Microfluidics-based diagnostics of infectious diseases in the developing world

Journal name:
Nature Medicine
Volume:
17,
Pages:
1015–1019
Year published:
DOI:
doi:10.1038/nm.2408
Received
Accepted
Published online

Abstract

One of the great challenges in science and engineering today is to develop technologies to improve the health of people in the poorest regions of the world. Here we integrated new procedures for manufacturing, fluid handling and signal detection in microfluidics into a single, easy-to-use point-of-care (POC) assay that faithfully replicates all steps of ELISA, at a lower total material cost. We performed this 'mChip' assay in Rwanda on hundreds of locally collected human samples. The chip had excellent performance in the diagnosis of HIV using only 1 μl of unprocessed whole blood and an ability to simultaneously diagnose HIV and syphilis with sensitivities and specificities that rival those of reference benchtop assays. Unlike most current rapid tests, the mChip test does not require user interpretation of the signal. Overall, we demonstrate an integrated strategy for miniaturizing complex laboratory assays using microfluidics and nanoparticles to enable POC diagnostics and early detection of infectious diseases in remote settings.

At a glance

Figures

  1. Schematic diagram and pictures of microfluidic device, and data on fluid handling of a POC ELISA-like assay.
    Figure 1: Schematic diagram and pictures of microfluidic device, and data on fluid handling of a POC ELISA-like assay.

    (a) Picture of microfluidic chip. Each chip can accommodate seven samples (one per channel), with molded holes for coupling of reagent-loaded tubes. (b) Scanning electron microscope image of a cross-section of microchannels, made of injection-molded plastic. Scale bar, 500 μm. (c) Transmitted light micrograph of channel meanders. Scale bar, 1 mm. (d) Schematic diagram of passive delivery of multiple reagents, which requires no moving parts on-chip. A preloaded sequence of reagents passes over a series of four detection zones, each characterized by dense meanders coated with capture proteins, before exiting the chip to a disposable syringe used to generate a vacuum for fluid actuation. (e) Illustration of biochemical reactions in detection zones at different immunoassay steps. The reduction of silver ions on gold nanoparticle–conjugated antibodies yields signals that can be read with low-cost optics (for quantification) or examined by eye. (f) Absorbance traces of a complete HIV-syphilis duplex test as reagent plugs pass through detection zones. High optical density (OD) is observed when air spacers pass through the detection zones, owing to increased refraction of light compared to in the liquid-filled channels. The train of reagents mimics the pipetting of reagents in and out of multiwell plates. This sample was evaluated (correctly against a reference standard) as HIV negative and syphilis positive. Ag, antigen.

  2. Results of immunoassays performed at Columbia University on commercial specimen panels.
    Figure 2: Results of immunoassays performed at Columbia University on commercial specimen panels.

    (a) Images of silver-enhanced signals on detection zones coated with HIV antigen (top group, left column), syphilis antigen (bottom group, left column), and antibodies to goat IgG (anti–goat IgG) (both groups, right columns) as a positive reference. Zones were exposed to positive and negative samples as judged by a reference standard (top and bottom rows, respectively). (b) Test results for HIV (left) and syphilis (right) antibodies. Vertical scatter plots of silver absorbance (normalized by cutoff values) for positive (Pos) and negative (Neg) serum or plasma specimens (each human sample is represented by one filled circle for HIV or cross for syphilis). Signal-to-cutoff values smaller than 0.1 are shown at 0.1 (with arrows). See Supplementary Tables 2–4 for raw data, cutoffs and specimen profiles. (c) Receiver-operating characteristic (ROC) curves for HIV (left) and syphilis (right), for illustrating changes in sensitivity and specificity depending on cutoff.

  3. Field results of the HIV immunoassay collected in Muhima Hospital in Rwanda using <1 [mu]l of unprocessed whole-blood sample.
    Figure 3: Field results of the HIV immunoassay collected in Muhima Hospital in Rwanda using <1 μl of unprocessed whole-blood sample.

    (a) Signal-to-cutoff ratios for positive (Pos) and negative (Neg) samples. (b) ROC curve.

  4. Field results of a HIV and syphilis duplex immunoassay collected in Projet Ubuzima in Rwanda, using 7 [mu]l of plasma or sera.
    Figure 4: Field results of a HIV and syphilis duplex immunoassay collected in Projet Ubuzima in Rwanda, using 7 μl of plasma or sera.

    (a) Signal-to-cutoff ratios of sera or plasma specimens that are positive (Pos) and negative (Neg) for HIV (circles) and syphilis (crosses). Signal-to-cutoff values >10 are shown at 10, and those <0.1 are shown at 0.1 (both with arrows). (b) ROC curves for HIV and syphilis.

