Label-Free and Ultrasensitive APE1 Detection Based on Hybridization Chain Reaction Combined with G-Quadruplex.

Abstract

Apurinic/apyrimidinic endonuclease 1 (APE1) selectively cleaves the apurinic/apyrimidinic site (AP site) in DNA, playing a critical role in base excision repair and genomic stability maintenance. Aberrant APE1 expression has been linked to numerous diseases, including cardiovascular disorders, neurological conditions, and various cancers. However, existing methods for detecting trace levels of APE1 remain suboptimal for certain applications. To address this limitation, we developed an innovative biosensing platform for ultrasensitive APE1 detection by integrating APE1-specific site recognition with hybridization chain reaction (HCR)-based signal amplification, enabling enzyme- and label-free bioassays. In this system, APE1 recognizes and cleaves the AP site-containing hairpin (HP) probe, releasing a single-stranded HCR initiator that triggers cascaded HCR amplification. Owing to the high efficiency of HCR, this method achieves ultrahigh sensitivity, with a calculated detection limit of 1.0 × 10^-8 U/mL. Furthermore, the biosensor demonstrates robust performance in cell lysates and is applicable for screening and evaluating APE1 inhibitors.