LOW-COST DIAGNOSTICS AND TOOLS FOR GLOBAL HEALTH
In low-resource settings (e.g., rural clinics in sub-Saharan Africa or a forward-deployed military unit), meeting the need for high-quality medical diagnostics requires overcoming four challenges: 1) harsh transportation and storage conditions, 2) minimally trained users who might be under high stress, 3) limited infrastructure for support and maintenance, and 4) an economy that cannot afford expensive solutions. An active area of research in our group involves the identification and development of tools and techniques that can overcome these barriers to create high-quality, low-cost diagnostics. Through our efforts, we have invented and developed new diagnostics for health conditions ranging from liver injury to sickle cell disease. We have also developed platform technologies, such as density-based analysis with magnetic levitation and aqueous multiphase systems, and microfluidics in paper-based devices. We are concerned not only with creating new ideas, but also bringing these technologies to reality. To this end, we have built partnerships with medical experts around the world, tested some of our technologies in the field in Africa, and established partnerships with companies like Diagnostics for All, Daktari Diagnostics, and Drummond Scientific to develop technologies into products that can benefit people around the world (Fig 1).1
Three-Dimensional Paper-based Microfluidic Devices
Paper is one of the oldest materials humans have made. This history and widespread use of paper has led to a well-developed industry around producing paper using high-throughput printing. Paper also has the property that it absorbs and wicks liquids. By printing hydrophobic patterns on the surface of paper (by wax printing with a desktop printer2 or photolithography3), we can define channels in paper to direct the flow of liquids. Stacking these channels and connecting them with tape allows the creation of 3D microfluidic devices (Fig 2).4 These devices allow complex fluid handling and multiplexing. Using these processes, we have created a low-cost liver function test with a material cost of a few cents.5 We are developing a host of other assays to provide low-cost diagnostic information at the point of care.6
Electrochemical Sensing on Paper-based Devices
In some types of diagnostics, such as those for pregnancy, a simple yes/no answer is sufficient. In other situations, such as the daily monitoring of glucose, a more quantitative measurement is needed. Using screen printing, we can create electrodes on paper-based microfluidic devices to produce strips that can be used for measuring glucose and other metabolites (Fig 3).7,8 We have also developed a paper-based reference electrode to enable potentiometry.9 By incorporating ion-selective membranes into our devices, we have measured the concentration of specific ions in solutions using paper devices (Fig 4).10
We have also developed methods to make the surface of paper hydrophobic by treating it with fluoroalkyl thrichlorosilanes.11 Such treatments allow the use of paper as a substrate for applications where wicking is not desirable, such as for use as a microwell to hold solutions. Hydrophobic treatments also allow the printing of high resolution electrodes using conductive inks.12 Combined with embossing to create wells, we have created paper-based systems to do electrochemical enzyme linked immunosorbent assays (ELISA).13
Point-of-Care Hematology with Aqueous Multiphase Systems
Density (the mass over the volume of an object) is a property of all matter. Measuring the density of cells can provide an aggregate measure of changes taking place metabolically or morphologically. Using aqueous multiphase polymers (AMPS)—mixtures of polymers in water that spontaneously form immiscible phases—we can separate cells by density. AMPS are thermodynamically stable, can be biocompatible, and can be tuned to have specific densities in each phase.14 The interfaces between phases are molecularly sharp, and define a step in density that can collect and concentrate cells within that range of densities.
Using AMPS, we have created a simple method to enrich reticulocytes (immature red blood cells) that can be used to grow malaria species better than conventional reticulocyte purification methods (Fig 5).15 In certain cases, the ability to separate cells by density can provide a means to diagnose disease. Sickle cell disease is a genetic disease that approximately 300,000 children are born with each year. Most of these children are undiagnosed and at high risk for childhood mortality even though simple and effective interventions exist to manage the disease. The morphological changes in sickle cell disease lead to the formation of very dense erythrocytes that can be separated using AMPS. Using capillary tubes, AMPS, and portable centrifuges, we have created a low-cost, visual test for sickle cell disease that can be performed in ~15 minutes (Fig 6).16 After initial validation in the U.S., we worked with partners in Zambia to test the device in a larger population and to assess feasibility of the design in rural health centers.17
Density-based ELISA with Magnetic Levitation
Another method to analyze density with minimal equipment is magnetic levitation. Using two opposing magnets, we create a magnetic field gradient. When a diamagnetic object is placed in a paramagnetic solution and within the magnetic field gradient, the paramagnetic fluid will be attracted to the highest field and will push diamagnetic object to point of lowest field in the middle of the two magnets. This force is opposed by the buoyant force which drives the object to the top or bottom of the solution. The balance of the magnetic and buoyant forces results in a stable levitation height within the paramagnetic fluid that is a function of density.18
Using antigens immobilized onto the surface of polymeric beads, we have created a density-based ELISA. When the beads are exposed to a sample, antibodies specifically bind the antigens. Anti-human antibodies conjugated with gold particles then bind to the captured antibody. We then chemically deposit silver or gold onto the beads. The deposition of metal leads to a change in density of the bead that indicates the amount of antigen that was captured.19 Using parallel vials, we can perform a multiplexed density-based ELISA (Fig 7).
