Exponential amplification-based one-pot method for sensitive and reliable non-small cell lung cancer-related MicroRNA detection mediated by multiple target-recycling

Abstract MicroRNAs (miRNAs) represent significant biomarkers because their aberrant expression or mutation frequently indicates the development of cancers, including non-small cell lung cancer. However, miRNA analysis is substantially hindered by their low abundance and high sequence homology within families. This study devised a one-pot, label-free method for the highly sensitive detection of miRNA-21. The approach integrates G-quadruplex/thioflavin T (ThT) complex formation with DNA polymerase/endonuclease activity within a multiple target-recycling exponential amplification (EXPAR) framework. Specific miRNA-21 target sequences induce self-priming of a “dumbbell probe”, initiating strand extension-driven recycling cycles catalyzed by DNA polymerase and endonuclease. This process generates numerous active G-quadruplex sequences. ThT dye binding to these G-quadruplexes produces a significantly amplified fluorescence signal, enabling ultra-sensitive detection across a dynamic range of 100 aM–100 pM, with a limit of detection of 35 aM. Innovatively, this method employs target recognition-mediated self-priming to induce strand extension and displacement, thereby minimizing non-specific primer-template hybridization inherent in conventional EXPAR strategies. Demonstrating high selectivity, the method was successfully applied to diluted serum samples. Consequently, this novel detection strategy presents significant potential for miRNA biomarker analysis in clinical diagnostics and biomedical research.