Ultrasensitive detection of circulating tumor DNA using a CRISPR/Cas9 nickase-driven 3D DNA walker based on a COF-AuNPs sensing platform.

A electrochemical biosensor was designed utilizing a CRISPR Cas9n-driven DNA walker combined with gold-nanosphere-like covalent organic frameworks (COFs-AuNPs) to detect breast cancer markers (PIK3CA E545K ctDNA). The DNA walker probe is activated only in the presence of circulating tumor deoxyribonucleic acid (ctDNA), binding to a support probe to form a double strand that is then specifically cleaved by the Cas9n/sgRNA complex. This cleavage produces numerous DNA fragments for signal amplification. The COF-AuNPs as electrode materials facilitate electronic transfer and provide additional active sites for the immobilization of nucleic acid probes. This setup achieves a detection limit of 1.76 aM, demonstrating high sensitivity. Additionally, Cas9n improves the specificity of the sensor, accurately distinguishing a pair of base-mismatched sequences, and reducing the occurrence of false positives. Overall, the sensor exhibits excellent selectivity, reproducibility, and potential for early diagnosis of breast cancer.