Assays and Tools for Biomolecular Analysis in Remote and Low-Resource Settings By Richard Harry Henrikson A dissertation submitted in partial satisfaction of the requirements for the degree of Joint Doctor of Philosophy With University of California, San Francisco in Bioengineering in the Graduate Division of the University of California, Berkeley Committee in Charge: Professor Luke P. Lee Professor Tejal Desai Professor George Sensabaugh Summer 2015 Assays and Tools for Biomolecular Analysis in Remote and Low-Resource Settings ©2015 By Richard Harry Henrikson Abstract Assays and Tools for Biomolecular Analysis in Remote and Low-Resource Settings By Richard Harry Henrikson Joint Doctor of Philosophy in Bioengineering with University of California, San Francisco University of California, Berkeley Professor Luke P. Lee, Chair The global population faces significant challenges in not only improving health care, but even just in maintaining it. Environmental and economic disparities are increasingly creating a massive population that lacks adequate medical attention. Traditional tools employed to detect, monitor, and treat sickness are woefully inadequate in the face of this grand challenge in medicine. However, a new generation of technologies is under development that promises to increase both the access and the quality of care available on a global scale. These tools provide simple but powerful means for both medical workers and patients to take a predictive and preventative approach to medicine – lowering the overall burden on the system and leading a new state of wellness. There is no shortage of problems to solve in this effort to vastly energize the medical system. However, at the core, molecular diagnostic tools hold particular promise for revolutionary gains. As the core machinery of life, proteins and nucleic acids tell the full story of health and illness through their rise and fall. The ability to capture these changes, and to draw meaning from them, will be a cornerstone of modern proactive medicine. Early detection of illness often leads to a far reduced burden on the medical system, and significantly improved outcomes for the patient. In this work, I focus on the development of several technologies aimed at bringing health care a step closer to the world that needs it most. In the first chapter, I provide a brief overview of the acute clinical needs in remote and low-resource settings, as well as an historical perspective on the periodic evolution of medicine over the centuries that has led us to the cusp of a molecular diagnostic world. I also review some of the enabling technologies that have been developed to facilitate point of care medicine. Chapter 2 focuses on new assays and methods for nucleic acid testing in low-resource settings. I particularly focus on the development of robust isothermal amplification to glean information about both the presence of infection and the proper course of treatment. Through genotypic analysis of the invading pathogen, drug susceptibility can be determined. Furthermore, these assays are incorporated into several microfluidic and electronic systems that enable inexpensive and rapid diagnosis. 1 Chapter 3 provides an insight into the world of protein analysis in point of care settings. In this work, I’ve focused on robust, DNA-based sensors to detect proteins and initiate a downstream signal that can be quantified through fluorescent or nanophotonic mechanisms. In both cases, there is a focus on maintaining a rapid and robust signal that can be delivered at a low cost. Chapter 4 deviates slightly, as it explores a means to revolutionize the traditional polymerase chain reaction by leveraging nanoplasmonic phenomena. I provide a description and initial empirical insights on a means to perform multiplexed nucleic acid quantification with little more than an LED driving both thermocycling and specific detection. While this technology certainly has applications in the developed world as well, the primary focus is on facilitating the use of gold standard assays with simple and affordable tools. The final chapter briefly reviews my work in this doctorate degree program, while providing some insights into next steps to move these tools even closer to applications in the developing world. 2 For my parents, who taught me curiosity, compassion, and courage. May they treasure this work as though they were alone in reading it. i Contents Chapter 1: Introduction to Molecular Diagnostics in Remote and Low-Resource Settings ......................... 1 1.1 Clinical Needs in Remote and Low-Resource Settings .............................................................................. 1 1.1.1 Design Criteria for Healthcare Technology in Remote and Low-Resource Settings .......... 1 1.1.2 Environments for Diagnostic Testing...................................................................................................... 2 1.1.3 Acute and Chronic Biomarkers of Disease ............................................................................................ 3 1.2 Traditional Clinical Diagnostic Methods ......................................................................................................... 5 1.2.1 History of Medical Diagnostics ................................................................................................................... 5 1.2.2 Symptomatic Analysis .................................................................................................................................... 6 1.2.3 Microscopy .......................................................................................................................................................... 6 1.2.4 Cell Culture .......................................................................................................................................................... 7 1.2.5 Agglutination ...................................................................................................................................................... 7 1.3 The Rise of Molecular Diagnostics ..................................................................................................................... 8 1.3.1 History of Molecular Diagnostics .............................................................................................................. 8 1.3.2 Protein Detection ............................................................................................................................................. 8 1.3.3 Nucleic Acid Detection ................................................................................................................................... 8 1.4 Adapting Molecular Diagnostics for the Point of Need ............................................................................. 9 1.4.1 Overview of Diagnostic Design for RLR Settings ................................................................................ 9 1.4.2 Sample Matrices ................................................................................................................................................ 9 1.4.3 Assay Containers and Substrates ............................................................................................................10 1.4.4 Fluidic Actuation.............................................................................................................................................11 1.5 Discussion ...................................................................................................................................................................13 1.6 References ..................................................................................................................................................................14 Chapter 2: Nucleic Acid Detection with a Microfluidic Biomolecular Amplification Reader ..................... 20 2.1 Introduction ...............................................................................................................................................................20 2.1.1 Nucleic Acid Detection and Quantification in Remote and Low-Resource Settings ......... 20 2.1.2 Current Nucleic Acid Diagnostic Technologies .................................................................................21 2.2 Loop-mediated Isothermal Amplification Assay .......................................................................................22 2.2.1 LAMP Assay Overview .................................................................................................................................22 2.2.2 LAMP Primer Design .....................................................................................................................................25 2.2.3 LAMP Reaction Mix Optimization ...........................................................................................................26 2.2.4 LAMP Analysis of Single Nucleotide Polymorphisms for Drug Resistance .......................... 29 2.2.5 LAMP Analysis of Clinical Sputum Samples........................................................................................37 2.2.6 Lyophilization of LAMP Reagents ...........................................................................................................39 2.3 Design of Microfluidic Chips for Multiplexed NA Assay .........................................................................39 2.3.1 LAMP Analysis Chip Design .......................................................................................................................39