Point-of-care multiplexed single-cell protein secretion analysis based on tyramide signal amplification

Secreted proteins are critical mediators in various biological systems, including the immune system, nervous system, and tumor microenvironment. Understanding profound cellular functional heterogeneity in secretion requires multiplexed secreted protein analysis at the single-cell level. However, most single-cell protein secretion analysis technologies rely on bulky, expensive instruments, preventing widespread use. Herein, we proposed using a portable and low-cost desktop scanner to detect multiplexed proteins secreted from single cells. Gold nanoparticle-enhanced silver staining and tyramide signal amplification are combined to construct a two-step amplification for single-cell secretion assay detection. We used this approach to investigate the heterogeneous immune response of macrophages at the single-cell level, which revealed that only a minority of cells could secrete 10-fold more MCP-1 and TNF-alpha than the averaged secretion. The reported platform eliminates bulky equipment for single-cell secretion analysis, making it affordable for basic and clinical research.