Target-independent hybridization chain reaction-fluorescence resonance energy transfer for sensitive assay of ctDNA based on Cas12a

Circulating tumor DNA (ctDNA) is a noninvasive biomarker which offer valuable information for cancer diagnosis and prognosis. In this study, a target-independent fluorescent signal system, Hybridization chain reaction-Fluorescence resonance energy transfer (HCR-FRET) system, is designed and optimized. Combined with CRISPR/Cas12a system, a fluorescent biosensing protocol was developed for sensing assay of T790 M. When the target is absent, the initiator remains intact, opens the fuel hairpins and triggers the following HCR-FRET. At presence of the target, the Cas12a/crRNA binary complex specifically recognizes the target, and the Cas12a trans-cleavage activity is activated. As a result, the initiator is cleaved and subsequent HCR responses and FRET processes are attenuated. This method showed detection range from 1 pM to 400 pM with a detection limit of 316 fM. The target independent property of the HCR-FRET system endows this protocol a promising potential to transplant to the assay of other DNA target in parallel.