Palindrome-Mediated Isothermal Cascade DNA Amplification and Effortless Nanosignal Transduction for One-Pot and Ultrasensitive miRNA Sensing

In this work, we developed an ultrasensitive method for microRNA (miRNA) sensing based on palindrome-mediated isothermal cascade nicking/polymerization for DNA amplification and fluorescent copper nanoparticle (CuNP) generation for signal transduction. With rational palindrome design, only two DNA hairpins and a pair of enzymes were needed to realize one-pot and two-step miRNA detection. In the first step, the target miRNA unfolded the nicking site-containing RNA Probe that subsequently hybridized with the palindromic sequence-engineered hairpin and initiated multiple isothermal cycles driven by polymerase and endonuclease to complete target recognition and signal amplification. Then, the abundant DNA templates accumulated in the previous step facilely and rapidly guided the in situ generation of fluorescent CuNPs for signal transduction. The efficient isothermal amplification and effortless signal transduction jointly achieved ultrasensitive miRNA-21 sensing with a low detection limit (9.7 fM) in a simple and convenient manner. In addition, thanks to its high selectivity and anti-interference ability, the method was able to unambiguously distinguish cancer cells from normal cells based on the test results of cellular miRNA-21. Moreover, this method also enables the detection of different miRNAs simply by modifying the probe sequence, which demonstrates high sensing versatility and application potential in advanced molecular diagnostics.