Hyperbranched RCA-mediated construction of a dendritic DNA nanostructure for ratiometric fluorescence biosensing

As a robust isothermal strand amplification technique, rolling circle amplification (RCA) is popularly adopted for constructing various homogeneous fluorescence biosensing strategies. However, the limited functional unit decoration on linear RCA products and the slow homogeneous reaction kinetics usually restrict their performance. Herein, by designing a target biorecognition-triggered hyperbranched RCA reaction to construct an Mg2+-dependent DNAzyme (MNAzyme)-decorated dendritic DNA nanostructure, a novel ratiometric fluorescence biosensing method was proposed. The catalyzed cleavage of MNAzymes toward a short stand labeled with the FAM fluorophore and its quencher realized a “signal-on” fluorescence output. Meanwhile, a “signal-off” fluorescence output of 2-aminopurine (2-AP) labeled at a DNA hairpin was realized through the DNA hybridization with the nanostructure. Besides efficient acceleration of homogeneous reaction kinetics, these fluorescence responses were dramatically amplified by the high decoration of MNAzymes on the dendritic nanostructure and its strong signal suppression toward 2-AP. Together with the exonuclease III-amplified target biorecognition reaction, a very low detection limit of 0.53 fg mL–1 and a six-order magnitude linear range were endowed for kanamycin antibiotic assay. This excellent analytical performance and its high selectivity, and good operability determine promising prospect of the method for practical applications.