Programmable G‐Quadruplex@DNA Nano‐Highway Network Platform Enables One‐Pot Electrochemical Detection of Exosomes for Breast Cancer Lymph Node Metastasis Evaluation

ABSTRACT Exosomes are promising biomarkers for early tumor diagnosis and metastasis assessment. However, current methods are unsuitable for routine use because of low accuracy and complexity. In this study, we developed a universal detection platform based on a G‐quadruplex@DNA nano‐highway network (G4@DNA‐NHWN). This platform used aptamer‐triggered hybridization chain reaction and streptavidin‐biotin crosslinking to construct a protein‐scaffolded DNA nanostructure enriched with split G4 via a one‐pot method at room temperature, enabling sensitive and rapid detection of exosomes. Vimentin, a protein overexpressed in exosomes during the progression and metastasis of breast cancer, was selected as an example. Vimentin binding to the aptamer in G4@DNA‐NHWN induces structural disassembly, preventing the split G4 from forming G4‐Pb 2+ complexes with Pb 2+ , thereby increasing the free Pb 2+ . Electrochemical (EC) detection enabled the homogeneous quantification of vimentin by directly distinguishing the EC signal differences between the inert G4‐Pb 2+ complex and Pb 2+ . The results demonstrated that this system achieved exosomes detection with a sensitivity as 30 particles/mL within 1 h, and exhibited excellent selectivity. Validation across 42 clinical samples demonstrated a concordance rate of > 90% with the clinical diagnoses. As a DNA‐functionalized nanomaterial platform, this system holds the promise of approaches in targeted drug delivery and other biomedical applications beyond biosensing.