A highly sensitive and specific biosensor for ligation- and PCR-free detection of microRNAs.

A highly sensitive microRNA (miRNA) biosensor for both ligation- and PCR-free detection of miRNAs is described in this work. The biosensor was made of a monolayer of long oligonucleotide capture probes (CPs) comprising of miRNA capturing segments at the top (3' termini) and detection probe capturing segments at the bottom (5' termini). Following hybridization with a target miRNA, a cocktail of Surveyor(®) and exonuclease I in pH 7.4 phosphate buffered saline was applied to the biosensor. Unhybridized CP strands and mismatched miRNA/CP duplexes were digested while complementarily hybridized ones remained intact. Thereafter, electrochemically activated glucose oxidase-tagged peptide nucleic acid detection probes (GOx-DP) were hybridized to the bottom segments of the CPs on the biosensor. The number of GOx molecules found at the biosensor surface coincides with the number of miRNA strands hybridized and therefore correlates directly to the concentration of the target miRNA. A detection limit of 10 fM and a linear current-concentration relationship up to 10 pM were attained under optimized conditions. The biosensor effectively eliminated the needs for chemical/biological ligation and PCR. It was showed that there is little cross-hybridization among closely related miRNA family members even at single-base-mismatched levels. Successful attempts were made in applying the biosensor to the detection of miRNAs in total RNA extracted from cell lines.