The development of co-reactant-free electrochemiluminescence (ECL) is of great significance for simplifying the analysis process, reducing background interference, and enhancing the detection sensitivity in complex biological matrices. In this work, a ternary CuFeV layered double hydroxide (LDH) was synthesized via a facile method and employed as an efficient ECL electrocatalytic material. Without the addition of any co-reactants, CuFeV-LDH could effectively catalyze the in-situ transformation of O2 and H2O into reactive oxygen species (ROS), thereby triggering the generation of a strong and stable ECL signal in the luminol system. Experimental characterization combined with density functional theory (DFT) calculations confirms that the introduction of Vanadium optimizes the spin-orbital distribution of the active Cu and Fe centers. This modulation significantly facilitates the charge transfer of oxygen species at the catalyst interface, inducing the generation of abundant ROS to drive ECL. Based on the effective scavenging effect of glutathione (GSH) on ROS, a sensitive ECL sensing platform for GSH detection was further constructed. The sensor exhibited a good linear relationship in the concentration range from 1 × 10−9 to 1 × 10−5 M, with a low detection limit of 0.3 nM. In addition, the proposed sensor demonstrated excellent selectivity, long-term stability, and practical applicability in real sample analysis, providing a new strategy for the development of novel co-reactant-free ECL biosensing platforms.
