Fluorometric determination of microRNA-122 by using ExoIII-aided recycling amplification and polythymine induced formation of copper nanoparticles

The authors describe a method for the determination of microRNA-122 by using terminal deoxynucleotidyl transferase (TdT). It is based on the use of polythymine and exonuclease III-aided cycling amplification. A 3′-phosphorylated hairpin probe 1 (H 1 ) and a hairpin probe 2 (H 2 ) were designed. In the presence of the microRNA, hybridization and enzymatic cleavage will occur and produce lots of 3′-hydroxylated ssDNA which can be tailed by TdT and converted into long polythymine (polyT) sequences. These can be used to synthesize copper nanoparticles (CuNPs) with fluorescence excitation/emission maxima at 350 nm/630 nm. This method shows good selectivity and high sensitivity with a linear response in the 1.00 × 10 2 fM and 1.00 × 10 6 fM microRNA concentration range and a 44 fM limit of detection. It was successfully applied to determination of microRNA in spiked serum samples. A label-free and highly sensitive fluorometric method is described for the assay of microRNA on the basis of target-triggered two-cycle amplification and combining with terminal TdT. It produces a series superlong polyT that can be used for synthesis of copper nanoclusters (CuNCs) displaying red fluorecence.