Annals of Biomedical Engineering

, Volume 42, Issue 11, pp 2303–2313 | Cite as

Immunological Analyses of Whole Blood via “Microfluidic Drifting” Based Flow Cytometric Chip

  • Ahmad Ahsan Nawaz
  • Ruth Helmus Nissly
  • Peng Li
  • Yuchao Chen
  • Feng Guo
  • Sixing Li
  • Yasir M. Shariff
  • Arooj Nawaz Qureshi
  • Lin Wang
  • Tony Jun HuangEmail author


Cost-effective, high-performance diagnostic instruments are vital to providing the society with accessible, affordable, and high-quality healthcare. Here we present an integrated, “microfluidic drifting” based flow cytometry chip as a potential inexpensive, fast, and reliable diagnostic tool. It is capable of analyzing human blood for cell counting and diagnosis of diseases. Our device achieves a throughput of ~3754 events/s. Calibration with Flow-Check calibration beads indicated good congruency with a commercially available benchtop flow cytometer. Moreover, subjection to a stringent 8-peak rainbow calibration particle test demonstrated its ability to perform high-resolution immunological studies with separation resolution of 4.28 between the two dimmest fluorescent populations. Counting accuracy at different polystyrene bead concentrations showed strong correlation (r = 0.9991) with hemocytometer results. Finally, reliable quantification of CD4+ cells in healthy human blood via staining with monoclonal antibodies was demonstrated. These results demonstrate the potential of our microfluidic flow cytometry chip as an inexpensive yet high-performance point-of-care device for mobile medicine.


Microfluidics Flow cytometry Point of care Diagnostics CD4+ counting Human blood analysis Mobile medicine 



We thank Joseph Rufo and Adem Ozcelik for helpful discussions. This research was supported by the National Institutes of Health (NIH) Director’s New Innovator Award (1DP2OD007209-01), National Science Foundation, and the Penn State Center for Nanoscale Science (MRSEC) under grant DMR-0820404. Components of this work were conducted at the Penn State node of the NSF-funded National Nanotechnology Infrastructure Network (NNIN).


