Double determination of long noncoding RNAs from lung cancer via multi-amplified electrochemical genosensor at sub-femtomole level

Abstract

An ultrasensitive electrochemical genosensor has been fabricated for the double determination of two different specific sequences deduced from the maternally expressed gene3 (MEG3) lncRNA (long noncoding RNA), which was demonstrated by coupling RNase A-aided target recycling with DNA supersandwich-induced signal enhancement, based on a composite interface of graphene-like tungsten disulfide/dendritic gold nanostructures (WS₂/DGN). Firstly, duple target sequences of T₁ and T₂ were captured by the primer probes of P₁/P₂ functionalized Fe₃O₄@C magnetic nanoparticles, via the DNA/RNA hybridization between T₁/T₂ and P₁/P₂. In the presence of RNase A, T₁ and T₂ were released to trigger the target recycling, accompanied by the generation of numerous intermediate DNAs designated as IT₁ and IT₂, respectively. After the magnetic separation, the IT₁ and IT₂ were liberated and hybridized with the capture probes of CP₁/CP₂ loaded DGN/WS₂ modified electrode. Subsequently, the stepwise DNA hybridization chain reactions (HCR) labeled with ferrocene (Fc) and methyleneblue (MB) were processed, respectively. The DPV current values of Fc and MB were recorded, which were proportional with the concentration of T₁ and T₂, respectively. Using the multiplexed amplification strategy, this newly designed genosensor provided a wide linear range from 1 fM to 100 pM with a low detection limit of 0.25 fM for T₁ and 0.3 fM for T₂. The application of the genosensor in real serum sample has also been studied, confirming the excellent selectivity and sensitivity for the application in bioanalysis and clinical diagnostics.

Keywords: Dual signal detection; Electrochemical genosensor; Enzyme-aided target recycling; Hybridization chain reaction; Long noncoding RNA; Multiplexed amplification.