Rolling circle amplification-triggered loop-mediated isothermal amplification and in-situ fluorescence signal transduction for one-tube APE1 sensing.

چکیده اصلی

BACKGROUND: Molecular diagnostics heavily depend on accurate and sensitive biomarker sensing via efficient signal amplification and facile signal transduction. As a promising amplification strategy, isothermal polymerization delivers excellent structural stability and amplification efficiency for biomedical analysis. Different from conventional fluorophores with exogenous signal regulation, in-situ DNA-templated copper nanoparticles (CuNPs) enable an endogenous signaling strategy. It is reasonable to infer that isothermal polymerization amplification is highly compatible with DNA-templated CuNPs-mediated generative signal transduction, which effectively suppresses signal leakage and improves detection reliability and fidelity. RESULTS: We developed a one-tube method for ultrasensitive apurinic/apyrimidinic endonuclease 1 (APE1) sensing based on rolling circle amplification (RCA)-triggered loop-mediated isothermal amplification (LAMP), and fluorescent DNA-templated CuNPs-enabled in-situ signal transduction. Once the target recognition probe was specifically cleaved by APE1, linear primers were released and immediately triggered RCA. The resulting long-chain products directed the polymerization of hairpin primers into seed DNAs, which subsequently initiated LAMP under continuous catalysis by the same polymerase. The yielded abundant double-stranded DNA products, facilitating efficient fluorescence output through in-situ formation of CuNPs on AT-rich sequences. This streamlined workflow enabled simple and sensitive APE1 detection in a one-tube format with a low detection limit of 3.1 × 10-4 U/mL. Moreover, the proposed method exhibited remarkable selectivity and robust anti-interference performance, allowing for the unambiguous differentiation between tumor cells and normal cells via cellular APE1 detection. SIGNIFICANCE: Owing to its modular design, the sensing strategy can be easily extended to detect other DNA repair enzymes by simply engineering the recognition module, demonstrating excellent sensing versatility and promising application potential in advanced molecular diagnostics. Overall, this work establishes a novel paradigm for the seamless integration of polymerization-driven signal amplification and generation-mediated signal transduction, offering fresh possibilities for simple and sensitive biosensing.

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