A new method for the detection of miRNAs making use of a competitive RNA/RNA hybridization configuration is described in this work. A biotinylated miRNA (biotin-miRNA) of identical sequence to that of the target miRNA is mixed with the samples to be analyzed allowing competition to be accomplished with the target miRNA for a thiolated RNA probe assembled onto a gold nanoparticles (AuNPs) modified screen-printed electrode. After labeling the hybridized biotin-miRNA with streptavidin-HRP conjugates, amperometric detection at -0.20V was carried out using the H2O2/hydroquinone (HQ) system. The decrease in the amperometric response was proportional to the concentration of model target miRNA-21 in the 100 fM to 25.0 pM range. The integrated sensor provided a very low detection limit (25 fM, 0.25 attomol in 10μL sample) for miRNA-21 without any amplification step, a complete discrimination against single nucleotide mismatched sequences under practical conditions and high storage stability. The usefulness of the developed method was demonstrated by determining the endogenous levels of the mature target miRNA in total RNA (RNAt) extracted from cancerous and non-cancerous cells.
Keywords: Amperometry; Attomole sensitivity; Cancer cells; Competitive hybridization; GNPs-SPCEs; MiRNAs; Single-base mismatch discrimination.
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