Mycobacterium tuberculosis urine ESAT-6 antigen homogeneous immunoassay through spatial confinement and cascade material amplification.

Tuberculosis (TB) remains one of the leading causes of death from infectious diseases. Early secreted antigenic target-6 (ESAT-6) is a specific biomarker for active Mycobacterium TB infection; however, its ultrasensitive detection in urine specimens remains challenging. Herein, a homogeneous immunoassay for ESAT-6 detection integrating spatial confinement-mediated DNA conformational switching with cascade signal amplification is reported. The system employs antibody-bound triplet DNA molecules stabilized by C-Ag+-C coordination; specific antigen binding induces conformational changes that disrupt complexes, releasing Ag+ that initiates a cation exchange reaction (CER) with CuS nanoparticles (NPs) to generate Cu2+, and potently quenches CdTe quantum dots (QDs) with a secondary CER. This homogeneous platform operates without washing steps or enzyme-based amplification, achieving a limit of detection of 30 ag/mL with a linear range of 0.1-100 fg/mL. Clinical validation using 80 urine specimens (40 TB-positive and 40 negative) demonstrated highly significant discrimination between groups (p < 0.001), with receiver operating characteristic analysis confirming robust diagnostic performance. ESAT-6 testing exhibited excellent concordance with imaging findings and correlated strongly with enzyme-linked immunosorbent assay measurements. This system provides a one-pot urine-based detection tool for rapid TB diagnosis, significantly reducing operational complexity to meet clinical demands.