  1. 1.
    Chen, Y., P. Li, P.-H. Huang, Y. Xie, J. D. Mai, L. Wang, N.-T. Nguyen, and T. J. Huang. Rare cell isolation and analysis in microfluidics. Lab Chip 14:626–645, 2014.PubMedCrossRefGoogle Scholar
  2. 2.
    Chen, Y., A. A. Nawaz, Y. Zhao, P.-H. Huang, J. P. McCoy, S. J. Levine, L. Wang, and T. J. Huang. Standing surface acoustic wave (SSAW)-based microfluidic cytometer. Lab Chip 14:916–923, 2014.PubMedCrossRefGoogle Scholar
  3. 3.
    Cheng, X., Y. Liu, D. Irimia, U. Demirci, L. Yang, L. Zamir, W. R. Rodríguez, M. Toner, and R. Bashir. Cell detection and counting through cell lysate impedance spectroscopy in microfluidic devices. Lab Chip 7:746–755, 2007.PubMedCrossRefGoogle Scholar
  4. 4.
    Chin, C. D., Y. K. Cheung, T. Laksanasopin, M. M. Modena, S. Y. Chin, A. A. Sridhara, D. Steinmiller, V. Linder, J. Mushingantahe, G. Umviligihozo, E. Karita, L. Mwambarangwe, S. L. Braunstein, J. van de Wijgert, R. Sahabo, J. E. Justman, W. El-Sadr, and S. K. Sia. Mobile device for disease diagnosis and data tracking in resource-limited settings. Clin. Chem. 59:629–640, 2013.PubMedCrossRefGoogle Scholar
  5. 5.
    Chiu, Y.-J., S. H. Cho, Z. Mei, V. Lien, T.-F. Wu, and Y.-H. Lo. Universally applicable three-dimensional hydrodynamic microfluidic flow focusing. Lab Chip 13:1803–1809, 2013.PubMedCrossRefPubMedCentralGoogle Scholar
  6. 6.
    Eyal, S., and S. R. Quake. Velocity-independent microfluidic flow cytometry. Electrophoresis 23:2653–2657, 2002.PubMedCrossRefGoogle Scholar
  7. 7.
    Goddard, G. R., C. K. Sanders, J. C. Martin, G. Kaduchak, and S. W. Graves. Analytical performance of an ultrasonic particle focusing flow cytometer. Anal. Chem. 79:8740–8746, 2007.PubMedCrossRefGoogle Scholar
  8. 8.
    Godin, J., C.-H. Chen, S. H. Cho, W. Qiao, F. Tsai, and Y.-H. Lo. Microfluidics and photonics for Bio-System-on-a-Chip: a review of advancements in technology towards a microfluidic flow cytometry chip. J. Biophotonics 1:355–376, 2008.PubMedCrossRefPubMedCentralGoogle Scholar
  9. 9.
    Godin, J., V. Lien, and Y.-H. Lo. Demonstration of two-dimensional fluidic lens for integration into microfluidic flow cytometers. Appl. Phys. Lett. 89:061106, 2006.CrossRefGoogle Scholar
  10. 10.
    Godin, J., and Y.-H. Lo. Two-parameter angular light scatter collection for microfluidic flow cytometry by unique waveguide structures. Biomed. Opt. Express 1:1472–1479, 2010.PubMedCrossRefPubMedCentralGoogle Scholar
  11. 11.
    Gossett, D. R., and D. Di Carlo. Particle focusing mechanisms in curving confined flows. Anal. Chem. 81:8459–8465, 2009.PubMedCrossRefGoogle Scholar
  12. 12.
    Gottwald, E., B. Lahni, G. Lüdke, T. Preckel, and C. Buhlmann. Intracellular HSP72 detection in HL60 cells using a flow cytometry system based on microfluidic analysis. Biotechniques 35:358–362, 364, 366–367, 2003.Google Scholar
  13. 13.
    Grafton, M., L. M. Reece, P. P. Irazoqui, B. Jung, H. D. Summers, R. Bashir, and J. F. Leary. Design of a multi-stage microfluidics system for high-speed flow cytometry and closed system cell sorting for cytomics. Proc. SPIE 6859:1–10, 2008.Google Scholar
  14. 14.
    Jayat, C., and M. H. Ratinaud. Cell cycle analysis by flow cytometry: principles and applications. Biol. Cell 78:15–25, 1993.PubMedCrossRefGoogle Scholar
  15. 15.
    Kiesel, P., M. Beck, and N. Johnson. Monitoring CD4 in whole blood with an opto-fluidic detector based on spatially modulated fluorescence emission. Cytometry A 79:317–324, 2011.PubMedCrossRefGoogle Scholar
  16. 16.
