Dual-Pathway Enhanced Single-Molecule Electrochemiluminescence Imaging of T Cell Early Activation Biomarkers

Immunotherapy using activated T cells as the weapon for controlling and eliminating tumor cells represents a promising therapeutic strategy for cancer treatment, in which accurate detection of T cell activation is critical for evaluating the immunotherapy efficacy. Activated T cells upregulate specific plasma membrane biomarkers, such as CD69, that can act as an indicator of activation status. In this work, we report a novel strategy of assessing T cell activation by electrochemiluminescence (ECL) imaging of CD69 expression on the plasma membrane. Mesoporous silica nanoparticles functionalized by polyethylenimine (PEI-SiO₂) were prepared as both "nanocoreactant" and "nanoreactor" to remarkably enhance the ECL reaction through a dual-pathway scheme, in which both the catalytic route and the low-oxidation-potential route contribute to produce an enhanced ECL signal. Based on this strategy, subsequent conjugation of PEI-SiO₂ with anti-CD69 antibodies allowed us to detect individual CD69 proteins by single-molecule ECL imaging, visualize their spatial distribution on the plasma membrane of activated T cell, and reveal their expression dynamics. The results establish a novel dark-field imaging strategy for assessing T cell activation, offering a low-background tool for investigating immune cell dynamics and immunotherapy efficacy through single-cell-level analysis of key biomarkers.