A sandwich electrochemical immunoassay for Salmonella pullorum and Salmonella gallinarum based on a AuNPs/SiO2/Fe3O4 adsorbing antibody and 4 channel screen printed carbon electrode electrodeposited gold nanoparticles

Abstract

A rapid and highly sensitive sandwich electrochemical immunoassay method was constructed for Salmonella pullorum and Salmonella gallinarum (S. pullorum and S. gallinarum) determination based on immune magnetic beads (MBs) and an enzyme labeled antibody. An abundance of gold nanoparticles (AuNPs) were attached to SiO₂ coated Fe₃O₄ nanoparticles (Fe₃O₄/SiO₂) via covalent binding between the –SH groups of Fe₃O₄/SiO₂ and the AuNPs. Antibodies against S. pullorum and S. gallinarum were immobilized on Fe₃O₄/SiO₂/AuNPs nanocomposites (AuMNPs) by automatic adsorption between thiol and the AuNPs. S. pullorum and S. gallinarum in the sample were captured by the AuMNPs and separated from the samples by applying an external magnetic field. The AuMNPs–Salmonella complexes (Ag/Ab₁/AuMNPs) were re-dispersed in a buffer solution then exposed to horseradish peroxidase labeled anti-S. pullorum and S. gallinarum (HRP-Ab₂) solution, forming a sandwich-type immune complex (HRP-Ab₂/Ag/Ab₁/AuMNPs). A 4 channel screen printed carbon electrode (4-SPCE) was modified by gold nanoparticles (AuNPs) through the electrodeposition method to prepare AuNPs/4-SPCE. After magnetically separating the sandwich immune complexes from solution, HRP-Ab₂/Ag/Ab₁/AuMNPs was anchored on AuNPs/4-SPCE by a magnet. A linear response to S. pullorum and S. gallinarum was obtained in the concentration range from 10² to 10⁶ CFU mL⁻¹, with a limit of detection of 3.2 × 10¹ CFU mL⁻¹ (at an SNR of 3). This nanoparticle-based immunoassay method offers sensitive, highly specific, and reproducible detection of S. pullorum and S. gallinarum. Given its low detection limit, it represents promising potential in the detection of other food-borne pathogens by exchanging the antibody.