    Kummrow, A., J. Theisen, M. Frankowski, A. Tuchscheerer, H. Yildirim, K. Brattke, M. Schmidt, and J. Neukammer. Microfluidic structures for flow cytometric analysis of hydrodynamically focussed blood cells fabricated by ultraprecision micromachining. Lab Chip 9:972–981, 2009.PubMedCrossRefGoogle Scholar
  17. 17.
    Laerum, O. D., and R. Bjerknes. Flow Cytometry in Hematology. London: Academic Press, 1992.Google Scholar
  18. 18.
    Lapsley, M. I., L. Wang, and T. J. Huang. On-chip flow cytometry: where is it now and where is it going? Biomark. Med. 7:75–78, 2013.PubMedCrossRefGoogle Scholar
  19. 19.
    Lee, G.-B., C.-H. Lin, and G.-L. Chang. Micro flow cytometers with buried SU-8/SOG optical waveguides. Sensors Actuators A Phys. 103:165–170, 2003.CrossRefGoogle Scholar
  20. 20.
    Maleki, T., T. Fricke, J. Quesenberry, P. Todd, and J. F. Leary. Point-of-care, portable microfluidic blood analyzer system. Proc. SPIE 8251:1–3, 2012.Google Scholar
  21. 21.
    Mao, X., and T. J. Huang. Microfluidic diagnostics for the developing world. Lab Chip 12:1412–1416, 2012.PubMedCrossRefGoogle Scholar
  22. 22.
    Mao, X., S.-C. S. Lin, C. Dong, and T. J. Huang. Single-layer planar on-chip flow cytometer using microfluidic drifting based three-dimensional (3D) hydrodynamic focusing. Lab Chip 9:1583–1589, 2009.PubMedCrossRefGoogle Scholar
  23. 23.
    Mao, X., S.-C. S. Lin, M. I. Lapsley, J. Shi, B. K. Juluri, and T. J. Huang. Tunable Liquid Gradient Refractive Index (L-GRIN) lens with two degrees of freedom. Lab Chip 9:2050–2058, 2009.PubMedCrossRefGoogle Scholar
  24. 24.
    Mao, X., A. A. Nawaz, S.-C. S. Lin, M. I. Lapsley, Y. Zhao, J. P. McCoy, W. S. El-Deiry, and T. J. Huang. An integrated, multiparametric flow cytometry chip using “microfluidic drifting” based three-dimensional hydrodynamic focusing. Biomicrofluidics 6:24113–241139, 2012.PubMedCrossRefGoogle Scholar
  25. 25.
    Mao, X., J. R. Waldeisen, B. K. Juluri, and T. J. Huang. Hydrodynamically tunable optofluidic cylindrical microlens. Lab Chip 7:1303–1308, 2007.PubMedCrossRefGoogle Scholar
  26. 26.
    Morris, K. Mobile phones connecting efforts to tackle infectious disease. Lancet Infect. Dis. 9:274, 2009.PubMedCrossRefGoogle Scholar
  27. 27.
    Nawaz, A. A., X. Zhang, X. Mao, J. Rufo, S.-C. S. Lin, F. Guo, Y. Zhao, M. Lapsley, P. Li, J. P. McCoy, S. J. Levine, and T. J. Huang. Sub-micrometer-precision, three-dimensional (3D) hydrodynamic focusing via “microfluidic drifting”. Lab Chip 14:415–423, 2014.PubMedCrossRefGoogle Scholar
  28. 28.
    Oakey, J., R. W. Applegate, E. Arellano, D. Di Carlo, S. W. Graves, and M. Toner. Particle focusing in staged inertial microfluidic devices for flow cytometry. Anal. Chem. 82:3862–3867, 2010.PubMedCrossRefPubMedCentralGoogle Scholar
  29. 29.
    Pop-Eleches, C., H. Thirumurthy, J. P. Habyarimana, J. G. Zivin, M. P. Goldstein, D. de Walque, L. MacKeen, J. Haberer, S. Kimaiyo, J. Sidle, D. Ngare, and D. R. Bangsberg. Mobile phone technologies improve adherence to antiretroviral treatment in a resource-limited setting: a randomized controlled trial of text message reminders. AIDS 25:825–834, 2011.PubMedCrossRefPubMedCentralGoogle Scholar
  30. 30.
    Shapiro, H. M. Practical Flow Cytometry. New York: Wiley-Liss, 2003.CrossRefGoogle Scholar
  31. 31.
    Shapiro, H. M., and N. G. Perlmutter. Violet laser diodes as light sources for cytometry. Cytometry 44:133–136, 2001.PubMedCrossRefGoogle Scholar
  32. 32.
    Shi, J., X. Mao, D. Ahmed, A. Colletti, and T. J. Huang. Focusing microparticles in a microfluidic channel with standing surface acoustic waves (SSAW). Lab Chip 8:221–223, 2008.PubMedCrossRefGoogle Scholar
  33. 33.
