Dual-Signal Amplification Strategy Based on Catalytic Hairpin Assembly and APE1-Assisted Amplification for High-Contrast miRNA Imaging in Living Cells

Early tumor diagnosis is crucial to successful treatment. Earlier studies have shown that microRNA is a biomarker for early tumor diagnosis. The development of highly sensitive miRNA detection methods, especially in living cells, plays an indispensable role for early diagnosis and treatment of tumor. Although the catalytic hairpin assembly (CHA)-based miRNA analysis strategy is commonly used for disease diagnosis, further application of CHA is hindered due to its low amplification efficiency and low tumor recognition contrast. To address these limitations, we propose a dual-signal amplification strategy based on CHA and APE1-assisted amplification, enabling highly sensitive and high-contrast miRNA imaging. The miR-221 was selected as a target model. This dual-signal amplification strategy has exhibited high amplification efficiency, which could analyze miRNA as low as 21 fM. This strategy also exhibited high specificity, which could distinguish target miRNA and nontarget with single-base differences. Moreover, this method showed significant potential for practical application, as it could successfully distinguish the expression difference of miR-221 in the plasma samples of normal people and patients. Most importantly, the expression level of the APE1 enzyme in tumor cells is higher than that in normal cells, allowing this strategy to sensitively and specifically image miRNA within tumor cells. This proposed method has also been successfully used to indicate fluctuations of intracellular miRNA and to distinguish miRNA expression between normal cells and cancer cells with high contrast. We anticipate that this method will provide fresh insights and can be a powerful tool for tumor diagnosis and treatment based on miRNA analysis.