Label-free detection of microRNA: two-stage signal enhancement with hairpin assisted cascade isothermal amplification and light-up DNA-silver nanoclusters

A method is described for the determination of microRNAs via two-stage signal enhancement. This is attained by combining hairpin (HP) assisted cascade isothermal amplification with light-up DNA-Ag nanoclusters. A rationally designed dual-functional HP is used, and microRNA-21 is chosen as a model analyte. At the first stage, upon the hybridization of the microRNA-21 with HP, microRNA recycling via polymerase-displacement reaction and a circulative nicking-replication process are achieved. This generates numerous G-abundant overhang DNA sequences. In the second stage, the above-released G-abundant overhang DNA sequences hybridize with the dark green Ag NCs, and this results in the appearance of bright red fluorescence. Thanks to the two signal enhancement processes, a linear dependence between the fluorescence intensity at 616 nm and the concentration of microRNA-21 is obtained in the range from 1 pM to 20 pM with a detection limit of 0.7 pM. The strategy clearly discriminates between perfectly-matched and mismatched targets. The method was applied to the determination of microRNA-21 in a spiked serum sample. Schematic representation of microRNA detection by integrating hairpin assisted cascade isothermal amplification with light-up DNA Ag nanoclusters. With microRNA, G-abundant overhang DNA sequences from amplification reaction hybridize with dark green Ag nanoclusters to produce a concentration-dependent bright red fluorescence