An enzyme-free and label-free fluorescence biosensor for microRNA detection based on cascade amplification of DNAzyme-powered three-dimensional DNA walker and hybridization chain reaction

Herein, an enzyme-free and label-free fluorescence microRNA biosensor based on cascade amplification of DNAzyme-powered three-dimensional DNA walker and hybridization chain reaction was developed. Firstly, the microRNA hybridized with the locking DNA and caused the locking DNA released from the blocked walking probes (BWPs) through toehold-mediated strand displacement reaction. The DNAzyme was exposed and then the walking probes automatically moved along the three-dimensional tracks and catalyzed the cleavage of hairpin DNA substrates (HDSs), releasing abundant triggers. Finally, the triggers initiated the hybridization chain reaction to generate amplified fluorescence signals. Compared with the three-dimensional DNA walker powered by protein enzyme, this DNAzyme-powered three-dimensional DNA walker was simpler and more stable. The high amplification efficiency of cascade amplification guaranteed a satisfactory sensitivity with a detection limit of 7.9 fM. The toehold-mediated strand displacement reaction guaranteed an admirable specificity. The microRNA analysis in real biological sample further indicated that this biosensor offered an alternative strategy for the quantification of microRNA.