Solvent-Engineered Fe-Doped CuCo Prussian Blue Analogue Nanozyme with Enhanced Catalytic Activity for Dual-Mode Colorimetric and Electrochemical Immunoassays

Early and accurate determination of human epidermal growth factor receptor 2 (HER2) is of great significance for the diagnosis and prognosis evaluation of breast cancer. Herein, a structurally transformed Fe-doped hexagon-like CuCo Prussian blue analogue (FeCuCoPBAH) with enhanced peroxidase (POD)-like activity was developed as a nanozyme signal amplifier for dual-mode immunosensing of HER2. Structural characterization indicated that the structural transformation was associated with changes in the local chemical environment of Cu sites and redistribution of Cu oxidation states, which correlated with improved apparent POD-like catalytic performance. Benefiting from the enhanced nanozyme activity, FeCuCoPBAH promoted the TMB/H2O2 chromogenic reaction and showed improved apparent catalytic kinetics compared with the parent materials. On the basis of a glucose oxidase (GOx)-mediated cascade catalytic reaction, a sandwich immunoassay was constructed in which glucose oxidase generated H2O2 in situ, followed by FeCuCoPBAH-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to generate two independently optimized colorimetric and electrochemical readouts based on the same TMB/oxTMB cascade chemistry. Under optimized conditions, the colorimetric mode exhibited a linear response toward HER2 over the range of 0.02-20 ng mL-1 with a detection limit of 8.8 pg mL-1, while the electrochemical mode achieved the linear range of 0.004-20 ng mL-1 with a lower detection limit of 3.8 pg mL-1. In addition, the proposed immunosensor demonstrated excellent selectivity and satisfactory analytical performance. This work provides an effective strategy for enhancing nanozyme activity through solvent regulation and offers a versatile dual-mode sensing platform for sensitive biomarker analysis.