An ultrasensitive biosensor for fast detection of Salmonella using 3D magnetic grid separation and urease catalysis

Screening of pathogenic bacteria plays a crucial role in preventing foodborne disease outbreaks. In this study, an ultrasensitive biosensor was developed for fast detection of Salmonella using self-assembled magnetic nanoparticle (MNP) chains for continuous-flow separation of Salmonella from large-volume sample, urease coated gold nanoparticles (GNPs) for specific labelling of Salmonella and efficient amplification of signal, and linear scan voltammetry for sensitive detection of catalysate. First, MNP chains were formed and distributed in a 3D spiral channel using mutually repelling cylindrical magnets and ring iron gears to control anti-Salmonella monoclonal antibody coated MNPs. After bacterial sample was continuous-flow drawn into the channel, bacteria-MNP complexes (magnetic bacteria) were formed on the chains, resulting in specific separation of target bacteria from sample background. Then, anti-Salmonella polyclonal antibodies and urease coated GNPs were drawn to label the magnetic bacteria, resulting in the formation of enzymatic bacteria. After washing to remove residual GNPs, urea was drawn and catalyzed by urease on enzymatic bacteria, resulting in the produce of catalysate (ammonium carbonate). Finally, the catalysate was transferred into a microfluidic chip with a thin-film Ag/AgCl reference electrode array for linear scan voltammetric measurement, and the resistance of catalysate was obtained to determine the amount of target bacteria. This biosensor could quantitatively detect Salmonella from 1.0 × 10¹ to 1.0 × 10⁶ CFU/mL in 1 h with low detection limit of 10¹ CFU/mL. The mean recovery for Salmonella in spiked milk was about 104.3%.