Isothermal RNA detection through the formation of DNA concatemers containing HRP-mimicking DNAzymes on the surface of gold nanoparticles

An enzyme-free signal amplification strategy for colorimetric detection of RNA molecules was developed. The detecting process was started by the hybridization of the target RNA via two helper oligonucleotides to bi-functionalized (initiator and linker oligonucleotides-modified) gold nanoparticles. Afterwards, in presence of two auxiliary oligonucleotides, the nanoparticle-confined initiators triggered the formation of DNA concatemers containing hemin-binding aptamers through a modified hybridization chain reaction [HCR] strategy. In addition to the mediatory role, the helper oligonucleotides, with a toehold-mediated strand displacement [TMSD] ability, helped unwind the secondary structures of the RNA molecule and provided a binding site for a biotinylated capture probe. Upon binding, the complex was harvested on the streptavidin-coated microwell, and subsequently the formation of HRP-mimicking DNAzymes was stimulated by adding hemin molecules. The assay was successfully employed for detecting target nucleic acids, which bears secondary structures, in isothermal conditions (room temperature) without heat denaturation. The assay detected DNA molecule with LOD value of 1 pM with the ability to differentiate a spurious target containing a single-nucleotide substitution. On real RNA samples, the assay truly discriminated Escherichia coli O157:H7's 16s rRNA from closely related bacteria with a detection limit of ≥5×105 CFU. Compared to enzyme-based signal amplifications, the assay can detect the target RNA with a sensitivity similar to the one already found for RT-PCR, NASBA, and RT-LAMP assays. Overall, the analytical platform eliminates the need for enzymatic reactions, heat denaturation, and complicated instruments during the detection process.