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Rational Hydrogel Design for Point-of-Care Bioassays MASSACHUSETTNSTITNTE by Sarah Jane Shapiro SEP 112019 B.S., University of Oklahoma (2013) LIBRARIES M.S., University of Oklahoma (2014) S.M., Massachusetts Institute of Technology (2017) Submitted to the Department of Chemical Engineering in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemical Engineering at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY September 2019 @Massachusetts Institute of Technology 2019. All rights reserved. Signature redacted Author ............................. .. ... Department of Chemical Engineering August 28, 2019 Certified by...............................Signatureredacted Patrick S. Doyle Robert T. Haslam (1911) Professor of Chemical Engineering Thesis Supervisor Accepted by ............................ Signatureredacted Patrick S. Doyle Robert T. Haslam (1911) Professor of Chemical Engineering Chairman, Committee for Graduate Students 2 Rational Hydrogel Design for Point-of-Care Bioassays by Sarah Jane Shapiro Submitted to the Department of Chemical Engineering on August 28, 2019, in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemical Engineering Abstract As the global disease burden shifts increasingly towards chronic diseases, there is a need for improved diagnosis and monitoring so that patients can get the care they need. This is particularly evident in the developing world, where many people live far from diagnostic laboratories. Point-of-care diagnostics are tests that can be run in doctor's offices, clinics, and in patient homes. These tests must be rapid, so that they can be run quickly while the patient waits for results. Established point-of-care technologies are largely centered on lateral flow assays. Hydrogel microparticles have been used extensively for bioassays due to their nonfouling nature and ability to be functionalized with different types of biomolecules. Here, we use polyethylene gly- col hydrogel particles to develop point-of-care bioassays. We focus on two different biomarkers: proteins and microRNA. Proteins are well established clinical biomarkers that are regularly tested to diagnose a number of different diseases. miRNA are emerging biomarkers that were discovered within the past thirty years and have dysregulation patterns that are implicated in a wide variety of diseases. The aim of this thesis is to enable hydrogel-based point-of-care detection of miRNA and proteins by developing and applying theory to aid in rational design of the bioassay. First, we establish a theoretical framework to investigate the key factors that influence bioassay signal for hydrogel-based rapid bioassays. By developing scaling arguments for the flux of target into the hydrogel, we find that the key factors that influence bioassay signal are the reaction rate constant, the diffusion coefficient of the target in the gel, the probe concentration, the target concentration, the assay time, and the shape of the hydrogel. By changing the hydrogel particle shape, we are able to decrease the limit of detection of a protein assay by a factor of six. We then apply the theory we developed for hydrogel signal to an assay for microRNA. Using the theory, we are able to design the hydrogels to enable muultiplexed detection of miRNA directly from serum in a 40-minute assay, with a clinically-relevant limit of detection. This assay only requires minimal preprocessing of the serum, making it useful for point-of-care applications. Leveraging our theoretical knowledge, we also develop a new assay format by incorporating hydrogels into fibrous substrates such as nitrocellulose, glass fiber membranes, and silk fabric and demonstrating their utility 3 for bioassays. We demonstrate that these constructs can be used for detection of both miRNA and proteins. This work combines the fields of flexible fibrous materials and lithographic patterning to directly pattern hydrogels of varying shape and function within fibrous substrates. The work presented in this thesis demonstrates the utility of hydrogels for point-of- care applications. We believe that this work can be leveraged in the future to develop tests for additional biomarkers and can be combined with advances in fluorescence imaging and portable heating to create point-of-care devices that can quickly and reliably quantify proteins and miRNA from complex samples, in order to enable earlier diagnosis of disease. Thesis Supervisor: Patrick S. Doyle Title: Robert T. Haslam (1911) Professor of Chemical Engineering 4 Acknowledgments A PhD is a very long road and I am grateful for the many people whose support has helped bring me to this point. First of all, I would like to thank my advisor, Professor Patrick Doyle, whose intelligence and piercing questions shaped who I am as a researcher. Pat, thanks for always asking me hard questions and for pushing me to be the best researcher I could be. Thank you also for building a supportive