MICROSCALE ELECTRONIC MEASUREMENT OF FUNCTIONAL CELLULAR IMMUNITY FOR POINT-OF-CARE DIAGNOSTICS

Introduction: Current point-of-care (POC) diagnostics for detection and monitoring of infectious diseases often focus on detecting the pathogens (e.g. microscopy, nucleic acid or antigen-based tests) or the host humoral immune response against it (e.g. antibodies), missing out on host cellular immunity which can offer a more sensitive and differentiated readout and therefore insight into the underlying disease state. Methods for monitoring T-cell immunity include measurement of secreted cytokines in ELISAs or single-cell scale measurement via enzyme-linked immunosorbent spot (ELISpot) assay or intracellular cytokine staining (ICS) followed by flow cytometry, all of which are infrastructure-hungry, costly, and have high sample requirements, making them incompatible with POC diagnostics, especially for resource-poor settings. Electronic detection offers an inexpensive alternative to optical instrumentation. We have earlier developed tools for electronic antibody detection on beads [1] and microelectrodes [2] using probe-directed enzymatic metallization. We develop here a method for direct electronic detection of immune cells and functional cellular immunity using probe-directed silver metallization on cells and intracellular or secreted cytokines captured on micro-interdigitated electrode (µIDEs) chips. We demonstrate this technique for a direct impedance readout of interferon-gamma (IFN-γ), a common tuberculosis (TB) biomarker, in stimulated peripheral blood mononuclear cells (PBMCs). This microscale electronic detection technology drastically decreases the sample requirements and necessary equipment to measure T-cell activity and paves the way for POC diagnostics for monitoring cellular immunity.

Materials and

Methods: To demonstrate probe-directed silver metallization and electronic detection of cells, THP-1 cells, used as a model, were labelled using biotinylated Anti-CD-45 antibody, then HRP-Streptavidin (Figure 1). Control cells were incubated in wash buffer, then HRP-Streptavidin. Silver deposition was performed using enzymatic metallization reagents for 30 minutes. Finally, cells were captured on a microchip containing an array of gold micro-interdigitated electrodes (µIDEs) (electrode width & gap: 5µm) [1]. A thin layer (0.254mm) of polydimethylsiloxane (PDMS) with laser-cut 4mm diameter wells was reversibly assembled on the chip. 7.5µL of the final cell suspension was pipetted into the wells, incubated for 10 minutes, dipped in DI water to remove excess enzymatic metallization substrate, and air dried. Multi-frequency impedance measurements were performed using a NF ZM2376 LCR meter.

For detection of intracellular IFN-γ, thawed fresh-frozen primary blood mononuclear cells (PBMCs), isolated earlier from human blood, were stimulated (PMA-Ionomycin) overnight, and after fixing and permeabilizing, stained with anti-IFN-γ HRP. Cells were metallized as above, and impedance measurements were performed (Figure 3).

For the electronic IFN-γ detection assay, gold nanoparticles were immobilized on µIDEs with a 10µm electrode width and gap. Anti-IFN-γ antibody was added to wells and incubated overnight at 4°C. Following blocking, washing, and drying, a serial dilution of recombinant IFN-γ was added. Target antibody and signal amplification probes were added with wash steps in between. Following addition of enzymatic metallization substrates, impedance measurements were performed. Optical readout of silver was obtained using a commercial office scanner.

Results, Conclusions, and Discussions: Figure 1 shows a schematic of the workflow including probe-directed metallization of cells, capture on µIDE chips and impedance measurement. Initial results using THP-1 cells stained with anti-CD45 followed by silver metallization show that the differences in metallized and non-metallized cells can be visualized under phase-contrast microscopy as well as SEM imaging (Figure 2a-d). In Figure 2a, metallized cells appear dark and opaque and in Figure 2b, a metallized cell exhibits characteristic “rosette” morphology of silver nanoparticles as seen in previous work with antibody-directed metallization [1]. Non-metallized cells appeared transparent and did not exhibit silver formation (Figures 2c,d). Impedance measurement showed that metallized cells (anti-CD45+) were consistently conducting by bridging the 5µm inter-electrode gaps while control cells (anti-CD45-) were consistently not conducting (Figure 2e). A serial dilution of metallized cells was performed and after capture on the chip, this demonstrates the decrease in impedance with increasing number of metallized cells (Figure 2f) with a low ( < 1kOhms) impedance measured up to ~15 cells captured while ~8 cells were also distinguishable from control cells. To measure intracellular cytokines from PBMCs, the metallization protocol was modified as shown in Figure 3b. Stimulated PBMCs intracellularly metallized for IFN-γ exhibited low impedance readings and were distinguishable from unstimulated cells. With recombinant IFN-γ reagents, for the detection of extracellular cytokine secretion, we observed a limit of detection (LOD) of 88pg/mL before lower concentrations become non-conducting (~10MOhms) (Figure 4d). In Figure 4f, optical darkness readout of the silver provided a LOD of 11pg/mL indicating that further optimization can be performed to improve the electronic LOD.

We show that using probe-directed silver metallization with cells and intracellular or secreted cytokines captured on µIDEs chips, we can directly electronically detect specific cell surface receptors or cytokine-secreting cells from a small sample volume ( < 10µL) without the use of any intermediate optical instrumentation. Detection of low numbers ( < 10) of cells and an IFN-γ measurement is demonstrated which, with further development and optimization, can be used to replace current resource-intensive IFN-γ release assays (IGRA) in TB diagnosis. Future work will involve integrating sample preparation [3] and testing with clinical samples.

Acknowledgements (Optional): [1] Rudge, J., Hoyle, M., Rafat, N., Spitale, A., Honan, M., & Sarkar, A. (2023). Electronic Immunoassay Using Enzymatic Metallization on Microparticles. ACS Omega, 8(25), 22934–22944. https://doi.org/10.1021/acsomega.3c01939

[2] Rafat N, Zhang H, Rudge J, et al. Enhanced Enzymatically Amplified Metallization on Nanostructured Surfaces for Multiplexed Point-of-Care Electrical Detection of COVID-19 Biomarkers. Small. 2022;18(49):e2203309. doi:10.1002/smll.202203309

[3] Sarkar A, Hou H W, Mahan A E, Han J, Alter G, Multiplexed Affinity-Based Separation of Proteins and Cells Using Inertial Microfluidics, Scientific Reports. 2016; 6:23589