    Shi, J., S. Yazdi, S.-C. S. Lin, X. Ding, I.-K. Chiang, K. Sharp, and T. J. Huang. Three-dimensional continuous particle focusing in a microfluidic channel via standing surface acoustic waves (SSAW). Lab Chip 11:2319–2324, 2011.PubMedCrossRefPubMedCentralGoogle Scholar
  34. 34.
    Skommer, J., J. Akagi, K. Takeda, Y. Fujimura, K. Khoshmanesh, and D. Wlodkowic. Multiparameter Lab-on-a-Chip flow cytometry of the cell cycle. Biosens. Bioelectron. 42:586–591, 2013.PubMedCrossRefGoogle Scholar
  35. 35.
    Telford, W. G., T. S. Hawley, and R. G. Hawley. Analysis of violet-excited fluorochromes by flow cytometry using a violet laser diode. Cytometry A 54:48–55, 2003.PubMedCrossRefGoogle Scholar
  36. 36.
    Tudos, A. J., G. J. Besselink, and R. B. Schasfoort. Trends in miniaturized total analysis systems for point-of-care testing in clinical chemistry. Lab Chip 1:83–95, 2001.PubMedCrossRefGoogle Scholar
  37. 37.
    Wang, J., B. Fei, R. L. Geahlen, and C. Lu. Quantitative analysis of protein translocations by microfluidic total internal reflection fluorescence flow cytometry. Lab Chip 10:2673–2679, 2010.PubMedCrossRefPubMedCentralGoogle Scholar
  38. 38.
    Wang, J., Y. Zhan, N. Bao, and C. Lu. Quantitative measurement of quantum dot uptake at the cell population level using microfluidic evanescent-wave-based flow cytometry. Lab Chip 12:1441–1445, 2012.PubMedCrossRefGoogle Scholar
  39. 39.
    Wang, J.-H., L. Cheng, C.-H. Wang, W.-S. Ling, S.-W. Wang, and G.-B. Lee. An integrated chip capable of performing sample pretreatment and nucleic acid amplification for HIV-1 detection. Biosens. Bioelectron. 41:484–491, 2013.PubMedCrossRefGoogle Scholar
  40. 40.
    Wang, S., F. Inci, G. De Libero, A. Singhal, and U. Demirci. Point-of-care assays for tuberculosis: role of nanotechnology/microfluidics. Biotechnol. Adv. 31:438–449, 2013.PubMedCrossRefPubMedCentralGoogle Scholar
  41. 41.
    Wei, F., R. Lam, S. Cheng, S. Lu, D. Ho, and N. Li. Rapid detection of melamine in whole milk mediated by unmodified gold nanoparticles. Appl. Phys. Lett. 96:133702, 2010.PubMedCrossRefPubMedCentralGoogle Scholar
  42. 42.
    Woods, J., and R. Hoffman. Evaluating fluorescence sensitivity on flow cytometers: an overview. Cytometry A 33:256–259, 1998.CrossRefGoogle Scholar
  43. 43.
    Xia, Y., and G. M. Whitesides. Soft Lithography. Annu. Rev. Mater. Sci. 28:153–184, 1998.CrossRefGoogle Scholar
  44. 44.
    Yager, P., G. J. Domingo, and J. Gerdes. Point-of-care diagnostics for global health. Annu. Rev. Biomed. Eng. 10:107–144, 2008.PubMedCrossRefGoogle Scholar
  45. 45.
    Zhao, C., Y. Liu, Y. Zhao, N. Fang, and T. J. Huang. A reconfigurable plasmofluidic lens. Nat. Commun. 4:2305, 2013.PubMedPubMedCentralGoogle Scholar

Copyright information

© Biomedical Engineering Society 2014

Authors and Affiliations

  • Ahmad Ahsan Nawaz
    • 1
  • Ruth Helmus Nissly
    • 2
  • Peng Li
    • 1
  • Yuchao Chen
    • 1
  • Feng Guo
    • 1
  • Sixing Li
    • 3
  • Yasir M. Shariff
    • 4
  • Arooj Nawaz Qureshi
    • 1
  • Lin Wang
    • 5
  • Tony Jun Huang
    • 1
    Email author
  1. 1.Department of Engineering Science and MechanicsThe Pennsylvania State UniversityUniversity ParkUSA
  2. 2.Microscopy and Cytometry Facility, The Huck Institutes of the Life SciencesThe Pennsylvania State UniversityUniversity ParkUSA
  3. 3.Cell and Developmental Biology (CDB) Graduate Program, The Huck Institutes of the Life SciencesThe Pennsylvania State UniversityUniversity ParkUSA
  4. 4.Mechanical Engineering DepartmentTaibah UniversityMadinaSaudi Arabia
  5. 5.Ascent Bio-Nano Technologies Inc.State CollegeUSA

Personalised recommendations