An “off-on” fluorescent sensor based on FRET and magnetic beads for APE1 activity detection in breast cancer cell lysates

Abstract

The activity of apurinic/apyrimidinic endonuclease 1 (APE1) is a key biomarker in breast cancer. We report an “off-on” fluorescent nanosensor (OOFN) that combines fluorescence resonance energy transfer (FRET) with magnetic bead (MB) enrichment for simple and sensitive APE1 detection. The platform employs a MB-anchored FAM-labeled DNA strand (D1) hybridized with a BHQ1-labeled strand containing an AP site (D2). In the absence of APE1, close donor–acceptor proximity quenches fluorescence via FRET. APE1 cleaves the AP site, releases the quencher, and restores fluorescence, while MBs enable easy separation to reduce background. The sensor achieves a detection limit of 0.03 U mL⁻¹ for purified APE1 and can detect activity from as few as 50 breast cancer cells. It reliably distinguishes APE1 levels between normal (MCF-10A) and cancerous (MCF-7, MDA-MB-231) cells, with >96% recovery in serum. This work provides a practical tool for activity-based APE1 detection in cancer research and diagnostics.