Electrochemical detection of S. typhimurium based on peroxidase-like activity of gold nanoparticle-doped CuZr-MOF nanozyme

Abstract

An electrochemical biosensor using gold nanoparticles (AuNPs)-doped bimetallic-organic framework (BMOF) with enhanced peroxidase-like activity was constructed to detect Salmonella Typhimurium (S. typhimurium). The BMOF of CuZr-MOF was synthesized via a two-step method and used as carrier to in situ immobilize AuNPs. Due to the stability of Zr-MOF, the good electrocatalytic ability of Cu (II), and the synergetic effects of AuNPs, Cu (II) and Zr (IV), the prepared AuNPs@CuZr-MOF nanozyme showed improved stability and catalytic activity to H₂O₂ oxidation. The oxidation reaction was found to be a surface-controlled process of electron transfer and a pH-dependent electron transfer process of oxidation reaction involving two electrons. Further, AuNPs@CuZr-MOF was biofunctionalized with signal DNA probe, forming sDNA-AuNPs@CuZr-MOF nanotags. The biosensing platform was constructed on a glassy carbon electrode modified sequentially with electrodeposited AuNPs, capture DNA probe (cDNA), and BSA. Finally, a sandwich-type detection structure was formed by hybridization reactions between cDNA and target invA gene of S. typhimurium, as well as between invA gene and the sDNA of sDNA@AuNPs@CuZr-MOF nanotags. Under optimized experimental conditions, the biosensor achieved a linear range of 1 × 10⁻¹⁶ to 1 × 10⁻⁸ mol L⁻¹ for the target invA gene with a detection limit (LOD) of 6.2 × 10⁻¹⁷ mol L⁻¹ using differential pulse voltammetry measurement (DPV). It was successfully applied to the direct and quantitative detection of invA gene segments in total DNA extracts of S. typhimurium, showing a linear range from 3.5 to 3.5 × 10⁶ CFU mL⁻¹ and a LOD of 0.82 CFU mL⁻¹. The fabricated biosensor exhibited good selectivity, reproducibility, and storage stability, enabling its use for the detection of invA gene segments in contaminated milk, with recoveries between 95.9% and 103.1%.

Graphical Abstract