References

  1. Whitesides, G.M. The origins and the future of microfluidics. Nature 442, 368373 (2006).
  2. West, J., Becker, M., Tombrink, S. & Manz, A. Micro total analysis systems: Latest achievements. Anal. Chem. 80, 44034419 (2008).
  3. Chin, C.D., Linder, V. & Sia, S.K. Lab-on-a-chip devices for global health: past studies and future opportunities. Lab Chip 7, 4157 (2007).
  4. Yager, P. et al. Microfluidic diagnostic technologies for global public health. Nature 442, 412418 (2006).
  5. Hay Burgess, D.C., Wasserman, J. & Dahl, C.A. Global health diagnostics. Nature 444 (suppl. 1), 12 (2006).
  6. Laxminarayan, R. et al. Advancement of global health: key messages from the Disease Control Priorities Project. Lancet 367, 11931208 (2006).
  7. Wootton, R.C.R. & deMello, A.J. Microfluidics: Exploiting elephants in the room. Nature 464, 839840 (2010).
  8. Becker, H. & Locascio, L.E. Polymer microfluidic devices. Talanta 56, 267287 (2002).
  9. Linder, V., Sia, S.K. & Whitesides, G.M. Reagent-loaded cartridges for valveless and automated fluid delivery in microfluidic devices. Anal. Chem. 77, 6471 (2005).
  10. Oh, K.W. & Ahn, C.H. A review of microvalves. J. Micromech. Microeng. 16, R13R39 (2006).
  11. Thorsen, T., Maerkl, S.J. & Quake, S.R. Microfluidic large-scale integration. Science 298, 580584 (2002).
  12. Sia, S.K., Linder, V., Parviz, B.A., Siegel, A. & Whitesides, G.M. An integrated approach to a portable and low-cost immunoassay for resource-poor settings. Angew. Chem. Int. Ed. 16, 498502 (2004).
  13. Nam, J.M., Thaxton, C.S. & Mirkin, C.A. Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins. Science 301, 18841886 (2003).
  14. Sia, S.K., Linder, V., Parviz, B.A., Siegel, A. & Whitesides, G.M. An integrated approach to a portable and low-cost immunoassay for resource-poor settings. Angew. Chem. Int. Edn Engl. 43, 498502 (2004).
  15. Laksanasopin, T. et al. Microfluidic point-of-care diagnostics for resource-poor environments. Proc. SPIE 7318, 10571059 (2009).
  16. Parsa, H. et al. Effect of volume- and time-based constraints on capture of analytes in microfluidic heterogeneous immunoassays. Lab Chip 8, 20622070 (2008).
  17. Patakfalvi, R., Papp, S. & Dekany, I. The kinetics of homogeneous nucleation of silver nanoparticles stabilized by polymers. J. Nanopart. Res. 9, 353364 (2007).
  18. Miranda, A.E., Alves, M.C., Neto, R.L., Areal, K.R. & Gerbase, A.C. Seroprevalence of HIV, hepatitis B virus, and syphilis in women at their first visit to public antenatal clinics in Vitoria, Brazil. Sex. Transm. Dis. 28, 710713 (2001).
  19. De Cock, K.M. et al. Prevention of mother-to-child HIV transmission in resource-poor countries — translating research into policy and practice. J. Am. Med. Assoc. 283, 11751182 (2000).
  20. Peeling, R.W., Mabey, D., Herring, A. & Hook, E.W. Why do we need quality-assured diagnostic tests for sexually transmitted infections? Nat. Rev. Microbiol. 4, S7S19 (2006).
  21. Mylonakis, E., Paliou, M., Lally, M., Flanigan, T.P. & Rich, J.D. Laboratory testing for infection with the human immunodeficiency virus: Established and novel approaches. Am. J. Med. 109, 568576 (2000).
  22. Herring, A. et al. Evaluation of rapid diagnostic tests: syphilis. Nat. Rev. Microbiol. 4, S33S40 (2006).
  23. Beelaert, G. et al. Comparative evaluation of eight commercial enzyme linked immunosorbent assays and 14 simple assays for detection of antibodies to HIV. J. Virol. Methods 105, 197206 (2002).
  24. Banoo, S. et al. Evaluation of diagnostic tests for infectious diseases: general principles. Nat. Rev. Microbiol. 4, S20S32 (2006).
  25. Gatarayiha, J.P. et al. Rwanda Demographic and Health Survey 2005. Institut National de la Statistique du Rwanda (INSR) and ORC Macro: Calverton, Maryland, USA (2006).
  26. Posthuma-Trumpie, G.A., Korf, J. & van Amerongen, A. Lateral flow (immuno) assay: its strengths, weaknesses, opportunities and threats. A literature survey. Anal. Bioanal. Chem. 393, 569582 (2009).
  27. Gray, R.H. et al. Limitations of rapid HIV-1 tests during screening for trials in Uganda: diagnostic test accuracy study. BMJ 335, 188190 (2007).
  28. Klarkowski, D.B. et al. The evaluation of a rapid in situ HIV confirmation test in a programme with a high failure rate of the WHO HIV two-test diagnostic algorithm. PLOS One 4, e4351 (2009).
  29. Pavie, J. et al. Sensitivity of five rapid HIV tests on oral fluid or finger-stick whole blood: a real-time comparison in a healthcare setting. PLOS One 5, e11581 (2010).
  30. Jani, I.V., Janossy, G., Brown, D.W.G. & Mandy, F. Multiplexed immunoassays by flow cytometry for diagnosis and surveillance of infectious diseases in resource-poor settings. Lancet Infect. Dis. 2, 243250 (2002).
  31. Yager, P., Domingo, G.J. & Gerdes, J. Point-of-care diagnostics for global health. Annu. Rev. Biomed. Eng. 10, 107144 (2008).
  32. Martinez, A.W. et al. Simple telemedicine for developing regions: camera phones and paper-based microfluidic devices for real-time, off-site diagnosis. Anal. Chem. 80, 36993707 (2008).
  33. Daar, A.S. et al. Top ten biotechnologies for improving health in developing countries. Nat. Genet. 32, 229232 (2002).
  34. Mabey, D., Peeling, R.W., Ustianowski, A. & Perkins, M.D. Diagnostics for the developing world. Nat. Rev. Microbiol. 2, 231240 (2004).
  35. Aledort, J.E. et al. Reducing the burden of sexually transmitted infections in resource-limited settings: the role of improved diagnostics. Nature 444 (suppl. 1), 5972 (2006).

