Cascade toehold-mediated strand displacement along with non-enzymatic target recycling amplification for the electrochemical determination of the HIV-1 related gene

The authors describe a dual-signal electrochemical biosensor for highly sensitive determination of the HIV-1 related gene. This method is based on the application of cascaded toehold-mediated strand displacement reactions (TMSDRs) in combination with non-enzymatic target recycling amplification (TRA). A DNA machine with two TMSDRs was designed, and this resulted in reusable target and an output of two oligonucleotides, referred to as strand A (AS) labeled with the redox tag methylene blue (MB) and as untagged strand B (BS). A ferrocene (Fc)-modified signal probe (Fc-P1) is immobilized on the gold electrode surface by hybridizing with a thiolated probe (P2). The labeled AS causes the dissociation of Fc molecules and the gathering of MB molecules via strand displacement reaction. The target gene triggers TMSDRs and TRA. This leads to an increase in the distance changes between the redox tags and the gold electrode. The assay works in the 1 pM to 10 nM concentration range. On account of target recycling and dual recognition, the limit of detection is as low as 0.88 pM (at an S/N ratio of 3). The assay also has a remarkable selectivity which is ascribed to the use of both cascaded TMSDRs and dual recognition. In our perception, this assay represents a robust means of wide scope in that it may be applied to the detection of various kinds of nucleic acid even in complex samples.