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Microfluidic differential immunocapture biochip for specific leukocyte counting

Nature Protocols volume 11, pages 714–726 (2016)Cite this article

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Abstract

Enumerating specific cell types from whole blood can be very useful for research and diagnostic purposes—e.g., for counting of CD4 and CD8 T cells in HIV/AIDS diagnostics. We have developed a biosensor based on a differential immunocapture technology to enumerate specific cells in 30 min using 10 μl of blood. This paper provides a comprehensive stepwise protocol to replicate our biosensor for CD4 and CD8 cell counts. The biochip can also be adapted to enumerate other specific cell types such as somatic cells or cells from tissue or liquid biopsies. Capture of other specific cells requires immobilization of their corresponding antibodies within the capture chamber. Therefore, this protocol is useful for research into areas surrounding immunocapture-based biosensor development. The biosensor production requires 24 h, a one-time cell capture optimization takes 6–9 h, and the final cell counting experiment in a laboratory environment requires 30 min to complete.

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Figure 1: The schematics of the differential immunocapture biochip29.
Figure 2: Electronic setup of the biochip.
Figure 3: Scatter plots of opacity versus pulse amplitude at low frequency29.
Figure 4: Capture chamber for CD4+ and CD8+ T cell capture.
Figure 5: Illustration of capture efficiency optimization29.
Figure 6: Percentage of error in specific cell counts.
Figure 7: Reagent loading mechanism into the biochip.
Figure 8: Anticipated results29.

References

  1. Global HIV/AIDS Response. Epidemic update and health sector progress towards Universal Access. World Health Organization (2011).

  2. Damhorst, G., Watkins, N.N. & Bashir, R. Micro and nanotechnology for HIV/AIDS diagnostics in resource-limited settings. IEEE Trans. Biomed. Eng. 60, 715–726 (2013).

    Article  Google Scholar 

  3. Damhorst, G., Murtagh, M., Rodriguez, W.R. & Bashir, R. Microfluidics and nanotechnology for detection of global infectious diseases. Proc. IEEE 103, 150–160 (2015).

    Article  CAS  Google Scholar 

  4. Rowley, C.F. Developments in CD4 and viral load monitoring in resource-limited settings. Clin. Infect. Dis. 58, 407–412 (2014).

    Article  Google Scholar 

  5. Pahwa, S. et al. CD4+/CD8+ T cell ratio for diagnosis of HIV-1 infection in infants: women and infants transmission study. Pediatrics 122, 331–339 (2008).

    Article  Google Scholar 

  6. Taylor, J.M., Fahey, J.L., Detels, R. & Giorgi, J.V. CD4 percentage, CD4 number, and CD4:CD8 ratio in HIV infection: which to choose and how to choose. J. AIDS 2, 114–124 (1989).

    CAS  Google Scholar 

  7. Wang, J.H., Wang, C.H., Lin, C.C., Lei, H.Y. & Lee, G.B. An integrated microfluidic system for counting of CD4+/CD8+ T lymphocytes. Microfluid. Nanofluid. 10, 531–541 (2011).

    Article  CAS  Google Scholar 

  8. Gohring, J.T. & Fan, X. Label free detection of CD4+ and CD8+ T lymphocytes with the optofluidic ring resonator biosensor. Sensors 10, 5798–5808 (2010).

    Article  CAS  Google Scholar 

  9. Carey, J.L., McCoy, J.P. Jr. & Keren, D.F. Flow Cytometry in Clinical Diagnosis 4th edn. American Society for Clinical Pathology, 2007.

  10. Srithanaviboonchai, K. et al. Novel low-cost assay for the monitoring of CD4 counts in HIV-infected individuals. J. Acquir. Immune Defic. Syndr. 47, 135–139 (2008).

    Article  Google Scholar 

  11. Crowe, S., Turnbull, S., Oelrichs, R. & Dunne, A. Monitoring of human immunodeficiency virus infection in resource-constrained countries. Clin. Infect. Dis. 37, S25–S35 (2003).

    Article  Google Scholar 

  12. Coulter, W.H. Means for counting particles suspended in a fluid. US Patent 2656508 (1953).

  13. Koch, M., Evans, A. & Brunnschweiler, A. Design and fabrication of a micromachined Coulter counter. J. Micromech. Microeng. 9, 159–161 (1999).

