Proximity sequence enhanced CRISPR-Cas12a connected through hybridization chain reaction for sensitive biosensing of dengue virus

Dengue caused by dengue virus (DENV) is a highly pathogenic viral disease. Early detection of pathogens is very important for patient treatment and pathogen control. This work designed a dual signal amplification strategy for sensitive fluorescent detection of DENV RNA. The dual signal amplification was achieved by using a target-triggered hybridization chain reaction (HCR) to connect multiplex proximity ligation activated CRISPR-Cas12a (pCRISPR-Cas12a). The HCR was triggered by the recognition of DENV RNA to a hairpin probe (H3), which led to a product to initiate the continuous hybridization of a couple of hairpins. Here one of the hairpins (H2) was designed to contain two domains (a and b), and domains a and b from two H2 formed a proximity ligation sequence (PLS) to connect the multiplex pCRISPR-Cas12a onto the HCR product, in which domain a activated the Cas12a, and domain b enhanced the enzymatic activity by about 5 times. Upon the enzymatic cleavage of single-stranded DNA labeled with a fluorophore and quencher at each end (ssDNA-FQ), a strong fluorescent signal was produced for DENV RNA analysis, which showed a linear range from 1 pM to 10 nM and a detection limit of 51 fM. The excellent performance of the proposed homogenous fluorescent assay with high sensitivity and superior accuracy endowed this strategy broad application prospects in clinical disease diagnosis.