Localized Cas12a-based cascade amplification for sensitive and robust detection of APE1.

APE1, an essential enzyme for DNA repair, is overexpressed in various cancers and has been identified as a potential biomarker for cancer diagnosis. However, detecting APE1 at low expression levels in the early stage of cancer presents a significant obstacle. Here, we introduced a novel localized Cas12a-based cascade amplification (LCas12a-CA) method. This method confined both the terminal deoxynucleotidyl transferase and the crRNA/Cas12a complex onto the surfaces of gold nanoparticles (AuNPs). This confinement not only boosts the stability of the multiple enzymes but also induces a substrate channeling effect. As a result, it significantly accelerates the reaction rate and enhances the sensitivity of APE1 detection. Upon the addition of APE1, the AP sites within the APE1 primer can be recognized and cleaved by APE1, exposing the 3'-OH ends. In the presence of LCas12a-CA, polyA sequences are generated at 3'-OH ends with the help of TdT and dATP. The sequences directly enter the Cas12a system, activating the trans-cleavage activity of Cas12a, thereby cutting the reporters on the surface of AuNPs and releasing fluorescence. Our platform demonstrates a detection limit (LOD) as low as 2.51 × 10 U/mL, which is more than 60 times lower than that of free Cas12a-CA. Furthermore, the LCas12a-CA exhibits enhanced resistance ability in extreme environments and has been proven effective for the detection of APE1 in clinical samples. Overall, this work offers a promising platform for robust biosensing in cancer diagnosis and prognosis.