Ψ-type hybridization and CRISPR/Cas12a-based two-stage signal amplification for microRNA detection

Abstract

A two-stage signal amplification strategy was designed for sensitive microRNA detection. It included nicking endonuclease free strand displacement amplification (SDA) and CRISPR/Cas12a-assisted fluorescence signal amplification. To achieve single-base specificity, a novel Ψ-type hybridization was proposed to recognize target and trigger the cyclic strand displacement amplification (CSDA) for the generation of target DNA product, which activated the trans-cleavage activity of Cas12a. In addition, an innovative fluorescence signal readout method based on mono-labeled ssDNA as reporter and tremella-like graphdiyne (GDY) as scavenger was presented, which eliminated the fluorescent background of Cas12a amplification system and led to an order of magnitude improvement in the detection limit. Combining the Ψ-type hybridization triggered CSDA and Cas12a amplification system, the fluorescence signal was linear with miR-214 concentration from 20.0 fM to 10.0 pM, and the detection limit was as low as 0.1 fM. The work provided new opportunity for miRNAs assay.