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

• Published in: Sensors and actuators. B, Chemical Chemical, 2022-09, Vol.366, p.132011, Article 132011
• Main Authors: Zhong, Min, Liu, Jinbo, Wu, Jie, Li, Jinqian, Luo, Nini, Zhu, Chuanlong, Liu, Rui, Xia, Qianfeng, Ju, Huangxian
• Format: Article
• Language: English
• Subjects: Amplification; Biosensors; CRISPR; CRISPR-Cas12a; Dengue fever; Dengue virus; Domains; Fluorescence; Fluorescent biosensing; Hybridization; Hybridization chain reaction; Multiplexing; Pathogens; Proximity; Ribonucleic acid; RNA; Signal amplification; Viral diseases; Viruses
• ISSN: 0925-4005; 1873-3077
• DOI: 10.1016/j.snb.2022.132011

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. [Display omitted] •A universal fluorescence biosensing strategy was developed for highly sensitive detection of Dengue virus RNA.•The parallel connection of proximity sequence enhanced CRISPR-Cas12a was achieved through target-triggered HCR.•This proposed homogenous detection method showed a wide detection range and 50 fM-level detection limit.•This proposed strategy possesses good accuracy, high precision, and good extendability for target analysis.