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bioRxiv · 10.1101/2025.10.17.682894

Microparticle-Enabled Single Cell Multiparameter Electronic Immunophenotyping for Selective Electroporation

Hoyle, M.·Rudge, J.·Rallapalli, Y.·Sarkar, A.
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Abstract

Electroporation (EP) is one of the leading non-viral intracellular delivery methods used in various applications across research and cell therapy development and manufacturing. Currently widely used bulk EP methods, while they offer scalability, cost efficiency and simplicity, cannot be used for targeted or selective delivery to a defined subset of a input cell population. Here, we present a Microparticle-Enabled Selectively Permeabilizing Impedance Cytometer (ME-SPICy), a microfluidic single-cell EP platform that enables targeted EP of selected cell subpopulations based on their surface markers. Antibody conjugated microparticles (MPs) are used to label selected cell subpopulations within a larger heterogenous sample. Using multifrequency impedance detection, ME-SPICy discriminates, in real-time, non-labeled and labeled cells within the mixed sample as they flow through a 3D printed biconical micro-aperture. This allows for the system to analyze if a cell is a target cell and selectively apply a low voltage (<16 V) for targeted single-cell EP. Simulations and experimental validation demonstrate that MP binding substantially alters cell impedance and phase signature, enabling accurate label-based discrimination. We demonstrated selective EP first using Jurkat cells by targeting either the labeled or non-labeled populations. Then we demonstrated targeted delivery to primary human lymphocytes within peripheral blood mononuclear cells. ME-SPICy achieved high precision, with 98% purity and >5 fold enrichment of lymphocytes in the electroporated cell population. This approach expands the capabilities of EP, offering a promising solution to decrease manufacturing complexity in both research and clinical cell engineering workflows

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Hoyle, M., Rudge, J., Rallapalli, Y., Sarkar, A.. 2025-10-17. Microparticle-Enabled Single Cell Multiparameter Electronic Immunophenotyping for Selective Electroporation. https://doi.org/10.1101/2025.10.17.682894

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