Herein, by directly introducing mismatched reactant DNA, high-reactivity and high-threshold enzyme-free target recycling amplification (EFTRA) is explored. The developed high-efficiency EFTRA (HEEFTRA) was applied as a programmable DNA signal converter, possessing higher conversion efficiency than the traditional one with perfect complement owing to the more negative reaction standard free energy (ΔG). Once traces of input target miRNA interact with the mismatched reactant DNA, amounts of ferrocene (Fc)-labeled output DNA could be converted via the EFTRA. Impressively, the Fc-labeled output DNA could be easily captured by the DNA tetrahedron nanoprobes (DTNPs) on the electrode surface to form triplex-forming oligonucleotide (TFO) at pH = 7.0 for sensitive electrochemical signal generation and the DTNPs could be regenerated at pH = 10.0, from which the conversion efficiency (N) will be accurately obtained, benefiting the selection of suitable mismatched bases to obtain high-efficiency EFTRA (HEEFTRA). As a proof of concept, the HEEFTRA as an evolved DNA signal converter is successfully applied for the ultrasensitive detection of miRNA-21, which gives impetus to the design of other signal converters with excellent efficiency for ultimate applications in sensing analysis, clinical diagnosis, and other areas.
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