Microfluidic Single Cell Electronic Immunophenotyping using Enzymatic Metallization

Introduction: Current immune-monitoring based point-of-care (POC) diagnostics focus almost exclusively on humoral immunity, such as antibody detection. Tools for monitoring cellular immunity are currently often incompatible with POC diagnostics since they are dependent on bulky and expensive instrumentation such as fluorescence microscopes or flow cytometers which use lasers and photomultipliers for cellular immunophenotyping using fluorophore-labeled antibodies. This impedes their use for cellular diagnostics and monitoring in resource-poor settings for endemic diseases e.g. for antigen-specific T-Cell activation assays for diagnosis of Tuberculosis. 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. Here we develop a device and scheme for microfluidic, in-flow, single-cell electronic immunophenotyping, capable of detecting selectively metallized cells using antibody probe-directed enzymatic metallization. Coupled to machine-learning based analytics, this enables sensitive detection of cells based on surface marker expression as well as intracellular targets such as cytokines (Figure 1 A, D).

Materials and

Methods: Cells were labeled with a biotinylated antibody directed against a selected surface marker, followed by streptavidin-polyHRP. Silver deposition on cell surface was performed by adding enzymatic silver metallization reagents, with an incubation of 30 mins. Control cells were incubated with the wash buffer followed by Streptavidin-PolyHRP. THP1 cells were labeled with anti-CD45 antibodies, and monocyte-depleted PBMCs were stained with anti-CD45, anti- CD19 or anti-CD3 to selectively metallize all cells, B cells or T cells respectively. The device consists of a 3D printed biconical micro-aperture, with a 25µm constriction (Figure 1B, E), sandwiched between electrodes on printed circuit boards (Figure 1C) and assembled with a cell reservoir and tubing connection to a syringe pump (Figure 1F). Each cell is detected in-flow as it passes through the constriction and impedance spectra of each passing cell is measured by a lock-in amplifier at a set of six measurement frequencies (f=45kHz, 150kHz, 750kHz, 1MHz, 2MHz and 5MHz).

Results, Conclusions, and Discussions: Results
Micrographs of control and metallized THP1 cells labeled with anti-CD45 are shown in Figure 2. Single-cell impedance measurement using these cells showed that there is a change in impedance spectra between metallized and non-metallized cells. At 1MHz and above, we observed that highly metallized cells have a negative impedance peak relative to the baseline conductivity of the solution, due to the deposition of silver (Figure 3). Linear Discriminant Analysis (LDA) applied to the impedance spectra, fully visualizes the change with better accuracy (AUC~0.9 between 0% and 100% metallized cells) (Figure 4 A, B). LDA also successfully differentiated different dilutions of CD45-labeled cells, as shown by the confusion matrix (Fig 4 C), where labeled and control cells were mixed before metallization in different ratios. This was further explored using CD3, CD19 and CD45 staining of primary blood mononuclear cells (PBMCs) identifying T cells, B cells and all PBMCs respectively (Figure 5 A, B).

Discussion
We showed that using antibody-directed selective silver metallization of cells, we can detect distinct subpopulations of cells expressing different surface markers using direct electronic detection in a simple 3-D printed microfluidic device without the use of any expensive or bulky optical instrumentation. With further development, this technology can provide enable identification of rare cell populations such as antigen-specific T cells which can be biomarkers of infection of disease state. This workflow can also be modified for intracellular staining to detect cytokine-secreting cells as well (Figure 1B) thus enabling monitoring of functional cellular immunity. Due to its in-flow, high throughput nature, we can process thousands of cells per minute while maintaining single-cell resolution. This device can thus be a low-cost and POC alternative to current state-of-the-art methods for cellular diagnostics such as flow cytometers.

Acknowledgements (Optional): References
[1] Electronic Immunoassay Using Enzymatic Metallization on Microparticles
Josiah Rudge, Madeline Hoyle, Neda Rafat, Alexandra Spitale, Margaret Honan, and Aniruddh Sarkar, ACS Omega 2023 8 (25), 22934-22944 DOI: 10.1021/acsomega.3c01939
[2] N. Rafat, H. Zhang, J. Rudge, Y. N. Kim, S. P. Peddireddy, N. Das, A. Sarkar, Enhanced Enzymatically Amplified Metallization on Nanostructured Surfaces for Multiplexed Point-of-Care Electrical Detection of COVID-19 Biomarkers. Small 2022, 18, 2203309. https://doi.org/10.1002/smll.202203309