Sensitive Chemiluminescent Immunoassay Based on Synergistic Antioxidative Behavior Between Atomically Dispersed Mn Active Centers and Defect-Rich CuFe-Layered Double Hydroxide

Single atom nanozymes (SAEs), featuring atomically dispersed metallic active centers and maximum atomic utilization efficiency, have demonstrated outstanding catalytic activity compared with conventional nanozymes. The interaction between metallic active centers and carrier may play a crucial role for the improvement of catalytic performance of SAEs. In this work, we anchored an atomically dispersed Mn element on CuFe-layered double hydroxide with Mn content of 2.15 wt % through a defect engineering strategy, creating a synergistic dual-site catalyst denoted as Mn SAE. Owing to the interaction between the carrier and the metallic active centers, Mn SAE exhibited significantly enhanced superoxide dismutase- and catalase-mimicking activities. Additionally, it also demonstrated remarkable capability for scavenging hydroxyl radicals (•OH) and singlet oxygen (¹O₂). Thus, Mn SAE with synergistic antioxidant activity can be a powerful quenching agent for reactive oxygen species-mediated luminol-H₂O₂ chemiluminescence. By using it as a sensitive probe, we developed a chemiluminescent immunoassay method for fentanyl, with a linear range of 0.001-25 ng/mL and a detection limit of 0.3 pg/mL. Its practicability was successfully demonstrated by detecting fentanyl in diverse real samples. This investigation provides a new pathway for the development of synergistic dual-site SAEs with multiple antioxidant activities that are suitable for application in biosensing.