Colorimetric and visual determination of microRNA via cycling signal amplification using T7 exonuclease

Abstract

A sensitive and specific bioassay based on cyclic enzymatic amplification was developed for determination of microRNA (miRNA) by taking advantage of the exodeoxyribonuclease activity of T7 exonuclease (T7 Exo). In the presence of miRNA, DNA/RNA duplexes are formed by hybridization of miRNA and capture probes (Cp). Then, the Cp is digested to release miRNA into the next amplification cycle assisted by T7 Exo. This leads to the digestion of numerous Cp molecules. The broken Cp does no longer hybridize with hairpin probes (Hp) to unveil G-quadruplex DNAzyme (GDNAs). However, in the absence of miRNA, the Hp hybridizes with Cp to unveil GDNAs. The generated GDNAs form assemblies with hemin to form the G-quadruplex/hemin DNAzyme complex which is capable of catalyzing the oxidation of the substrate ABTS by H2O2. Upon cyclic enzymatic amplification, the output signal is reduced accordingly, this resulting in a “signal-off” signal best acquired at a wavelength of 418 nm. The lower detection limit is 0.69 pM (at an S/N ratio of 3). The assay involves efficient signal amplification, is homogeneous and isothermal, and enables visual detection. It provides a simple, rapid and sensitive platform for use in clinical diagnostics.