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Paper-based Microfluidics for Disease Diagnostics An Integrated, Portable Approach to Digital Medicine Bill Bedell | Travis Heinze | Meghan Keck | Spencer Saunders Manish Giri & James Stasiak Hewlett-Packard & Oregon State University School of Chemical, Biological, Environmental Engineering Corvallis, Oregon – 2012 HIV Rapid Test Kits ($0.60 - $4.00) ( + ) Robust and inexpensive ( - ) Lacking in sensitivity and specificity Existing Technologies Dried blood tests ($10.00) ( + ) Connects remote areas with central labs, accurate ( - ) Notoriously slow Diagnostics For All ® (~$0.50) ( + ) Inexpensive and versatile ( - ) Qualitative and in limited circulation μPAD Design Small size requires microliters of blood and nanoliters of reagents, decreasing pain and costs. diagnostic solutions Data Matrix Unique ID on each μPAD, allowing information to be associated with it. Color Reference For robust colorimetric analysis in different lighting conditions Wicking Material Draws blood to test point; covered in agglutination factor to separate plasma from red blood cells Assay Site Location of reagent printing & color response Hydrophobic Barriers Borders wicking channel, direct serum towards assay site The Data Ecosystem HP Device - Prints assay reagents - Analyzes color response - Reads μPAD data code -Assigns: -Assay type -Response -Patient ID Migration of color to edge of droplets creates a “coffee ring” effect that affects analysis Smart Phone - Displays and allows interaction w/ - Assay results - Patient information - Medical history cloud cloud Informatics Short-Range Wireless Internet Connection Uses of aggregate, non-identifying health data: · Establishing health demographics · Monitoring the spread of infectious disease · Determining disease morbidity rates · Epidemiological studies · Assessing pharmacological priorities · Planning medical humanitarian organizations’ campaigns - Organizes patient health information - Can analyze assay pictures/scans Red blood cells (RBC) interfere with color response - Normally separated from plasma w/centrifuge Acknowledgements Dr. Jean Hall – OSU College of Veterinary Medicine Dr. Sue Tornquist – OSU College of Veterinary Medicine Dr. Phil Harding – OSU, Linus Pauling Chair in Chemical Engineering Lectin proteins bind to antigens on RBC, agglutinating the cells, allowing plasma to wick to the reagent site Microfluidics 180 μm 500 μm Image Analysis Our MATLAB script identifies assay droplets and runs colorimetric analysis. Assay Scan Greyscale Cleaned B/W Filled Drops Object Outlines Outlines + Original Image Average Color in each Outline The Problem Our Solution Location: Rural population center in Sub-Saharan Africa Print liver function assays on ‘blank’ test strip at point-of-care Distribute prepared test kits to patients Collect tests; analyze responses with internet- connected scanner Compare result with electronic patient history; adjust drug regimen as necessary Prepare Assay Analyze Decide · ‘In-field reagent printing’ -- Nanoliter dispensing of precious active reagents at the point-of-care, lowering cost, reducing contamination, and improving adaptability. Patients: Suffering from HIV, tuberculosis, or malaria The Test: Determine if dosage of drug cocktails are causing liver failure Infrastructure: No laboratories or trained personnel, but internet is available · ‘Small footprint system’ -- inexpensive, hygienic, and portable; bringing quality diagnostics to remote communities. · ‘Digital integration’ -- tests are analyzed quickly and accurately, and patients are connected with doctors and health organizations A high-throughput paper-based medical test kit printed on-site · Developing countries struggle with HIV, tuberculosis, and malaria · Necessarily-strong pharmaceuticals can pose risk to patients’ livers · Communities lack the diagnostic measures necessary to prevent liver failure Issue Objective · Develop an inexpensive test kit that can perform liver function assays · Research point-of-care diagnostics market opportunities for Hewlett-Packard · Propose a business model for HP that enables diagnostics and informatics in developing countries Input - Assay Image - # of Droplets Output - Analyte - Concentrations With Standard Curve MATLAB - Indexes droplet outlines in image - Finds ave. color in each outline - Converts where L* is color intensity - Relates L* to analyte concentraion RGB L*a*b* Barriers must completely permeate through paper media to prevent leaks <- Counter example Plasma Dry paper Wet Blood Plasma RBC aggregates Assays consist of reagent mixes in buffers that react with proteins or enzymes in biological samples to produce quantifiable colorimetric responses (Alanine Aminotransferase) Elevated levels Definite liver damage Elevated levels Jaundice Possible liver damage Printing Assays Standard Curves Wicking media needs: fast wicking & good colorimetric response Intensity = -0.2396[Bilirubin] + 68.075 R² = 0.9908 60 61 62 63 64 65 66 67 68 69 0 5 10 15 20 25 30 35 Reflected LIght Intensity [%] Bilirubin Standard Concentration [μM] Bilirubin Assay Intensity = -0.7059[ALT] + 64.166 R² = 0.9789 57 58 59 60 61 62 63 64 65 0 1 2 3 4 5 6 7 8 9 Reflected Light Intensity [%] Pyruvate Standard Concentration [nM] ALT Assay Bilirubin Assay ALT Assay Color Intensity Values No need for bulky centrifuge Surfactant smoothed printing of aq. solutions Optimal print order: Reagent, then buffer/sample Known drying time vs drop volume allowed control of reaction time Findings We printed 50 – 400 nL droplets of assay solutions on proprietary textured paper media, designed for bright color response Our HP D300 Digital Dispenser printed large matrices of assay droplets in < 1 min, allowing for quick implementation of planned experiments. http://www.ral.ucar.edu/projects/ghana/ http://journik.posterous.com/serendipity-how-your-life-can-flourish-in-jus Diagnostics For All Hewlett-Packard http://www.business-clipart.com http://www.ncbi.nlm.nih.gov/pubmed/22094609 http://www.ncbi.nlm.nih.gov/pubmed/22094609 May 18 th 2012 2012 OSU Engineering Fair References Xiaoxi Yang, et al. Integrated separation of blood plasma from whole blood for microfluidic paper-based analytical devices. RSC. (2012) Andres W. Martinez, et al. Diagnostics for the Developing World: Microfluidic Paper-Based Analytical Devices. Anal. Chem. (2010) Health Research Institute. Needles in a haystack: Seeking knowledge with clinical informatics. PWC. (Feb. 2012) I love you, Mom
