High‐Throughput Single‐Cell Biochip System for Functional Interrogation of Protein Interactions in Living Cells

ABSTRACT In live cells, investigating protein–protein interactions (PPIs) with single‐cell resolution provides critical insights into endogenous regulatory mechanisms underlying heterogeneous cellular behaviors, such as migration and proliferation. Conventional live‐cell PPI detection techniques, which rely on the delivery of high‐molecular‐weight fluorescent protein‐tagged plasmids into large cell populations, are constrained by low delivery efficiency and inadequate single‐cell analytical capability. To address these, we report “Pixar,” for p rotein i nteraction e xa mination and r eal‐time behavior monitoring. This platform enables efficient PPI detection in living cells through focused electric field‐based delivery of peptide‐tagged protein plasmids and specific probes (both delivery efficiency and cell viability > 90%), avoiding the steric interference associated with conventional bulky labeling approaches. Its high‐throughput single‐cell capture array allows for the dynamic profiling of the tumor‐associated AKT‐mTOR protein interaction in regulating heterogeneous cell migration and proliferation across thousands of individual cells. The Pixar system establishes a versatile strategy for exploring protein function and cellular behavioral phenotypes with single‐cell precision.