A novel CRISPR/Cas13a biosensing platform comprising dual hairpin probe and traditional lateral flow assays

Abstract

Recently, CRISPR/Cas13a-based biosensors have been combined with non-traditional lateral flow assays (N-TLFAs) successfully used to clinical detection of target RNA due to their simplicity, portability, and cost-effectiveness. However, N-TLFAs used in such biosensors have several disadvantages, such as inversion of test and control zones, in which color intensity depends on reporter probe cleavage. Herein, we propose a novel biosensor in which CRISPR/Cas13a is integrated with traditional lateral flow assays (TLFAs) to facilitate the sequential direct location of test/control zones by dual hairpin probes, namely DNA hairpin reporter probe B-HPs (5′ Biotin-hairpins, B-HPs, loop containing 5 rU) and F-HPs (5′ FAM-hairpins, F-HPs). Cas13a activated by crRNA-targeted RNA duplexes can indiscriminately cleave B-HPs reporter probes to release short sequences to open hairpin F-HPs reporter probes to form Biotin-dsDNA-FAM reporter probes. The advantage of this platform is that detection results only rely on the formed Biotin-dsDNA-FAM reporter probes, which can be used for SARS-CoV-2 RNA detection after preliminary detection. Under optimized conditions, the SARS-CoV-2 RNA detection range was 10 aM to 1 µM and the limit of detection was as low as 5.34 aM. This proof-of-concept study demonstrates that dual-hairpin reporter-probe binding to TLFAs opens up new opportunities for CRISPR/Cas13a activity detection and bioanalytical applications.