A combinatorial screening platform for in situ gene delivery to adherent cells via digital microfluidics and flexible...

Conventional electroporation techniques are often limited by low throughput, high reagent consumption, and, critically, the requirement for cell suspension. The latter necessitates enzymatic detachment, subjecting cells to significant physiological stress. To address these challenges, we present a combinatorial screening platform, the programmable digital electroporation system (PDES), which integrates digital microfluidics (DMF) with disposable, flexible interdigitated electrode (IDE) arrays. To enhance system versatility and optical monitoring, we implemented two distinct top-electrode configurations: a flexible polyimide electrode and a large-area transparent quartz electrode. This design enables the fully automated generation of reagent gradients and parallel in situ electroporation of adherent cells under low-voltage conditions. By decoupling fluidic actuation from electrical stimulation, the system facilitates high-throughput parameter optimization while reducing reagent consumption by an order of magnitude. We validated the platform by automatically screening eGFP mRNA transfection conditions, achieving a transfection efficiency of over 90% with cell viability exceeding 95%. Furthermore, we demonstrated the platform's utility in reprogramming human dermal fibroblasts into induced pluripotent stem cells (iPSCs) via circular RNA (circRNA) delivery. Crucially, the PDES performs this process entirely in situ, circumventing the detrimental effects of enzymatic digestion (e.g., trypsinization) inherent to bulk electroporation, the step that often compromises viability and efficiency in sensitive cell types. This work offers a powerful, automated solution for non-viral gene editing, particularly for engineering fragile cells in regenerative medicine and synthetic biology.