Generic Neutravidin Biosensor for Simultaneous Multiplex Detection of MicroRNAs via Electrochemically Encoded Responsive Nanolabels.

Current electrochemical biosensors for multiple miRNAs require tedious immobilization of various nucleic acid probes. Here, we demonstrate an innovative approach using a generic neutravidin biosensor combined with electrochemically encoded responsive nanolabels for facile and simultaneous multiplexed detection of miRNA-21 and miRNA-141. The selectivity of the biosensor arises from the intrinsic properties of the electrochemically encoded responsive nanolabels, comprising biotinylated molecular beacons (biotin-MB) and metal nanoparticles (metal-NPs). The procedure is a simple one-pot assay, where the targeted miRNA causes the opening of biotin-MB followed by capturing of the biotin-MB-metal-NPs by the neutravidin biosensor and simultaneous detection of the captured metal-NPs by stripping square-wave voltammetry (SSWV). The multiplexed detection of miRNA-21 and miRNA-141 is achieved by differentiation of the electrochemical signature (i.e., the peak current) for the different metal-NP labels. The biosensor delivers simultaneous detection of miRNAs with a linear range of 0.5-1000 pM for miRNA-21 and a limit of detection of 0.3 pM (3σ/sensitivity,  n = 3), and a range of 50-1000 pM for miRNA-141, with a limit of detection of 10 pM. Furthermore, we demonstrate multiplexed detection of miRNA-21 and miRNA-141 in a spiked serum sample.

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

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.