Co-Cu bimetallic MOF-derived nanozyme-based colorimetric immunoassay with smartphone-based RGB readout for sensitive detection of alpha-fetoprotein

Nanocatalysts based on metal-organic frameworks exhibit limited catalytic activity due to their symmetrical inactive structures, thereby restricting their application in nanozyme-linked immunoassays (NLISA). This work constructed geometrically structure expanded Cu-Co bimetallic metal-organic frameworks (GS-CCBM) nanozymes through a two-step hydrothermal and alkali treatment, enabling a specific nanozyme-based immunoassay for the target alpha-fetoprotein (AFP). The constructed GS-CCBM nanozyme demonstrated excellent substrate affinity (Km = 1.79 mM) and H2O2 reaction rate (Vm = 8.569 μmol min-1). Based on this, a signal probe was constructed that enables changes in the absorbance and color of the catalytic system in the presence of the target AFP. Semi-quantitative and quantitative analyses were performed using a self-developed color value extraction system. Under optimized conditions, the constructed NLISA method demonstrated an acceptable working range (0.01–50 ng mL⁻¹) and a sensitive detection limit (LOD = 4.7 pg mL⁻¹) for AFP. This approach innovatively proposes a novel strategy for constructing highly efficient MOF-based nanozymes with catalytic activity, offering a new method for developing novel clinical detection techniques.