An Electrochemiluminescence Biosensor Based on Ti₃C₂/AuNPs/CsPbBr₃ Nanocomposite and PAM-Regulated CRISPR-Cas12a Signal Amplification for Highly Sensitive Detection of APE1

Apurinic/apyrimidinic endonuclease 1 (APE1), a key enzyme in the base excision repair pathway, is frequently overexpressed in multiple cancers and has emerged as an important biomarker for early diagnosis and therapeutic response evaluation. In this work, a highly sensitive electrochemiluminescence (ECL) biosensor was developed for APE1 detection by integrating a PAM-regulated CRISPR-Cas12a activation mechanism with a multifunctional nanocomposite-modified electrode. A Ti₃C₂/AuNPs/CsPbBr₃ ternary nanocomposite was fabricated through ultrasonic assembly to provide enhanced conductivity, water stability, and probe-loading capacity. Meanwhile, a DNA duplex containing an AP site within the PAM region was engineered to regulate Cas12a activation through APE1-mediated cleavage and polymerase-assisted repair, thereby minimizing background interference and enabling highly specific signal generation. Ferrocene-labeled DNA tetrahedrons (TDNs) were further introduced as a spatially ordered probe scaffold to improve hybridization efficiency and facilitate Cas12a trans-cleavage–mediated signal release. This work presents three key innovations: (1) Construction of a Ti₃C₂/AuNPs/CsPbBr₃ nanocomposite that simultaneously enhances ECL stability, conductivity, and electron-transfer efficiency, addressing the inherent water-instability of perovskite emitters. (2) Coupling of a PAM-regulated APE1–KF repair mechanism with Cas12a activation to achieve highly specific AP-site recognition and selective signal generation. (3) Introduction of a TDN-based probe architecture that provides rigid spatial orientation and high-density probe loading, markedly improving hybridization efficiency and Cas12a trans-cleavage performance. Benefiting from these synergistic innovations, the biosensor achieves a detection limit of 6.28 × 10⁻⁸ U μL⁻¹ and a wide linear range (1 × 10⁻⁷ to 1 × 10⁻ 1 U μL⁻¹). The platform also demonstrates excellent specificity, reproducibility, and reliable performance in diluted serum, highlighting its promise for clinical and biochemical applications.