Highly sensitive electrochemical assay based on strand displacement polymerization-assisted CRISPR/Cas12a collateral cleavage

Abstract

In this work, a novel strand displacement polymerization strategy is designed with a single hairpin-structured DNA probe acting as both of primer and template. After target miRNA-induced structural transitions, extension and displacement reactions occur at the dimer with recycled target miRNA. The generated double-stranded products further activate CRISPR/Cas12a collateral cleavage of substrate DNA at the electrode surface, which can be reflected by the silver stripping peak variations. By analyzing the reduced peak intensity, the concentration of miRNA can be determined. This method combines the strand displacement amplification and CRISPR/Cas12a’s shearing capability. Thus a highly sensitive electrochemical biosensor is established with the limit of detection as low as 31 aM. It may also inspire new ideas of electrochemical platforms integrating CRISPR/Cas system for biological studies and clinical applications.

Keywords

• miRNA
• CRISPR/Cas12a
• Strand displacement amplification
• Silver nanoparticles
• Hairpin assembly