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

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 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 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.