Nanozyme-driven fenton-RAFT polymerization for highly sensitive PEC–CL dual-mode biosensing of caspase-3

Reversible addition–fragmentation chain-transfer (RAFT) polymerization offers an effective strategy for signal amplification in sensitive biosensing. Herein, we developed a novel photoelectrochemical–colorimetric (PEC–CL) dual-mode biosensing platform for the highly sensitive detection of caspase-3 activity, based on the RAFT polymerization initiated by nanozyme-driven Fenton reaction. Specifically, a magnetic CuFe₂O₄ nanozyme was modified with chain transfer agents (CPAD) to form CPAD-CuFe₂O₄, which was immobilized on the sensing interface via a caspase-3-cleavable DEVD peptide linkage. Upon the introduction of caspase-3, the functionalized CuFe₂O₄ nanozyme was released and catalyzed the Fenton reaction to generate hydroxyl radicals (•OH), thereby initiating RAFT polymerization of Hemin-fixed vinyl monomers (Hemin-VM). The resulting poly(Hemin-VM) (pHemin-VM) was in situ grafted onto the surface of nanozyme, forming a pHemin-VM/CuFe₂O₄ composite that enhanced •OH generation through a positive feedback mechanism, consequently further amplifying the sensing signals. Finally, caspase-3 was sensitively detected through PEC responses arising from polarity switching at the SnS₂/MITO electrode and CL signals generated by the oxidation of 3,3’,5,5’-tetramethylbenzidine (TMB). The proposed PEC–CL dual-mode sensing platform exhibited a wide linear range (PEC: 10⁻¹⁶–10⁻⁸ g mL⁻¹, CL: 10⁻¹⁵–10⁻⁸ g mL⁻¹) and low detection limit (5.0 × 10⁻¹⁷ g mL⁻¹ for PEC, and 2.7 × 10⁻¹⁶ g mL⁻¹ for CL) for caspase-3 activity assay. This study introduced a nanozyme-driven Fenton chemistry-initiated RAFT polymerization signal amplification strategy, and expanded its application in biosensing, offering a robust platform for dual-mode detection of disease biomarkers and advancing next-generation biosensor development.