ENGINEERING ANALYTICAL TOOLS FOR THE FIELD
A Universal Mobile Electrochemical Detector (uMED)
Electrochemistry is a powerful method used to quantify analytes in complex mixtures. To access the analytical power of electrochemistry at the point-of-care, a low-cost, portable electrochemical reader is necessary to read strips developed for various diagnostics. The glucose meter provides an example of an electrochemical reader designed for home use, but it is limited to only a subset of electrochemical methods (i.e., amperometry). We have created a universal Mobile Electrochemical Detector (uMED) capable of performing nearly all the functions of a benchtop potentiostat (e.g., amperometry, cyclic voltammetry, square wave voltammetry, and potentiometry) (Fig 8).20 The handheld device can be used to read commercially available electrodes as well as the paper-based electrodes we develop in the lab. In addition to providing quantitative analysis at the point-of-care, the device provides connectivity to the cloud by connecting through the headphone jack of any phone over any wireless network.
Low-cost Containers for Biological Samples
In some cases where analysis at the point-of-care is not possible, health workers may want to store samples and send them to a separate location for analysis. Sterile containers suitable for biological samples can be expensive. We have demonstrated that bubble wrap can provide a low-cost, sterile material for the storage and analysis of biological samples (Fig 9).21 The transparency of the bubble wrap also enables samples to be analyzed by spectroscopy without the need to transfer the sample to a different container.
1. Kumar, A. A.; Hennek, J. W.; Smith, B. S.; Kumar, S.; Beattie, P.; Jain, S.; Rolland, J. P.; Stossel, T. P.; Chunda-Liyoka, C.; Whitesides, G. M. "From the Bench to the Field in Low-Cost Diagnostics: Two Case Studies", Angewandte Chemie International Edition 2015, 54, 5836–5853.
2. Carrilho, E.; Martinez, A. W.; Whitesides, G. M. "Understanding Wax Printing: A Simple Micropatterning Process for Paper-Based Microfluidics", Analytical chemistry 2009, 81, 7091–7095.
3. Martinez, A. W.; Phillips, S. T.; Butte, M. J.; Whitesides, G. M. "Patterned Paper as a Platform for Inexpensive, Low-Volume, Portable Bioassays", Angewandte Chemie (International ed. in English) 2007, 46, 1318–1320.
4. Martinez, A. W.; Phillips, S. T.; Whitesides, G. M. "Three-Dimensional Microfluidic Devices Fabricated in Layered Paper and Tape", Proceedings of the National Academy of Sciences of the United States of America 2008, 105, 19606–19611.
5. Vella, S. J.; Beattie, P.; Cademartiri, R.; Laromaine, A.; Martinez, A. W.; Phillips, S. T.; Mirica, K. A.; Whitesides, G. M. "Measuring Markers of Liver Function Using a Micropatterned Paper Device Designed for Blood from a Fingerstick", Analytical Chemistry 2012, 84, 2883–2891.
6. Martinez, A. W.; Phillips, S. T.; Whitesides, G. M.; Carrilho, E. "Diagnostics for the Developing World: Microfluidic Paper-Based Analytical Devices", Analytical Chemistry 2010, 82, 3–10.
7. Nie, Z.; Nijhuis, C. A.; Gong, J.; Chen, X.; Kumachev, A.; Martinez, A. W.; Narovlyansky, M.; Whitesides, G. M. "Electrochemical Sensing in Paper-Based Microfluidic Devices", Lab on a chip 2010, 10, 477–483.
8. Nie, Z.; Deiss, F.; Liu, X.; Akbulut, O.; Whitesides, G. M. "Integration of Paper-Based Microfluidic Devices with Commercial Electrochemical Readers", Lab on a Chip 2010, 10, 3163–9.
