An integrated dual functional recognition/amplification bio-label for the one-step impedimetric detection of Micro-RNA-21

Sawsen Azzouzi, Cheung Mak, Kamalodin Kor, Anthony P.F. Turner, Mounir Ben Ali, Valerio Beni

Alteration in expression of miRNAs has been correlated with different cancer types, tumour stage and response to treatments. In this context, a structurally responsive oligonucleotide-based electrochemical impedimetric biosensor has been developed for the simple and sensitive detection of miRNA-21. A highly specific biotinylated DNA/LNA molecular beacon (MB) probe was conjugated with gold nanoparticles (AuNPs) to create an integrated, dual function bio-label (biotin-MB-AuNPs) for both biorecognition and signal generation. In the presence of target miRNA-21, hybridisation takes place resulting in the "activation" of the biotin-MB; this event makes the biotin group, which was previously "protected" by the steric hindrance of the MB stem-loop structure, accessible. The activated biotin-MB-AuNPs/miRNA complexes become available for capture, via supramolecular interaction, onto a nentravidin-modified electrode for electrochemical transduction. The binding event results in a decrease of the charge transfer resistance at the working electrode/electrolyte interface. The biosensor responded linearly in the range 1-1000 pM of miRNA-21, with a limit of detection of 0.3 pM, good reproducibility (Relative Standard deviation (RSD) =3.3%) and high selectivity over other miRNAs (i.e. miRNA-221 and miRNA-205) sequences. Detection of miRNA-21 in spiked serum samples at clinically relevant levels (low pM range) was also demonstrated, thus illustrating the potential of the biosensor for point-of-care clinical applications. The proposed biosensor design, based on the combination of a neutravidin transducing surface and the dual-function biotin-MB-AuNPs bio-label, provides a simple and robust approach for detection of short-length nucleic acid targets, such as miRNAs.