Multiselective Recognition of Metal Ion–Nucleic Acid Complexes by CRISPR/Cas12a and Quantum Dots Enables the Profiling of Circulating Tumor DNA in Breast Cancer

The rapid, noninvasive detection of circulating tumor DNA (ctDNA) is vital for the diagnosis and staging of breast cancer (BC). In this study, we developed a homogeneous CRISPR/Cas12a fluorescent platform using a hierarchical grape-cluster rolling circle amplification (GCRCA) nanomaterial to detect the PIK3CA E542K mutation. A pivotal discovery of this study is that activated Cas12a efficiently cleaves metal ion-mediated cytosine-Ag⁺-cytosine base pairs, which enables direct coupling between enzymatic activity and signal transduction. GCRCA, assembled by precise hybridization of long-chain RCA concatemers with auxiliary circular DNA, features Ag⁺-bridged dual-ring units that sequester both target sequences and Ag⁺ reporters within a self-shielding framework. Upon target recognition, activated Cas12a dismantles the GCRCA architecture, initiating an autocatalytic feedback loop that releases caged Ag⁺ to quench the quantum dot fluorescence. This label-free assay achieved attomolar sensitivity within 30 min without enzymatic preamplification or complex nucleic acid extraction. Importantly, the platform exhibits excellent sequence selectivity, enabling precise discrimination of single-base mutations against closely related sequences. Validation of 42 clinical plasma samples achieved 100% diagnostic specificity for BC. For staging, the platform yielded a sensitivity of 100%, a specificity of 92.3%, and an area under the curve of 0.978. With its exceptional sensitivity and operational simplicity, this platform offers a promising approach for precise ctDNA-based BC detection and staging, demonstrating significant translational clinical potential.