Dual MicroRNA Simultaneous One-Step Profiling via Filler-Mediated Quantum Dot DNA Nanospheres for Bladder Cancer Screening

Rapid and multiplexed detection of urinary microRNAs (miRNAs) is crucial for the noninvasive diagnosis and monitoring of bladder cancer. In this study, we develop a dual-channel fluorescence platform based on filler-mediated quantum dot-DNA nanospheres for simultaneous detection of miRNA-21 and miRNA-96. The nanospheres are constructed via a streptavidin-biotin cross-linked bidirectional hybridization chain reaction and compacted by phenyl-lactic acid, enabling high-density encapsulation of distinct QDs. Upon target miRNA recognition, a strand-displacement cascade triggers nanosphere disassembly and QDs release, restoring well-resolved fluorescence at 500 and 624 nm. This enzyme-free, extraction-free assay is completed in 40 min. It achieves attomolar-level sensitivity, with detection limits of 30 aM for miRNA-21 and 40 aM for miRNA-96, while maintaining high selectivity against mismatched and nontarget miRNAs. Clinical validation using urine samples from 45 patients with bladder cancer, 22 patients with other urological diseases, and 20 healthy controls indicated strong concordance with quantitative polymerase chain reaction. Integrated dual-miRNA analysis significantly enhanced diagnostic performance, yielding 91.0% sensitivity, 80.0% specificity, and an area under the curve of 0.91. The platform provides a rapid tool for noninvasive miRNA profiling, with potential to distinguish bladder cancer from healthy individuals and other urological diseases, thereby improving bladder-cancer screening, stratification, and recurrence monitoring.