Heat, Fluid, and Sample Control in Point-Of-Care Diagnostics Joshua
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Heat, Fluid, and Sample Control in Point-of-Care Diagnostics Joshua Ronald Buser A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2016 Reading Committee: Paul Yager, Chair Barry Lutz Wendy Thomas Program Authorized to Offer Degree: Bioengineering © Copyright 2016 Joshua Ronald Buser University of Washington Abstract Heat, Fluid, and Sample Control in Point-of-Care Diagnostics Joshua Ronald Buser Chair of the Supervisory Committee: Professor Paul Yager Bioengineering Point-of-care diagnostics have transformed healthcare workflow, perhaps most notably in the wide-reaching impact of home pregnancy tests. Lateral flow strips are simple and inexpensive, but limited in their application to relatively simple diagnostic tasks. Nucleic acid amplification assays on the other hand are more accurate and sensitive, but currently restricted to use in well-equipped laboratories due to reliance on external supplies, power, and trained users. The Multiplexable Autonomous Disposables for Nucleic Acid Amplification Tests in Limited Resource Settings (MAD NAAT) Project, led by Paul Yager, uses instrument-free, disposable, paper-based fluidics for multiple pathogen detection with DNA/RNA. The end goal is to create a sample in, answer out isothermal nucleic acid amplification device simple enough for untrained users, with low enough cost to enable wider use of nucleic acid testing. My contributions to this project, combining to form my thesis, include advances in sample preparation for nucleic acid amplification, isothermal chemical temperature control, and fluid transport in paper microfluidics. Progress in these subject areas will help advance the development of nucleic acid tests and other advanced assays compatible with point-of-care usage. 1 Table of Contents 1 Table of Contents..................................................................................................................... 1 2 Acronyms............................................................................................................................... 14 3 Summary: Heat, fluid, and sample control in point-of-care diagnostics ............................... 19 4 Overview of thesis sections ................................................................................................... 22 4.1 Background .................................................................................................................... 22 4.2 Aim 1) Fluid flow in paper microfluidics: parameter measurement and flow modeling22 4.3 Aim 2) Isothermal temperature control using chemical heaters .................................... 22 4.4 Aim 3) Develop an electromechanical sample preparation methodology for hard-to-lyse pathogens compatible with point-of-care deployment .............................................................. 23 4.5 Aim 4) Utilize the techniques developed in Aims 1-3 to design and build integrated systems ...................................................................................................................................... 23 4.6 Integrated MAD NAAT ................................................................................................. 23 4.7 Appendices ..................................................................................................................... 24 4.8 Future directions ............................................................................................................. 24 4.9 Publications .................................................................................................................... 25 4.10 Patent applications.......................................................................................................... 26 4.11 Posters and presentations ............................................................................................... 27 5 Introduction: The Need for Diagnostics in Low-resource Settings ....................................... 28 5.1 Importance of Diagnostic Testing .................................................................................. 28 5.2 Limitations in Low-resource Settings ............................................................................ 28 5.3 Scope of Section ............................................................................................................. 29 5.4 Types of Diagnostic Testing Needed in Low-resource Settings .................................... 30 5.5 Diagnosing Disease ........................................................................................................ 31 5.6 Monitoring Disease ........................................................................................................ 37 5.7 Counterfeit Drug Testing ............................................................................................... 41 5.8 Environmental Testing ................................................................................................... 43 5.9 Overview of Microfluidic Diagnostics for Use at the Point of Care .............................. 44 5.9.1 Channel-based Microfluidics .................................................................................. 45 5.9.2 Paper-based Microfluidics ...................................................................................... 47 5.10 Enabling All Aspects of Diagnostic Testing in Low-resource Settings: Examples of and Opportunities for Microfluidics (Channel-based and Paper-based) .......................................... 56 5.10.1 Transportation and Storage of Devices in Low-resource Settings .......................... 57 5.10.2 Specimen Collection ............................................................................................... 59 5.10.3 Sample Preparation ................................................................................................. 60 1 5.10.4 Running the Assay .................................................................................................. 62 5.10.5 Signal Read-out ....................................................................................................... 67 5.10.6 Data Integration into Health Systems ..................................................................... 69 5.10.7 Disposal................................................................................................................... 71 5.11 Conclusions .................................................................................................................... 72 5.11.1 Acknowledgements ................................................................................................. 73 6 Aim 1: Fluid flow in paper microfluidics: necessary parameter measurements and flow modeling ....................................................................................................................................... 74 6.1 Background .................................................................................................................... 75 6.2 Materials and Methods ................................................................................................... 81 6.2.1 Porosity measurements ........................................................................................... 81 6.2.2 Thickness measurements ........................................................................................ 81 6.2.3 Wicking time measurements ................................................................................... 82 6.2.4 Saturated permeability measurements .................................................................... 82 6.2.5 Water retention curve measurements ...................................................................... 85 6.2.6 Unbacked membrane preparation ........................................................................... 86 6.2.7 Backed membrane preparation ............................................................................... 87 6.2.8 Time required to reach equilibrium ........................................................................ 87 6.2.9 Water retention curve model fitting ........................................................................ 87 6.2.10 Finite element modeling of the Richards Equation................................................. 88 6.3 Results and Discussion ................................................................................................... 88 6.3.1 Permeability measurements .................................................................................... 88 6.3.2 Capillary pressure vs saturation: water retention curves ........................................ 91 6.3.3 Hydraulic conductivity vs saturation: water retention curve-fitting ....................... 92 6.3.4 Relative permeability vs. saturation ........................................................................ 93 6.4 Conclusions .................................................................................................................... 97 6.5 Acknowledgements ........................................................................................................ 98 7 Aim 2) Precision heating in electricity-free diagnostic devices ............................................ 99 7.1 Introduction .................................................................................................................. 101 7.2 General design considerations for chemical heaters .................................................... 103 7.2.1 ASSAY