Cryo-deposited Au shells amplify nanozyme catalysis: A Km-enhanced immunoassay for mycotoxin screening.

This study introduces an innovative freeze-induced Au deposition-assisted catalytic enhancement strategy addressing the critical challenge of nanozyme activity loss in enzyme-linked immunosorbent assay (ELISA). Conventional biomolecule immobilization was found to dramatically inhibit Au@Pt nanoparticle activity, increasing the Michaelis constants (Km) value for H2O2 from 22.4 ± 1.8 mM to 84.2 ± 2.9 mM. Our cryogenically aligned ice-templated assembly strategy achieved uniform gold deposition on Au@Pt nanoparticles, thereby not only restored but also enhanced catalytic performance (Km = 18.8 ± 1.3 mM). First-principles calculations reveal the atomic-scale enhancement mechanism. Interfacial charge redistribution (0.09e vs. 0.32e in Au@Pt) creates electron-deficient sites that lower reaction barriers by 16.7% (-1.8 eV vs. -1.5 eV) while maintaining Pt 5d orbital dominance. Furthermore, projected density of states analysis confirms Au-mediated O-2p/H-1s hybridization that stabilizes reaction intermediates. The engineered freeze-induced gold enhancement Au@Pt-based ELISA (Au@Pt/Au-ELISA) achieved an ultra-sensitive limit of detection (LOD) of 5.2 pg/mL for zearalenone (a 7-fold improvement over conventional ELISA) while concurrently enabling 16-fold higher probe dilution and a 48-fold reduction in antibody consumption, markedly reducing assay cost. The same enhancement trend was observed for sterigmatocystin (ST) and aflatoxin B1, with the ST assay showing an impressive 35-fold LOD improvement. Validation with real coix seed samples demonstrated strong correlation with regulatory LC-MS/MS confirmatory results (r = 0.9811). This study provides a significant tool for nanozyme activity preservation in immunoassays, which is essential for biosensing applications. Meanwhile, the observed influence of ice crystallization on metal atom assembly behavior may provide new mechanistic insights into the fabrication of noble metal nanozymes.