Microfluidic Studies of Biological and Chemical Processes by Ethan Tumarkin A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Chemistry University of Toronto © Copyright by Ethan Tumarkin, 2012 Microfluidic Studies of Biological and Chemical Processes Ethan Tumarkin Doctor of Philosophy Department of Chemistry University of Toronto 2012 Abstract This thesis describes the development of microfluidic (MF) platforms for the study of biological and chemical processes. In particular the thesis is divided into two distinct parts: (i) development of a MF methodology to generate tunable cell-laden microenvironments for detailed studies of cell behavior, and (ii) the design and fabrication of MF reactors for studies of chemical reactions. First, this thesis presented the generation of biopolymer microenvironments for cell studies. In the first project we demonstrated a high-throughput MF system for generating cell-laden agarose microgels with a controllable ratio of two different types of cells. The MF co-encapsulation system was shown to be a robust method for identifying autocrine and/or paracrine dependence of specific cell subpopulations. In the second project we studied the effect of the mechanical properties on the behavior of acute myeloid leukemia (AML2) cancer cells. Cell-laden macroscopic agarose gels were prepared at varying agarose concentrations. A modest range of the elastic modulus of the agarose gels were achieved, ranging from 0.62 kPa to 20.21 kPa at room temperature. We observed a pronounced decrease in cell proliferation in stiffer gels when compared to the gels with lower elastic moduli. The second part of the thesis focuses on the development of MF platforms for studying chemical reactions. In the third project presented in this thesis, we exploited the temperature dependent solubility of CO2 in order to: (i) study the temperature mediated CO2 transfer between the gas and the various liquid phases on short time scales, and (ii) to generate bubbles with a dense layer of colloid particles (armoured bubbles). The fourth project involved the fabrication of a multi-modal MF device with integrated analytical probes. The MF device comprised a pH, temperature, and ATR-FTIR probes for in- situ analysis of chemical reactions in real-time. Furthermore, the MF reactor featured a temperature controlled feedback system capable of maintaining on-chip temperatures at flow rates up to 50 mL/hr. Key words: microfluidics, microenvironments, high-throughput, encapsulation, co-culture, biopolymers, elastic modulus, multi-modal, modular, integrated analytical probes, titrations III Imagination is more important than knowledge. Knowledge is limited. - Albert Einstein IV Acknowledgments First and foremost I want to give my outmost gratitude to my supervisor, mentor, and friend, Professor Eugenia Kumacheva. Without a doubt, her guidance, wisdom, enthusiasm, and constant support have shaped me into the person, teacher, and researcher that I am today. I am forever grateful for her insights about science and life. I am also extremely grateful to my committee members Professor Axel Guenther, Professor Gilbert Walker, and Professor Aaron Wheeler. In addition, I want to give my utmost appreciation to my collaborators Professor Axel Guenther, Professor Gilbert Walker, Professor Peter Zandstra, Professor Barbara Sherwood Lollar, Professor Mark Ungrin, Professor Zhihong Nie, and Professor Jesse Greener for their support and helpful discussions over the past 4 years. I want to thank all the persons I have had the pleasure of interacting with since I was a young undergraduate summer student during the summer of 2005. In no particular order, I want to say thank you to Dr. Hong Zhang, Dr. Mallika Das, Ilya Gourevich, Dr. Zhixiang Wei, Dr. Alla Petukhova, Dr. Chantal Paquet, Dr. Wei Li, Dr. Minseok Seo, Dr. Lindsey Fiddles, Andrew Paton, Dr. Ryan Simms, Michael Debono, Dr. Daniele Fava, Neta Raz, Dr. Raheem Peerani, Dan Voicu, Lsan Tzadu, Milad Abolhasani, Pasquale Benvenuto, Jörg Fochtmann, and Dr. Jemma Vickery. In the past 7 years I have had the pleasure of working with people which have become like family to me. Dr. Anna Lee, Yannick Bohren, Dr. Jai Il Park, Dr. Diego Velasco, Dr. Kun Liu, Dr. Slava Dubinsky, Miguel Neves, and Ivan Gorelikov have been like family watching over my shoulders and stepping in during difficult times. My life has been enriched because of them. Finally, I want to give thanks to my family. To my sister, I want to say thank you for always being supportive and helpful. Always know that I am very proud of you. To my father, I want to say thank you for your unparalleled belief in me, for your constant curiosity and willingness to listen to my research and my ideas, and for pushing