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@SiO2) for detecting tumor necrosis factor-α (TNF-α) secreted by activated T cells. The AuNR@SiO2 nanoparticles served as efficient nanoaccelerators, increasing the ECL photon emission rate of the tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)32+)/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 SiO2 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.