CRISPR/Cas12a cleavage-mediated isothermal amplification lights up the dimeric G-quadruplex signal unit for ultrasensitive and label-free detection of circulating tumor DNA

Circulating tumor DNA (ctDNA) is a valuable and potential cancer biomarker for real-time monitoring, and plays a vital role in cancer early diagnosis. In this work, we developed an ultrasensitive and label-free fluorescent biosensor for ctDNA detection. A well-designed RNA-DNA chimeric primer (RD) with phosphate groups modified at the 3-terminal was cleaved by target-initiated CRISPR/Cas12a, and then the subsequent exponential amplification reaction (EXPAR) was started to produce numerous G-rich sequences, which could be assembled with Thioflavin T to form a dimeric G-quadruplex (dimer-G4/ThT) and significantly enhance the fluorescence. Due to the innovative combination of the specificity of CRISPR/Cas12a and RD primers, the high amplification efficiency of EXPAR, and the strong fluorescence response of dimer-G4/ThT, this strategy demonstrated excellent sensitivity at the aM level. In addition, the sensing system showed satisfactory anti-interference ability and reliable accuracy in complex serum environment and tumor cell lysate. More importantly, this strategy avoided the complicated and expensive labeling process of traditional signal probes, making the construction of fluorescent biosensor simpler and more practical, offering practical insights for molecular diagnostics and clinical applications.