Catalytic boost in dual-active sites hierarchical hollow nanozymes: An enhanced robust and sensitive immunochromatographic assay for Aspergillus flavus

The practical application of nanozyme-mediated Point-of-Care Testing (POCT) is always constrained by insufficient sensitivity, slow development time, and limited robustness. Here, we developed a dual-active site hierarchical hollow CuS@MIL-100(Fe) (CuS@MIL) nanozyme through an easy-to-operate process. The resultant CuS@MIL nanozyme exhibited a 5- and 3-fold increase in catalytic efficiency relative to CuS and MIL-100, while the color development time was shortened by more than 6-fold. Meanwhile, CuS@MIL retained 91.4% of its catalytic activity after six months of storage, indicating its robustness. Density functional theory (DFT) revealed that the improved catalytic activity of CuS@MIL nanozyme was associated with low energy barriers for •OH generation and efficient interfacial electron transfer at Cu-Fe dual-active sites. Leveraging their high and stable catalytic performance, a sensitive colorimetric lateral flow immunoassay (LFA) was applied for detection of Aspergillus flavus (A. flavus), achieving a 200- and 5.6-fold increase in sensitivity compared to conventional and nanozyme-based colorimetric methods. Attractively, the LFA effectively detected A. flavus in peanuts with results closely matching those of ELISA, demonstrating the potential for rapid, efficient, sensitive and robust colorimetric POCT.