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

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 protein interaction examination and real-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.