Electrochemical immunoassay for Salmonella Typhimurium based on magnetically collected Ag-enhanced DNA biobarcode labels

Abstract

We describe a sensitive electrochemical immunoassay for Salmonella enterica serovar Typhimurium, a common foodborne pathogen which can cause infection at extremely small doses. The assay is based on the recognition of DNA biobarcode labels by differential pulse anodic stripping voltammetry (DPASV), following Ag enhancement. The biobarcodes consist of latex spheres (mean diameter 506 nm ± 22 nm) modified by ferromagnetic Fe3O4 particles. Each biobarcode is loaded by adsorption with approx. 27 molecules of mouse monoclonal antibody against S. Typhimurium and 3.5 × 10(5) molecules of 12 mer ssDNA. The assay is performed by adding the biobarcode, S. Typhimurium cells, and biotin-conjugated rabbit polyclonal antibody against Salmonella into well plates. After antigen-antibody binding, magnetic collection enables the excess polyclonal antibody to be washed off. Exposure to avidin-coated screen printed electrodes, and formation of the avidin-biotin bond, then enables the excess biobarcode to be removed. The biobarcode remaining on the electrode is quantified by DPASV measurement of Ag(+) ions following catalytic Ag deposition. The assay showed a negligible response to 10(7) CFU mL(-1)E. coli and had a limit of detection of 12 CFU mL(-1) in buffer, and 13 to 26 CFU mL(-1) for heat-killed and whole cell S. Typhimurium in plain milk, green bean sprouts and raw eggs. To the best of our knowledge, this is the lowest reported limit of detection for Salmonella by an electrochemical immunoassay not requiring sample pre-enrichment.

Electrochemical Immunoassay for Salmonella Typhimurium Based on an Immuno‐magnetic Redox Label

“…Table lists the electrochemical labels which have been used for Salmonella immunoassays since 2000 . It can be seen that the platform described here is highly competitive with previous methods, having a lower LOD than all but one than all but one assay (which required a longer time) and being faster than all but one assay (which reported a higher LOD).…”

Highly sensitive electrochemical detection of genomic DNA based on stem loop probes structured for magnetic collection and measurement via metalised hollow polyelectrolyte shells

“…For this situation to be realised the magnetised particle needs to be relatively large. To meet this criteria we adapted a form of magnetic labeling previously developed for a biobarcode immunoassay (Pratiwi et al, 2013). This used magnetic Fe 3 O 4 particles coated with the silane APTS.…”

Electrochemical Biosensors for Rapid Detection of Foodborne Salmonella: A Critical Overview

“…An immunoassay format was used to electrochemically detect S . Typhimurium [ 50 ], according with the biobarcode method developed by Mirkin and co-workers [ 51 ].…”

“…In the presence of the target bacterium, the aptamer was displaced from the capture probe by binding the pathogen and, successively, the addition of a detection probe/gold nanoparticles/alkaline phosphatase structure allowed the capture probe to be hybridized. As for their previous work [ 50 ], the electrochemical signal was recorded following the oxidation of the alkaline phosphatase by-product in presence of α-naphthyl phosphate (substrate). A linear dynamic range from 20 to 2 × 10 6 CFU/mL and a detection limit of 20 CFU/mL were obtained.…”