me harder than anyone else. To my sister-in- law, thank you for always being there during good times and bad times; and of course for giving V me the most amazing niece in the world. To my brother, you are the glue that holds everything together. Many times I would have crumbled without your support. Brothers for life. To my mother, you are the guiding star I follow every day. Your light shines as bright today as it did when I was a child. It is with your love and wisdom that I am who I am today. To you I owe this manuscript. I promise you that I will always aspire for greatness, with my family and my life. I love you. VI Preface This thesis has been organized as a series of manuscripts (see the list below) which have been published in peer-reviewed scientific journals. As identified by primary authorship, all manuscripts were written by Ethan Tumarkin with critical comments and revision by Eugenia Kumacheva and corresponding collaborators. The contributions of other authors are provided in detail below. Chapter 1 Microfluidic Studies of Biological Phenomena The results in this chapter are mainly from manuscripts published in 1Chemical Society Reviews, 2009, 8, 2161, and 2Small, 2012, In Press Authors: 1Ethan Tumarkin, and Eugenia Kumacheva 2Diego Velasco, Ethan Tumarkin, and Eugenia Kumacheva Contributions: 1E. Tumarkin contributed to the article writing and figure design and preparation. 2E. Tumarkin contributed to the article writing and figure design and preparation. D. Velasco wrote the manuscript and helped with figure preparation. Chapter 3 High-Throughput Combinatorial Cell Co-Culture Using Microfluidics The results in this chapter are mainly from manuscripts published in Integrative Biology, 2011, 3, 653. Authors: Ethan Tumarkin, Lsan Tzadu, Elizabeth Csaszar, Minseok Seo, Hong Zhang, Anna Lee, Raheem Peerani, Kelly Purpura, Peter Zandstra, and Eugenia Kumacheva Contributions: E. Tumarkin contributed to the paper by designing and carrying out experiments, data analysis and interpretation, and article writing. L. Tzadu, M. Seo, and H. Zhang helped with microfluidic experiments. E. VII Csaszar, R. Peerani, and K. Purpura helped with cell culture, cell analysis, and interpretation. A Lee helped with the preparation of the manuscript. P. Zandstra provided guidance and suggestions on experimental design, and article writing. Chapter 6 Temperature-Controlled 'Breathing' of Carbon Dioxide Bubbles The results in this chapter are mainly from manuscripts published in 1Lab on a Chip, 2011, 11, 3545 and 2Chemical Communications, 2011, 47, 12712. Authors: 1Ethan Tumarkin,n, Zhihong Nie, Jai Il Park, Milad Abolhasani, Jesse Greener, Barbara Sherwood Lollar, Axel Guenther, and Eugenia Kumacheva 2Ethan Tumarkin, Zhihong Nie, Jai Il Park, and Eugenia Kumacheva Contributions: 1E. Tumarkin contributed to the paper by designing and carrying out experiments, data analysis and interpretation, and article writing. Z. Nie and J. Park help with experimental design and microfluidic experiments. M. Abolhasani assisted with simulation design and analysis. J. Greener performed ATR-FTIR experiments. B. Sherwood Lollar and A. Guenther provided guidance and suggestions on experimental design, data interpretation, and article writing. 2E. Tumarkin contributed to the paper by designing and carrying out experiments, data analysis and interpretation, and article writing. Z. Nie and J. Park help with experimental design and microfluidic experiments. Chapter 7 Development and Applications of Microfluidic Reactors with Multiple Reconfigurable Analytical Probes The results in this chapter are mainly from manuscripts published in 1Analyst, 2012, 137, 444 and 2Lab on a Chip, 2012, 12, 696. Authors: VIII 1Jesse Greener*, Ethan Tumarkin*, Michael Debono, Chi-Hang Kwan, Milad Abolhasani, Axel Guenther, and Eugenia Kumacheva, Co-First Author 2Jesse Greener, Ethan Tumarkin, Michael Debono, A. Dicks, and Eugenia Kumacheva Contributions: 1E. Tumarkin contributed to the paper by designing and carrying out experiments, data analysis and interpretation, and article writing. J. Greener contributed to the paper by designing and carrying out experiments, data analysis and interpretation, and article writing. M. Debono assisted with experiments. C.H. Kwan and M. Abolhasani assisted with simulation design and analysis. A. Guenther provided helpful guidance on experimental design and, as well as assisted with data interpretation and article writing. 2E. Tumarkin contributed to the paper by designing experiments, data analysis and interpretation, and supervision of M. Debono (undergraduate student). J. Greener contributed to the paper by designing experiments, data analysis and interpretation, and article writing. M. Debono performed