Aptakiss-guided CRISPR/Cas13a signal amplification for ultrasensitive FEN1 detection using CsPbBr₃@PDA@AuNPs-based...

Sensitive and reliable detection of DNA repair enzymes is critical for early cancer diagnosis and therapeutic monitoring. Herein, we report a novel electrochemiluminescence (ECL) biosensor for ultrasensitive detection of Flap Endonuclease 1 (FEN1), integrating an aptakiss-assisted CRISPR/Cas13a signal amplification strategy with a perovskite-based nanocomposite sensing interface. Specifically, a hybrid nanomaterial composed of CsPbBr₃ nanocrystals coated with polydopamine (PDA) and decorated with gold nanoparticles (AuNPs) was constructed to form a core-shell-satellite structure (CsPbBr₃@PDA@AuNPs). The PDA coating enhanced the aqueous stability of CsPbBr₃ and introduced functional groups for probe attachment, while the AuNPs facilitated electron transfer and signal amplification. Upon recognition and cleavage of a flap-structured DNA substrate by FEN1, a downstream transcription reaction was initiated to generate RNA triggers, which in turn activated the Cas13a system. Cas13a cleaved a surface-tethered RNA strand required for forming the aptakiss complex, thereby removing ferrocene-based ECL quenching and restoring strong luminescence. The proposed biosensor exhibited an excellent detection limit of 1.73 fM, with high selectivity against non-specific nucleases, and demonstrated remarkable reproducibility and long-term stability. This study not only presents a powerful biosensing platform for FEN1 but also highlights the potential of CsPbBr₃@PDA@AuNPs as a versatile ECL-active material. The modular design and tunable functionality of the nanocomposite enable broad applicability in detecting various biomolecules, paving the way for the development of advanced perovskite-based ECL biosensors for clinical and environmental diagnostics.