An Ultrasensitive Colorimetric Nucleic Acid Detection Method Using Crispr/Cas12a Mediated Strand Displacement/Hybridization Chain Reaction Based on Toehold Regulating the Trans-Cleavage Activity

Accurate, rapid and sensitive nucleic acid detections play an increasingly important role in clinical diagnosis, forensic medicine and environmental monitoring. Herein, we developed a novel CRISPR/Cas12a mediated strand displacement/hybridization chain reaction (CSDHCR ) for ultrasensitive DNA colorimetric detection. A biotinylated single-strand DNA is coupled on avidin magnetic beads (MBs) and acts as an initiator strand to trigger the SDHCR. The SDHCR amplification allowed the formation of long hemin/G-quadruplex-based DNAzyme products on MBs to catalyze the TMB-H 2 O 2 reaction for colorimetric detection. In the presence of the DNA targets, the trans-cleavage activity of CRISPR/Cas12a was activated to cleave the initiator DNA, resulting in the failure of SDHCR and no formation of hemin/G-quadruplex on MBs. Thereby, the TMB could not be catalyzed to change color from colorless to blue. Meanwhile, the DNA toehold was well-designed to regulate the trans-cleavage activity of CRISPR/Cas12a to further improve the sensitivity of the method. Under optimal conditions, the limit of detection for DNA targets was determined as 4.54 fM and the practical application was also validated using Vibrio vulnificus. In conclusion, the CSDHCR with ultrasensitive and specific detection performance for nucleic acid detection holds a promise application for point-of-care diagnostic.