The rapid, sensitive, and multiplexed detection of circulating tumor DNA (ctDNA) is crucial for improving breast cancer diagnosis. Here, we present a homogeneous dual-signal sensing platform that integrates rolling circle amplification (RCA)-based DNA hydrogels and DNA@Cu2+ nanospheres (NS) for the simultaneous detection of dual ctDNA markers. This design enables a fully enzyme-free detection process after initial material synthesis, operating isothermally within 40 min and achieving an attomolar-level detection limit. The mechanism relies on target-triggered hydrogel disruption that exposes G-quadruplex sequences to enhance ThT fluorescence, while the concomitant release of Cu2+ quenches quantum dot emission, together enabling a self-referencing fluorescent output for improved accuracy. Clinical proof-of-concept validation was performed on a cohort of 58 breast cancer patients and 30 healthy controls, showing a sensitivity of 88.0% and a specificity of 93.3%, with high concordance to clinical and qPCR results. The platform effectively differentiated not only cancer patients from healthy individuals but also showed significant signal differences between early- and late-stage disease. With its minimal sample processing, low instrumental requirements, and reliable performance, this assay represents a promising concept for rapid, cost-effective prescreening and stratification in breast cancer management.
Keywords: DNA nanohydrogel; DNA@Cu2+ nanoshpere; breast cancer profiling; dual circulating tumor DNA; homogeneous fluorescence.