lab culture; it has been an honor to work in your lab for the past four and a half years and I count many of my labmates among my close friends. Next, I would like to thank my thesis committee members: Professor Hadley Sikes and Professor Joel Voldman. Hadley, thanks for always being supportive of my career development and for asking insightful questions about the applications of the technology I was building. Joel, thanks for always being excited about the research I have done and for pushing me to set concrete goals for completion. The questions you both asked during committee meetings made this thesis better and your support helped me navigate the challenges of graduate school. To the Doyle Lab: Thanks for not only being labmates but also becoming some of my closest friends. I appreciate the way you are not only supportive of successes but are also always willing to exchange stories of failures. The lab you choose defines much of your graduate school experience and I am very fortunate to have spent the past five years in such a supportive place. In particular, I would like to thank Hyewon Lee, Beatrice Soh, Lynna Chen, Maxwell Nagarajan, Li-Chiun Cheng, Augusto Tentori, Nidhi Juthani, Alexander Klotz, Jae Jung Kim, Ankur Gupta, Vivek Narsimhan, Lillian Hsaio, Hyundo Lee, Jeremy Schieferstein, Liam Chen, George Kapellos, Yuan Tian, Trystan Domenech, Signe Lin Kuei Vehusheia, Abu Zayed Md Badruddoza, Doug Godfrin, Gaelle Le Goff, Ben Renner, Seung Goo Lee, Alona Birjiniuk, and Rathi Srinivas. A special thanks to Hyewon and Rathi for teaching me when I joined the lab. I would like to offer a special thanks to the Tata Center at MIT that provided funding for the majority of my PhD. They provided me with a unique experience 5 to not only work on developing diagnostics but also to partner with a startup in India, Achira Labs, and travel to India many times to gain an understanding of the state of diagnostics there and work on building and testing technology. I also thank Dr. Dhananjay Dendukuri for supporting me on many visits to Achira and providing many useful suggestions as well as the many people at Achira who made my travels there so enjoyable, including Mithila Ja, Dr. Bhavna Goyal, Dr. Purbasha Halder, Jayeeta Pai, Dr. Lokanathan Arcot, Raghavendra Katti, Gokul Rajamanickam, Tr- isha Manna, Rakesh Sharma, Dr. Satish Kalme, Sakul, Chethana, and Damu among others. Everyone at Achira was very hospitable during my visits and answered my frequent questions. I would like to thank my family and friends for being so supportive during my time in graduate school. Thanks for always being there when I needed to laugh, cry, or vent about the stressors of graduate school. Thanks also to all my colleagues on the Graduate Student Advisory Board and the Graduate Student Council for working with me to try to make the graduate student experience better. And a thank you to the Chemical Engineering Communication Lab for being great colleagues and helping me improve my communication skills. I would like to thank my practice school buddies for remaining close friends throughout the graduate school experience: J. C., T.-C. (J.) C., S. D., Y. K., M. L., Y. M., Y. R., L. V., and S. V. Particular thanks to the many friends who have helped me destress over the past year, including Teresa and Chad Ratashak (and George!), Jennifer Herrmann, Lisa Volpatti, Mark Goldman, Stephanie Doong, and Yamini Krishnan. A special thanks to Barbara and Tom Herrmann, who have become a second family to me. Finally, I would like to thank my family, who have been incredibly supportive my entire time in graduate school. To my parents, Kendall and LinMarie Stephenson: Dad, thanks for encouraging a love of science and math at a young age and for being the best teacher I have ever had. Mom, thanks for being a female engineer role model for me and so many others. Thank you both for always encouraging me to pursue my dreams, even when it took me far away from you. I am grateful to my sister Rebecca Kaufman and her husband Ryan for always being willing to listen and for encouraging 6 me to keep going when life was difficult. To Nicole Stephens, thanks for letting me invade your space so many times and for being a patient listener. To my brother, Matthew Stephenson, thanks for the many long phone calls where you listened to me vent, for letting me crash at your apartment so many times, and for distracting me with hiking and camping trips. "The one who called you is faithful, and he will do it." SDG 7 8 Contents 1 Introduction 21 1.1 Biomolecules ofinterest ................ .......... 22 1.1.1 P roteins ... ....................... .... 22 1.1.2 microRNA .. ....................... .... 22 1.2 Point-of-care detection methods ........ ............ 24 1.2.1 Point-of-Care Detection of Proteins ............... 26 1.2.2 Point-of-Care Detection of microRNA .............. 27 1.3 Hydrogel-based detection of biomolecules .........

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