng, and Mr. Guangyao Zhao for their time and valuable critique of my thesis. To my lab mates, Dr. Lin Jia, Dr. Gerald Guerin, Mr. Peng Liu, Mr. Meng Zhang, Mr. Lemuel Tong, Mr. Issacc Herrera, Mr. Graeme Cambridge and Mr. Hang Zhou for interesting discussions on science. I feel a deep appreciation and gratefulness for my family members (my parents, my grandparents, and my parents-in-law) for their encouragement and unconditional love. This thesis was made possible by their full support and encouragement and is dedicated to them. I would like to express my love and gratitude to my husband Dr. Ruirui (Raymond) Huang, who has been my spiritual support through times of frustration and joy. Heartfelt thanks to my lovely son Alexander, whose smile and calling me “Mommy” simply make my world full of sunshine each and every day. iv Table of Contents Abstract……………………………………………………………………………ii Acknowledgements……………………………………….……………………....iv Table of Contents …………………………………………………………...…….v List of Tables…………………………………………………………...……...….xi List of Figures and Schemes …………………...………...…………………….xiv List of Appendices……………………………………………………………xxviii Abbreviations………...………………………………………………………...xxix Chapter 1. General Introduction……………………………………………...…1 1.1 Multiplexed Bioassays………………………………………………………………………..1 1.1.1 Cell-Based Assays………………………….………………………………………….....1 1.1.2 Bead-Based Assays…………………………………………………………………..…...3 1.1.2.1 Bead-based Assays Based on Flow Cytometry……………………………………...3 1.1.2.2 Bead-based Assays Based on Mass Cytometry……………………………………...4 1.2 Introduction to Microgels.………………………………...……...………………………...…8 1.2.1 Definition of Microgels………………………………………………………………….. 8 1.2.2 Microgel Synthesis………………………………………………………………………11 1.2.2.1 Precipitation Polymerization………………………………………………………..11 1.2.2.2 Emulsification………………………………………………………………………13 1.2.2.3 Photolithography……………………………………………………………………14 1.2.2.4 Microfluidic Synthesis………………………………………………...……………14 1.2.2.5 Micromolding………………………………………………………………………15 1.2.3 Functional Group Distribution in Polyelectrolyte Microgels…………………….……..16 1.2.3.1 Microgels from Homogeneous Nucleation……………..…………………………..17 1.2.3.2 Core-Shell Polymer Microgels…………………………………………………..…19 v 1.2.4 Swelling Thermodynamics of Microgel Particles………………..……………………...20 1.2.4.1 Swelling in Neutral Gels……………………………………..……………………..20 1.2.4.2 Swelling in Ionic Gels……………………………………...………………….……23 1.2.5 Microgel Containing Nanoparticles………………………………..……………………25 1.2.5.1 Growing Nanoparticles Using Microgels as Microreactor………………….……...25 1.2.5.2 Growing Microgels in the Presence of Preformed Nanoparticles Acting as Seeds...26 1.2.5.3 Absorbing Nanoparticles into Microgels………………………………...…………27 1.2.5.4 Layer-by-Layer and Core-Shell Assembly………………………………………....28 1.3 Lanthanide Properties………...………………………………………………………….......29 1.3.1 Lanthanide Complexes with Organic Ligands…………………………………………..29 1.3.2 Inorganic Lanthanide Nanoparticles (LnF3, LnPO4, LnVO4, LnBO3)……………..……31 1.4 Research Objectives…………………………………………………………………...……..33 1.5 Thesis Outline……………………………………………………………………………......33 Reference……………………………………………………………………………………...…35 Chapter 2. Experimental: Materials, Protocols, Characterization and Data Analysis…………………………………………………………………………...41 2.1 Materials………………………………………………………………………………….….41 2.1.1 Reagents for Particle Synthesis……………………..………………………………..….41 2.1.2 Reagents for Bioconjugation…………………………………………………………….42 2.1.3 Reagents for Bead-Based Bioassays……………..………………………..…………….42 2.2 Synthesis of Lanthanide Encoded Microgels as Classifier Beads…………………………...42 2.2.1 Synthesis of Polyelectrolyte Microgels by Precipitation Polymerization………...……..42 2.2.1.1 Poly(NIPAm/VCL/MAA) Microgels……………………………………...…….…43 2.2.1.2 Poly(NIPAm/MAA/PEGMA) Microgels………………………..........................…44 2.2.2 Lanthanide Encoded Microgels........................................................................................45 2.2.2.1 Ln3+ Ions-Containing Microgels................................................................................45 2.2.2.2 LnF3 Nanoparticle-Containing Microgels..................................................................49 2.2.2.3 LnPO4 Nanoparticle-Containing Microgels...............................................................51 2.3 Characterization Methods and Data Analysis..........................................................................53 2.3.1 Titration………………………………………………………………………………….53 2.3.2 Light scattering………………………………………………………………………….55 vi 2.3.2.1 Dynamic Light Scattering…………………………………..………………………55 2.3.2.2 Static Light Scattering……………………..………………………………………..59 2.3.3 Mass Cytometry………………...……………………………………………………….61 2.3.4 ICP-MS………………………………………………………………………………….62 2.3.5 Electron Microscopy……………………………………………………………….……63 2.3.6 Energy-Dispersive X-Ray Spectroscopy……………………….……………………….63 2.3.7 UV-Vis Spectrometry………………………………………………………..………….63 2.3.8 Zeta Potential () and Electrophoretic Mobility (e)……………………………………65 2.3.9 NMR…………………………………………………………………………...………..66 Reference………………………………………………………………………………………...67 Chapter 3. Ln(III) ions and LnF3 Nanoparticle Containing Microgels as Classifier Beads for Mass Cytometric Analysis…………………….………….68 3.1 Introduction…………………………………………………………………………………..68 3.2 Experimental Procedures…………………………...……………………………………..…71 3.3 Results and Discussions…………………………………………………………………...…72 3.3.1 Polyelectrolyte Microgels…………………………………………………...……….….72 3.3.1.1 Synthesis of Polyelectrolyte Microgels………………………………...…….…….72 3.3.1.2 Microgel Characterization……………………………………………………...…..74 3.3.1.3 Polyelectrolyte Properties of Copolymer