    Article  CAS  Google Scholar 

  14. Holmes, D. et al. Leukocyte analysis and differentiation using high speed microfluidic single cell impedance cytometry. Lab Chip 9, 2881–2889 (2009).

    Article  CAS  Google Scholar 

  15. Hassan, U. Microfluidic sensor for white blood cell counting and flow metering. M.S. thesis, Electrical & Computer Engineering, University of Illinois at Urbana-Champaign (2013).

  16. Ellappan, P. & Sundararaian, R. A simulation study of a electrical model of a biological cell. J. Electrostat. 63, 297–309 (2005).

    Article  Google Scholar 

  17. Hughes, M.P. AC electrokinetics: applications for nanotechnology. Nanotechnology 11, 124–132 (2000).

    Article  CAS  Google Scholar 

  18. Gawad, S., Schild, L. & Renaud, P. Micromachined impedance spectroscopy flow cytometer for cell analysis and particle sizing. Lab Chip 1, 76–82 (2001).

    Article  CAS  Google Scholar 

  19. Pierzchalski, A., Hebeisen, M., Mittag, A., Berardino, M.D. & Tarnok, A. Label-free single cell analysis with a chip-based impedance flow cytometer. Proc. SPIE 75681B doi:10.1117/12.840865 (2010).

  20. Cheung, K.C. et al. Microfluidic impedance-based flow cytometry. Cytometry A 77, 648–666 (2010).

    Article  Google Scholar 

  21. Cheng, X. et al. Enhancing the performance of a point-of-care CD4+ T cell counting microchip through monocyte depletion for HIV/AIDS diagnostics. Lab Chip 9, 1357–1364 (2009).

    Article  CAS  Google Scholar 

  22. Beck, M. et al. On-chip sample preparation by controlled release of antibodies for simple CD4 counting. Lab Chip 12, 167–173 (2012).

    Article  CAS  Google Scholar 

  23. Smith, Z.J. et al. Single-step preparation and image based counting of minute volumes of human blood. Lab Chip 14, 3029 (2014).

    Article  CAS  Google Scholar 

  24. Boyle, D.S., Hawkins, K.R., Steele, M.S., Singhal, M. & Cheng, X. Emerging technologies for point-of-care CD4 T-lymphocyte counting. Trends Biotechnol. 30, 1 (2012).

    Article  Google Scholar 

  25. Schade-Kampmann, G., Huwiler, A., Hebeisen, M., Hessler, T. & Di Berardino, M. On-chip non-invasive and label-free cell discrimination by impedance spectroscopy. Cell Prolif. 41, 830–840 (2008).

    Article  CAS  Google Scholar 

  26. Cheng, X. et al. Cell detection and counting through cell lysate impedance spectroscopy in microfluidic devices. Lab Chip 7, 746–755 (2007).

    Article  CAS  Google Scholar 

  27. Watkins, N.N., Venkatesan, B.M., Toner, M., Rodriguez, W. & Bashir, R. A robust electrical microcytometer with 3-dimensional hydrofocusing. Lab Chip 9, 3177–3184 (2009).

    Article  CAS  Google Scholar 

  28. Watkins, N.N. et al. microfabricated electrical differential counter for the selective enumeration of CD4+ T lymphocytes. Lab Chip 11, 437–447 (2011).

    Article  Google Scholar 

  29. Watkins, N.N. et al. Microfluidic CD4+ and CD8+ T lymphocyte counters for point-of-care HIV diagnostics using whole blood. Sci. Transl. Med. 5, 214ra170 (2013).

    Article  Google Scholar 

  30. Hassan, U., Watkins, N., Edwards, C. & Bashir, R. Flow metering characterization within an electrical cell counting microfluidic device. Lab Chip 14, 1469 (2014).

    Article  CAS  Google Scholar 

  31. Hassan, U. & Bashir, R. Coincidence detection of heterogeneous cell populations from whole blood with coplanar electrodes in a microfluidic impedance cytometer. Lab Chip 14, 4370–4381 (2014).

