Cascaded confinement-encapsulated fluorescence nano-immunomachine for noninvasive urinary monitoring of bladder cancer.

Urinary protein biomarker analysis holds immense promise for noninvasive tumor monitoring; however, current assays are limited by multi-step operations and matrix interference. Herein, we developed a one-pot fluorescence nano-immunomachine for ultrasensitive quantification of urinary bladder cancer-specific nuclear matrix protein 4 (BLCA-4), driven by cascaded confinement-mediated quantum dots encapsulation in DNA nanospheres. Programmed DNA self-assembly and phenyllactic acid-mediated framework condensation jointly established hierarchical structural confinement to cage QDs with minimal signal leakage. Upon streptavidin-biotin-directed antibody functionalization, the resulting QDs@DNS@Ab platform specifically recognized BLCA-4, triggering localized structural disassembly and spontaneous fluorescence recovery. This washing-free homogeneous assay was completed within 40 min, achieving a dynamic range of 10-5 × 104 ag/mL and a detection limit of 3.6 ag/mL. Clinical validation of 72 urine samples, including bladder cancer patients (n = 30), healthy controls (n = 20), and urological disease controls (n = 22), demonstrated strong diagnostic discrimination and good concordance with enzyme-linked immunosorbent assay measurements, achieving an area under the curve of 0.93, sensitivity of 93.3%, and specificity of 90.5%. Longitudinal paired analysis effectively tracked post-resection biomarker clearance, highlighting the responsive nanoplatform as a rapid, sensitive, and operationally simple adjunct tool for noninvasive bladder cancer risk stratification, postoperative surveillance, and future point-of-care diagnostics.