Fluorometric determination of microRNA using arched probe-mediated isothermal exponential amplification combined with DNA-templated silver nanoclusters.

Abstract

A highly sensitive fluorometric method is described for the determination of microRNA-141. It is based on the use of arched probe-mediated isothermal exponential amplification reaction (EXPAR) and of DNA-templated silver nanoclusters (DNA-AgNCs). The EXPAR utilizes microRNA-141 as the trigger, polymerases and endonucleases as amplification activators, and two arched probes as exponential amplification templates. This enables the conversion of microRNA to a large number of reporter sequences under isothermal conditions within minutes. The generated reporter sequences act as scaffolds for the synthesis of fluorescent DNA-AgNCs by reduction of Ag (I) with NaBH4. The DNA-AgNCs function as signalling fluorophores with excitation/emission maxima at 540/610nm. The method exhibits high sensitivity for microRNA-141 with a detection limit as low as 0.87 fM and a dynamic range from 1 fM to 500 fM. The method can distinguish nucleotides in the microRNA-200 family. Graphical abstract Schematic representation of a fluorometric method for sensitive detection of microRNA based on arched probe-mediated isothermal exponential amplification combined with DNA-templated silver nanoclusters.