Microfluidic differential immunocapture biochip for specific leukocyte counting

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.

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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.

Author information

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.

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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