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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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.
The authors declare no competing financial interests.
Integrated supplementary information
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.
(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.
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.
(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.
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.
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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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