Homogeneous Extraction-Free Dual-ctDNA Detection via DNA Nanomaterial Fusion for Rapid Breast Cancer Diagnosis

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.