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Author information

Affiliations

  1. Department of Biomedical Engineering, Columbia University, New York, New York, USA.

    • Curtis D Chin,
    • Tassaneewan Laksanasopin,
    • Yuk Kee Cheung,
    • Hesam Parsa,
    • Jennifer Wang,
    • Hannah Moore,
    • Robert Rouse &
    • Samuel K Sia
  2. Claros Diagnostics, Woburn, Massachusetts, USA.

    • David Steinmiller &
    • Vincent Linder
  3. Rwanda Zambia HIV Research Group, Projet San Francisco, Kigali, Rwanda.

    • Gisele Umviligihozo &
    • Etienne Karita
  4. Projet Ubuzima, Kigali, Rwanda.

    • Lambert Mwambarangwe &
    • Janneke van de Wijgert
  5. Mailman School of Public Health, International Center for AIDS Care and Treatment Programs, Columbia University, New York, New York, USA.

    • Sarah L Braunstein,
    • Ruben Sahabo,
    • Jessica E Justman &
    • Wafaa El-Sadr
  6. Academic Medical Center of the University of Amsterdam, Department of Internal Medicine, Center for Poverty-related Communicable Diseases and Center for Infection and Immunity, Amsterdam, The Netherlands.

    • Janneke van de Wijgert

Contributions

S.K.S. initiated the study; C.D.C. and S.K.S. designed and conducted the study; C.D.C., T.L., J.W., H.M. and R.R. performed microfluidic immunoassays at Columbia; Y.K.C. developed the compact reader; D.S. and V.L. advised on assay development and provided materials and reagents; H.P. performed computational analysis; L.M. performed reference testing of clinical samples; S.L.B., J.v.d.W., R.S., J.E.J. and W.E.-S. acquired clinical samples and assisted with field studies; C.D.C. and T.L. performed microfluidic immunoassays in Rwanda; C.D.C., T.L. and S.K.S. analyzed data; C.D.C. and S.K.S. wrote the paper; all co-authors edited the paper.

Competing financial interests

S.K.S. is a co-founder of Claros Diagnostics.

Corresponding author

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Supplementary information

PDF files

  1. Supplementary Text and Figures (5M)

    Supplementary Figures 1–7, Supplementary Tables 1–9 and Supplementary Methods

Movies

  1. Supplementary Movie 1 (10M)

    Movie of HIV-syphilis duplex test (complete assay). Time lapse over 20 minutes (1200 s) for two duplex immunoassays, one with a sample which is negative for HIV antibodies and positive for syphilis antibodies (top) and another with a sample which is positive for HIV antibodies and negative for syphilis antibodies (bottom). Meandering zones are functionalized with HIV antigen (left), syphilis antigen (middle), and anti-goat IgG antibody (right, positive control) as described in Supplementary Methods.

  2. Supplementary Movie 2 (2M)

    Movie of whole blood passing through microchannel. The mChip can test whole blood samples without pre-processing or clogging of microchannels.

Additional data