A highly sensitive electrochemical sandwich immunoassay is described for determination of Escherichia coli O157:H7 (E. coli O157:H7). Silica coated magnetite nanoparticles (FeO) were modified with primary antibody to capture E. coli O157:H7. Gold-platinum core/shell nanoparticles (Au@Pt NPs) with different Pt shell thicknesses were prepared via changing the molar ratio of HPtCl to HAuCl in the precursor solution. The optimized Au@Pt NPs exhibit enhanced activity in the electrocatalytic reduction of hydrogen peroxide (HO). The Au@Pt NPs were modified with graphene that was functionalized with Neutral Red, and then used as an electrochemical label for secondary antibodies and horseradish peroxidase (HRP). The sandwich immunocomplexes were magnetically absorbed on a 4-channel screen printed carbon electrode. Due to the catalysis of the Au@Pt NPs and HRP, the signal is strongly amplified in the presence of HO when using thionine as the electron mediator. Under optimal conditions, the immunoassay has a linear response in the 4.0 × 10 to 4.0 × 10 CFU·mL concentration range, with a limit of detection of 91 CFU·mL (at an S/N ratio of 3). Graphical abstract Preparation of Au@Pt core/shell nanoparticles with different Pt shell thickness (A), rGO-NR (B), rGO-NR-Au@Pt-Ab-HRP (C) and the preparation and the detection process of the immunoassay (D). rGO: reduced graphene oxide, GO: graphene oxide, NR: Neutral Red, HRP: horseradish peroxidase, AuNPs: gold nanoparticles, FeO@SiO: Silica coated magnetite nanoparticles, 4-SPCE: 4-channel screen printed carbon electrode.
The authors describe a sandwich-type of electrochemical immunoassay for rapid determination of the foodborne pathogen Cronobacter sakazakii (C. sakazakii). Polyclonal antibody against C. sakazakii (anti-C. sakazakii) and horseradish peroxidase were immobilized on a nanocomposite consisting of reduced graphene oxide, thionine and gold nanoparticles (AuNPs) that was placed on a screen-printed carbon electrode (SPCE). Thionine acts as an electron mediator which also shortens the electron transfer pathway from the conjugated HRP to the electrode surface and amplifies the electrochemical signal. The AuNPs, in turn, improve the electron transfer rate and increase the surface area for capturing antibody. The morphologies of the electrodes were characterized by means of field emission scanning electron microscopy. The electrochemical performance of the immunoassay was evaluated by cyclic voltammetry and differential pulse voltammetry. Under optimal experimental conditions, the electrochemical immunoassay, best operated at a woking potential of -0.18 V (vs. Ag/AgCl) and scan rate of 20 mV/s has a linear response that covers the 8.8 × 10 to 8.8 × 10 CFU·mL C. sakazakii concentration range, with a 1.0 × 10 CFU·mL detection limit (at an S/N ratio of 3). The assay was applied to the detemination of C. sakazakii in spiked infant milk powder and gave recoveries ranging from 92.0 to 105.7%. Graphical abstract A sandwich-type electrochemical immunosensor was designed for C. sakazakii based on the use of rGO. TH, HRP, antibody and AuNPs were anchored on rGO. The nanocomposites were used as traces tag and HO as enzyme substrates. AuNPs were modified on SPCE by electrodeposition.
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