    Article  CAS  Google Scholar 

  32. Hassan, U. & Bashir, R. Electrical cell counting process characterization in a microfluidic impedance cytometer. Biomed. Microdevices 16, 697–704 (2014).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank A. Vaid at Champaign-Urbana Public Health District (CUPHD) for providing the HIV-infected blood samples; and C. Edwards, L. Orlandic and C. Yang for PDMS device fabrication. The authors acknowledge the support of Center for Integration of Medicine and Innovative Technology (CIMIT)'s Point-of-Care Technology Center in Primary Care (POCTRN) Grant and funding from University of Illinois at Urbana-Champaign.

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Author notes

  1. Nicholas N Watkins

    Present address: Present address: Lawrence Livermore National Laboratory, Livermore, California, USA.,

Authors and Affiliations

  1. Department of Electrical and Computer Engineering, University of Illinois at Urbana-Champaign, William L. Everitt Laboratory, Urbana, Illinois, USA

    Umer Hassan, Nicholas N Watkins & Rashid Bashir

  2. Micro and Nanotechnology Laboratory, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

    Umer Hassan, Bobby Reddy Jr, Gregory Damhorst & Rashid Bashir

  3. Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

    Umer Hassan, Bobby Reddy Jr, Gregory Damhorst & Rashid Bashir

  4. Biomedical Research Center, Carle Foundation Hospital, Urbana, Illinois, USA

    Umer Hassan, Bobby Reddy Jr, Gregory Damhorst & Rashid Bashir

Authors
  1. Umer Hassan
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  2. Nicholas N Watkins
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  4. Gregory Damhorst
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Contributions

U.H., N.N.W., B.R. and R.B. designed the study. U.H., N.N.W. and G.D. performed the experiments. U.H. wrote the paper and R.B. edited and proofread the paper.

Corresponding author

Correspondence to Rashid Bashir.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Differential counting Biochip

Image of the integrated biochip for specific leukocyte counting by differential immuno-capture technique. The footprint of the chip is 3 cm x 4 cm.

Supplementary Figure 2 Schematic of the electrical counter module.

(a) Schematic shows both entrance and exit counters. Holes can be punched in circular spots to be inlets and outlets of counters. The spacing and width of the counter pads are 2500 μm and 1500 μm respectively. (b) Shows the zoomed-in dotted region in (a). The top view of the counter with dimensions of the electrodes is shown. The width of the electrodes and the spacing in between the electrodes is 15 μm. (c) The cross-sectional view of the counter. The counting height is 15 μm, however rest of the fluidic channel height is 115 μm.

Supplementary Figure 3 Electrical cell pulse.

The counting channel cross-sectional view showing the cell flowing over the electrodes. The corresponding voltage pulse generated as a result of a cell transitioning through the counting channel over the electrodes. Adapted from Hassan, U. & Bashir, R. Coincidence detection of heterogeneous cell populations from whole blood with coplanar electrodes in a microfluidic impedance cytometer. Lab Chip 14, 4370–4381 (2014).

Supplementary Figure 4 The voltage pulses generated as a result of a cell passage over the electrodes through the counting channel.

Supplementary Figure 5 The simulation represents the shear stress along the posts with 3D representation of the capture chamber.

Supplementary Figure 6 Flow cytometry analysis of the labeled cells.

(a) The Side scatter (SSC) vs. CD45 plot shows a distinct population of lymphocytes, which can be gated out in the plot. (b) The gated lymphocyte population in (a) is plotted in a CD3 vs. CD4 fluorescent plot. The top right quadrant gives the CD4 lymphocytes. Similarly, bottom right gives CD4 monocytes.

Supplementary Figure 7 Schematic of individual modules of the biochip.

1. The cell-lysing module. Quenching, lysing and blood will be infused at a, b and c inlets ports respectively as shown. 2. The electrical counters module. 3. Capture chamber module. The inlet and outlet ports of each module are shown too.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Table 1 (PDF 1297 kb)

Supplementary Data

AutoCAD design file (BiochipDesign_NP.dwg); Lock-in amplifier settings file (lockin.zicfg); LabVIEW file for Data Acquisition (Counter_Differential_NP.vi) (ZIP 18372 kb)

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Hassan, U., Watkins, N., Reddy, B. et al. Microfluidic differential immunocapture biochip for specific leukocyte counting. Nat Protoc 11, 714–726 (2016). https://doi.org/10.1038/nprot.2016.038

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