Development of a multiplex fully automated assay for rapid quantification of CD4+ T cells from whole blood

Abstract

The development of cost-effective and rapid assays for the accurate counting of CD4 cells has remained prime focus for disease management. The lack of such assays has severely affected people living in resource-limited disease prevalent areas. CD4 count information plays a vital role in the effective management of HIV disease. There is an unmet need to develop rapid, cost-effective, portable and user-friendly point-of-care (POC) disease diagnostic platform technology for CD4⁺ T cell counting. Here, we have developed a flow-free magnetic actuation platform that uses antibody-coated magnetic beads to efficiently capture CD4⁺ T cells from a 30 μL drop of whole blood. On-chip cell lysate electrical impedance spectroscopy has been utilized to quantify the isolated CD4 cells. The developed assay has a limit of detection of 25 cells per μL and provides accurate CD4 counts in the range of 25-800 cells per μL. The whole immunoassay along with the enumeration process is very rapid and provides CD4 quantification results within 5 min time frame. The assay does not require off-chip sample preparation steps and minimizes human involvement to a greater extent. The developed impedance-based immunoassay has potential to significantly improve the CD4 enumeration process especially for POC settings.

Keywords: Antiretroviral therapy; CD4+ T cells; Human immunodeficiency virus; Rapid immunoassays; Resource-constrained settings.

Figure 1.

Pictorial representation of specific capture of CD4 cells from whole blood with antibody conjugated magnetic beads and their subsequent washing steps.

Figure 2.

Overview of the screen-printing process. (a) Emulsion coated screen. (b) Printing of sensors on transparency sheet. (c) Final version of screen ready for printing various biosensors. (d) 5 IDE based microfluidic device printed on paper. (e) 6 IDE based microfluidic device printed on paper. (f) 6 IDE printed on PMMA.

Figure 3.

(a) Schematic showing the conjugation of antibody to neutravidin coated (b) Flow-free fully automatic magnetic actuation platform loaded with a disposable microfluidic device with screen-printed electrodes.

Figure 4.

(a) Magnitude of impedance Vs frequency graphs for various concentrations of CEM cells mixed in buffer solution. (b) Magnitude of electrical impedance for various concentrations of CEM cells at 10 kHz

Figure 5.

(a) Change in the magnitude of impedance vs frequency graphs for various concentrations of CD4+ T cells. (b) Change in the magnitude of electrical impedance for various concentrations of CD4+ T cells at 10 kHz. (c) Images of the microfluidic chips after the successful completion of the immunoassay.