Target-triggered substantial stacking of electroactive indicators based on digestion-to-growth regulated tandem isothermal amplification for ultrasensitive miRNA determination

Abstract Ultrasensitive detection of microRNA (miRNA) has well-demonstrated values for cancer diagnosis, prognosis and therapy. In this work, taking advantages of the designed versatility of oligonucleotides and the robustness of electrochemistry, a unique digestion-to-growth regulated tandem isothermal amplification (DG-TIS) based electrochemical biosensor has been developed for miRNA-21. In DG-TIS system, two hairpin probes (HP1 and HP2) are used as the enzymatic reaction unit. Target miRNA firstly hybridize with HP1 to induce the T7 exonuclease aided-digestion amplification (T7 Exo-ADA) for HP1 digestion. The produced hydrolysis residue strand then captures HP2 to induce the Klenow polymerase promoted-growth amplification (Klenow Poly-PGA) for HP2 growth. The tandem connection of T7 Exo-ADA and Klenow Poly-PGA makes the DG-TIS finally contribute considerably to the electrochemical responses by stacking tremendous electrochemical active tags. This biosensor with the unique digestion-to-growth regulation for miRNA sensing, as a proof of concept, showed a wide linear response range over 4 orders of magnitude, a lower detection limit down to 34.8 fM, and an excellent specificity to easily distinguish even one-base mismatched analyte. Impressively, this electrochemical biosensor also offered an acceptable practical applicability to interrogate the expression levels of miRNA-21 in cancer cells and spiked human serum. These features do make DG-TIS based electrochemical biosensor an ideal analytical tool for diagnostic applications.