CRISPR/Cas12a bio-assay integrated with metal-organic framework based enhanced fluorescent labels for ultrasensitive detection of circulating tumor DNA

Circulating tumor DNA (ctDNA) is an important biomarker for liquid biopsy. Unfortunately, the detection of ctDNA is still challenging unless these obstacles, such as unsatisfactory sensitivity, time-consuming processes, and high costs, are solved. Herein, the first paradigm of nanomaterial’s application in CRISPR/Cas12a system with enhanced fluorescence readout was reported in this work. We designed and prepared a class of metal-organic framework (MOF) based fluorescent labels with stimulus response to control the release of fluorescent molecules. Notably, the MOF-based fluorescent labels was able to optimize traditional CRISPR/Cas12a detection system with fluorescence readout, which improved the detection sensitivity over four orders of magnitude to meet the requirements of trace ctDNA detection. We integrated the MOF-based fluorescent labels and CRISPR/Cas12a system to achieve ultrasensitive and convenient detection of target ctDNA. Aiming at Kirsten rat sarcoma-2 virus point mutations, the proposed strategy exhibited ultrahigh sensitivity for detecting target ctDNA as low as 5.6 fM, which had been successfully employed to quantify target ctDNA in clinical serum samples. The quantitative results could distinguish lung cancer patients from healthy person with high specificity and sensitivity, which had a broad application prospect in clinical diagnosis.