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While detection of microRNA with or without signal amplification is highly informative, nanosensors with high specificity for cell-specific RNA detection are rare. Methods: In this study, a tetrahedral DNA nanostructure (TDN) with a specific function was combined with gold nanoparticles (Au-NP) possessing fluorescence quenching effects and a large surface area to fabricate a fluorescence resonance energy transfer based nanosensor (Au-TDNN). The presence of miR-21 (target) can separate the fluorescent dye-labeled detection probe on Au-TDNNs from Au-NPs, which separates the donor and acceptor, thus inducing an intensive fluorescence signal. High specificity for discerning point mutation targets was achieved by rationally designing the nucleic acid strand displacement reaction to occur spontaneously with ΔG 0 ≈ 0 based on thermodynamic parameters; under this condition, slight thermodynamic changes caused by base mismatch exert significant effects on hybridization yield. Results: Chemically synthesized DNA of three single-base-changed analogues of target, let-7d, and miR-200b were tested. A discrimination factor (DF) of 15.4 was produced by the expected detection probe on Au-NPs for proximal single-base mismatch. As the control group, the DF produced by an ordinary detection probe on Au-NPs only reached 2.4. The feasibility of the proposed strategy was also confirmed using hepatocyte cancer cells (HepG2). Conclusion: This improved nanosensor opens a new avenue for the specific and easy detection of microRNA in live cells.

High-Discrimination Factor Nanosensor Based on Tetrahedral DNA Nanostructures and Gold Nanoparticles for Detection of MiRNA-21 in Live Cells