A high-throughput modular microfluidic platform for versatile functional assays at single-cell level

Understanding how individual cells behave is essential for uncovering the complexity of biology and disease, yet most single-cell technologies provide only static molecular snapshots. We present the High-throughput Single-Cell Omni-functional Profiling Engine (HiSCOPE), a microfluidic platform that directly investigates functional dynamics of ≥10,000 single cells. HiSCOPE integrates a hydrodynamic single-cell trapping array with customized modular assay chambers to reproducibly trap single cells, pairs, and trios of cells originating from distinct populations and to support diverse functional assays-including viability, proliferation, cytotoxicity, cytokine secretion, and contact-dependent and -independent cell-cell interactions. Importantly, live-cell retrieval enables downstream analysis of selected cells. We demonstrate HiSCOPE's utility by evaluating leukemia cells' drug susceptibility, profiling natural killer (NK) cell cytotoxicity and cytokine secretion, and dissecting T cell-macrophage crosstalk. Our results reveal striking functional diversity that is masked in bulk assays. HiSCOPE offers a scalable solution for functional profiling of single cells, with broad implications for biology, immunotherapy, and personalized medicine.