CRISPR/Cas9 cleavage triggered ESDR for circulating tumor DNA detection based on a 3D graphene/AuPtPd nanoflower biosensor.

Circulating tumor DNA (ctDNA) plays an important role in the early diagnosis and prognosis of several cancers and is a credible biomarker for predicting the response to therapy. Additionally, the fact that the strategy used to detect ctDNA is non-invasive also adds to the advantages of using ctDNA for predicting disease diagnosis and prognosis. However, low abundance in peripheral blood and the high background of wild-type DNA impair the precise and specific measurement of ctDNA. In this study, we developed a novel 3D GR/AuPtPd nanoflower sensing platform based on CRISPR/Cas9 cleavage-triggered entropy-driven strand displacement reaction (ESDR) for the effective detection of ctDNA. Low levels of ctDNA could be detected using this method as the ESDR amplification does require complicated operation procedures and stringent reaction conditions. By combining the advantages of the site-specific cleavage by "gene magic scissors," Cas9/sgRNA, with those of the rapid amplification kinetics of entropy-driven strand displacement, our method resulted in amplification efficiency as well as high specificity for discriminating single-nucleotide mismatches. The 3D GR/AuPtPd nanoflower-based electrochemical biosensor displayed high specificity and worthy performance in assays with human serum. Therefore, this pioneered method provides a new paradigm for efficient ctDNA detection and shows great potential for use in clinical and diagnostic applications.