An enzyme-free colorimetric biosensing strategy for ultrasensitive and specific detection of microRNA based on mismatched stacking circuits

A novel colorimetric biosensing strategy, employing coupling spontaneous cascade catalytic hairpin assembly (CHA) circuits and mismatched hairpin, has been developed successfully for ultrasensitive and specific detection of target microRNA (miRNA). This system is composed of two layers of CHA. While target miRNA exists, the toehold-based assembly of the first-layer hairpins comes into being, followed by the generation of the first-layer CHA products and the cyclic release of the target miRNA. Then the first-layer CHA products can act as initiator for the assembly of the second-layer hairpins to produce the second-layer CHA products, with the first-layer CHA products recovered simultaneously. Finally, the second-layer CHA products can combine with hemin to form G-quadruplex/hemin DNAzyme, a well-known horseradish peroxidase (HRP) mimic, for catalyzing a colorimetric reaction. Under the optimal experimental conditions, the established biosensor can detect target miRNA down to 36.2 fM (S/N = 3) with a linear range from 100 fM to 10 nM, and discriminate target miRNA from mismatched miRNA with high selectivity. It was also applied to test the concentration of miRNA spiked into salmon sperm samples. Therefore, this biosensing strategy may become an alternative tool for the detection of miRNA in biomedical research.