9. Lan, W.; Maxwell, E. J.; Parolo, C.; Bwambok, D. K. "Paper-Based Electroanalytical Devices with an Integrated, Stable Reference Electrode", Lab on a Chip 2013, 13, 4103–4108.
10. Lan, W.; Zou, X. U.; Hamedi, M. M.; Hu, J.; Parolo, C.; Maxwell, E. J.; Bu, P.; Whitesides, G. M. "Paper-Based Potentiometric Ion Sensing", Analytical Chemistry 2014, 86, 9548–9553.
11. Glavan, A. C.; Martinez, R. V.; Subramaniam, A. B.; Yoon, H. J.; Nunes, R. M. D.; Lange, H.; Thuo, M. M.; Whitesides, G. M. "Omniphobic “rF Paper” Produced by Silanization of Paper with Fluoroalkyltrichlorosilanes", Advanced Functional Materials 2014, 24, 60–70.
12. Lessing, J.; Glavan, A. C.; Walker, S. B.; Keplinger, C.; Lewis, J. A.; Whitesides, G. M. "Inkjet Printing of Conductive Inks with High Lateral Resolution on Omniphobic “rF Paper” for Paper-Based Electronics and MEMS", Advanced Materials 2014, 26, 4677–4682.
13. Glavan, A., Christodouleas, D. C., Mosadegh, B., Yu, H.-D., Smith, B., Lessing, J., Fernandez-Abedul, M. T. and Whitesides, G. M. "Folding Analytical Devices for Electrochemical ELISA in Hydrophobic RH Paper", Analytical Chemistry, 2014, 86, 11999-12007.
14. Mace, C. R.; Akbulut, O.; Kumar, A. A.; Shapiro, N. D.; Derda, R.; Patton, M. R.; Whitesides, G. M. "Aqueous Multiphase Systems of Polymers and Surfactants Provide Self-Assembling Step-Gradients in Density", Journal of the American Chemical Society 2012, 134, 9094–9097.
15. Kumar, A. A.; Lim, C.; Moreno, Y.; Mace, C. R.; Syed, A.; Tyne, D. Van; Wirth, D. F.; Duraisingh, M. T.; Whitesides, G. M. "Enrichment of Reticulocytes from Whole Blood Using Aqueous Multiphase Systems of Polymers", American Journal of Hematology 2015, 90, 31–36.
16. Kumar, A. A.; Patton, M. R.; Hennek, J. W.; Lee, S. Y. R.; D’Alesio-Spina, G.; Yang, X.; Kanter, J.; Shevkoplyas, S. S.; Brugnara, C.; Whitesides, G. M.; Wang, X. "Density-Based Separation in Multiphase Systems Provides a Simple Method to Identify Sickle Cell Disease", Proceedings of the National Academy of Sciences of the United States of America 2014, 111, 14864–14869.
17. Kumar, A. A.; Chunda-Liyoka, C.; Hennek, J. W.; Mantina, H.; Lee, S. Y. R.; Patton, M. R.; Sambo, P.; Sinyangwe, S.; Kankasa, C.; Chintu, C.; Brugnara, C.; Stossel, T.; Whitesides, G. M. "Evaluation of a Density-Based Rapid Diagnostic Test for Sickle Cell Disease in a Clinical Setting in Zambia", PLOS ONE 2014, 9, e114540.
18. Mirica, K. A.; Shevkoplyas, S. S.; Phillips, S. T.; Gupta, M.; Whitesides, G. M. "Measuring Densities of Solids and Liquids Using Magnetic Levitation: Fundamentals", Journal of the American Chemical Society 2009, 131, 10049–10058.
19. Subramaniam, A. B.; Gonidec, M.; Shapiro, N. D.; Kresse, K. M.; Whitesides, G. M. "Metal-Amplified Density Assays, (MADAs), Including a Density-Linked Immunosorbent Assay (DeLISA)", Lab Chip 2015, 15, 1009–1022.
20. Nemiroski, A.; Christodouleas, D. C.; Hennek, J. W.; Kumar, A. A.; Maxwell, E. J.; Fernandez-Abedul, M. T.; Whitesides, G. M. "Universal Mobile Electrochemical Detector Designed for Use in Resource-Limited Applications", Proceedings of the National Academy of Sciences 2014, 111, 11984–11989.
21. Bwambok, D. K.; Christodouleas, D. C.; Morin, S. a.; Lange, H.; Phillips, S. T.; Whitesides, G. M. "Adaptive Use of Bubble Wrap for Storing Liquid Samples and Performing Analytical Assays", Analytical Chemistry 2014, 86, 7478–7485