Dual DNA Nanomaterial-Regulated Homogeneous Immunoreaction-Enabled One-Step Electrochemical Analysis of Multiple Biomarkers in Clinical Samples

Homogeneous immunoassays offer a streamlined alternative to traditional heterogeneous methods by eliminating separation and washing steps. However, they are often hampered by low sensitivity, matrix interference, and limited multiplexing capability. Herein, we reported a dual DNA nanomaterial-based homogeneous electrochemical immunoassay that achieved ultrasensitive, one-step, simultaneous detection of multiple biomarkers. The system comprised a cyclic DNA nanomaterial (C-DNM) and a Y-shaped DNA nanosphere (Y-DNM) that coordinated Ag+ and Hg2+ via base mismatching, and antibodies were conjugated by the biotin–streptavidin system. Specific antigen–antibody binding could exert spatial steric hindrance on C-DNM, thereby triggering cascade metal ions release from Y-DNM and producing a second burst of signal amplification. This mechanism enabled detection limits down to the ag/mL level. Utilizing this strategy, we successfully quantified urinary ESAT-6 and LAM for tuberculosis, as well as PD-L1/MUC1-positive small extracellular vesicles (SEVs) and circulating tumor cells for lung cancer. The whole detection could be completed within 30 min. Clinical validation was taken in 60 samples, and the receiver operating curve analysis demonstrated good diagnostic accuracy, which showed strong correlation with clinical imaging findings. This dual DNA nanomaterial strategy provided a universal, rapid, and ultrasensitive tool for homogeneous immunoassay-based precision diagnostics of diseases.