CHA-mediated split G-quadruplex assembly: a ratiometric AIE strategy for label-free detection of APE1

Apurinic/apyrimidinic endonuclease 1 (APE1), a critical enzyme in the base excision repair pathway, is a potential biomarker for numerous cancer types. Traditional APE1 activity detection methods are hindered by complex protocols, expensive labeling requirements, and vulnerability to environmental factors. Herein, we develop a label-free ratiometric fluorescent biosensor (TCHG) that integrates APE1-mediated catalytic hairpin assembly (CHA) amplification with split G-quadruplex/malachite green (MG)-mediated aggregation-induced emission (AIE) signaling for sensitive activity analysis of APE1. Our system operates through APE1's specific cleavage of apurinic/apyrimidinic (AP) site-containing double-stranded substrates, which releases trigger strands that initiate a CHA cascade. This cascade drives the assembly of split G-quadruplex structures. Upon reconstruction, the complete G-quadruplex binds MG, generating amplified near-infrared fluorescence with AIE characteristics. Simultaneously, Hoechst 33258 (HOE) embedded within double-stranded DNA provides a stable reference signal, enabling self-calibrated ratiometric output that effectively minimizes signal fluctuations from instrumental and environmental variations. The TCHG biosensor demonstrates high selectivity for APE1 with a detection limit of 2.4 × 10^-3 U/mL. Importantly, it maintains reliable analytical performance in complex biological matrices, including fetal bovine serum and various cell lysates, successfully differentiating endogenous APE1 activity between normal and cancer cells. The TCHG system offers a tool for APE1 activity analysis in biological samples, with potential applications in biomedical research.