Graphene oxide-based fluorometric determination of microRNA-141 using rolling circle amplification and exonuclease III-aided recycling amplification

A graphene oxide-based method has been developed for ultrasensitive and selective determination of microRNA-141 by means of rolling circle amplification (RCA) and exonuclease III (Exo III)-assisted recycling amplification. The method uses (a) a padlock probe with a hybrid sequence that is complementary to the target microRNA-141 at both the 5'- and the 3'-end, and (b) a long binding region of a signalling reporter strand. On addition of microRNA-141, it acts as the primer for triggering the RCA reaction following ligation. This results in the formation of a repeatedly concatenated sequence of the padlock probe. Subsequently, the RCA product hybridizes with the fluorescein-labelled signal strand to form the duplex DNA containing the blunt 3'-termini of signal strand. Addition of Exo III causes signal strand digestion and leads to RCA product recycling and liberation of fluorescein. Added graphene oxide does not absorb the fluorescein liberated because of the poor mutual interaction. Therefore, microRNA-141 can be quantified by measurement of the green fluorescence under excitation/emission wavelengths of 470/520 nm. The method has a 100 aM detection limit towards microRNA-141 and works in the wide range from 1 fM to 1 nM. It can discriminate even single-mismatched microRNA and shows good selectivity and sensitivity when applied to spiked human serum.