Electrochemiluminescence Imaging Digital Immunoassay of Cytokines Secreted by Activated T Cells

High-sensitivity cytokine detection is essential for predicting immunotherapy efficacy and monitoring treatment. Conventional methods such as ELISA suffer from limited sensitivity, while single-molecule immunoassays, although highly sensitive, often require physical compartmentalization, resulting in high cost and limited clinical applicability. Herein, we report a compartmentalization-free electrochemiluminescence (ECL) digital immunoassay based on silica-coated gold nanorod (AuNR@SiO 2 ) for detecting tumor necrosis factor-α (TNF-α) secreted by activated T cells. The AuNR@SiO 2 nanoparticles served as efficient nanoaccelerators, increasing the ECL photon emission rate of the tris(2,2′-bipyridine)ruthenium(II) (Ru(bpy) 3 2+ )/tri -n -propylamine (TPrA) system by 17–30-fold. Mechanistic investigations suggest that the enhancement originates from two complementary effects: plasmonic modulation associated with the localized surface plasmon resonance (LSPR) of the AuNR core, and nanoconfinement provided by the mesoporous SiO 2 shell, which promotes local enrichment of ECL reactants and increases the effective reaction frequency around individual nanoparticles. Using this platform, TNF-α was detected with approximately one-order-of-magnitude higher sensitivity than conventional ELISA and a wide linear range of 10–50,000 pg/mL. The method also allowed direct analysis of TNF-α in cell culture supernatants without pretreatment, revealing activation-dependent secretion kinetics and confirming a positive feedback circuit in T cell cytokine production. Compared with existing single-molecule immunoassays, this ECL platform eliminates the need for precisely fabricated microchambers or time-consuming signal amplification, enabling straightforward digital readout while remaining compatible with standard immunoassay workflows. This work provides a simple and practical strategy for isolation-free ECL digital immunoassays and demonstrates promising potential for clinical translation.