iSECRETE: Integrating Microfluidics and DNA Proximity Amplification for Synchronous Single‐Cell Activation and IFN‐γ Secretion Profiling

Cytokines, crucial in immune modulation, impact disease progression when their secretion is dysregulated. Existing methods for profiling cytokine secretion suffer from time‐consuming and labor‐intensive processes and often fail to capture the dynamic nature of immune responses. Here, iSECRETE, an integrated platform that enables synchronous cell activation, wash‐free, and target‐responsive protein detection for single‐cell IFN‐γ cytokine secretion analysis within 30 min at room temperature is presented. By incorporating a DNA proximity assay (DPA) into a multifunctional microfluidic system, one‐pot homogenous cytokine signal amplification, with a limit of detection of ≈50 secreted molecules per cell is achieved. iSECRETE can robustly handle various sample types that are shown. Two distinct immune activation assay modalities are demonstrated on iSECRETE. Finally, the detection of single‐cell IFN‐γ secretion as an activation hallmark of chimeric antigen receptor T cells within 6 h of exposure to cancer targets is shown. iSECRETE represents the fastest single‐cell sample‐to‐result cytokine secretion assay to date, providing a powerful tool for advancing the understanding of biological phenotypes, functions, and pathways under in vivo‐like conditions. iSECRETE combines the advantages of integrated microfluidics and isothermal DNA proximity assay for a one‐step homogeneous single‐cell cytokine release assay. The assay can analyze whole blood or undiluted cell culture samples and detect activation‐induced cytokine release in live immune cells. iSECRETE is performed on chimeric antigen receptor (CAR) T cells cytotoxicity assay showing CAR T functional cytokine secretion.