G-wire-based self-quenched fluorescence probe combining with target-activated isothermal cascade amplification for ultrasensitive microRNA detection

Abstract

Herein, we reported the G-wire-based self-quenched fluorescence probe and its application in ultrasensitive microRNA (miRNA) detection by combining with target-activated isothermal cascade amplification. The terminal-single-fluorescein (FAM)-labeled G-rich oligonucletides self-assembled into G-wire nanostructures (G-wires) with K⁺ and Mg²⁺. Thereafter, the G-wires brought terminal-labeled FAM into close proximity, as a result, the self-quenched signal probe formed. Besides, when there was the target miRNA, target-activated isothermal cascade amplification converted miRNA into the copious trigger DNA. After hybridization between trigger DNA and the self-quenched probe, the G-wires were splited and forced the apart of proximate FAM, and then the self-quenched probe displayed an "on" mechanism. Therefore, the approach gave a limit of detection (LOM) of 0.82 aM to miRNA-21 and could be implemented within a wide linear range of 2 aM to 2 nM. This approach was able to distinguish the single-mismatched miRNA-21, which was selective and sensitive in detecting human spiked serum samples.

Keywords: G-Wire nanostructures; Isothermal cascade amplification; MicroRNA; Self-quenched; Signal on; Ultrasensitive detection.