Magnetic Nanoparticles Enhanced Surface Plasmon Resonance Biosensor for Rapid Detection of Salmonella Typhimurium in Romaine Lettuce

Abstract

Salmonella is one of the major foodborne pathogens responsible for many cases of illnesses, hospitalizations and deaths worldwide. Although different methods are available to timely detect Salmonella in foods, surface plasmon resonance (SPR) has the benefit of real-time detection with a high sensitivity and specificity. The purpose of this study was to develop an SPR method in conjunction with magnetic nanoparticles (MNPs) for the rapid detection of Salmonella Typhimurium. The assay utilizes a pair of well-characterized, flagellin-specific monoclonal antibodies; one is immobilized on the sensor surface and the other is coupled to the MNPs. Samples of romaine lettuce contaminated with Salmonella Typhimurium were washed with deionized water, and bacterial cells were captured on a filter membrane by vacuum filtration. SPR assays were compared in three different formats-direct assay, sequential two-step sandwich assay, and preincubation one-step sandwich assay. The interaction of flagellin and MNPs with the antibody-immobilized sensor surface were analyzed. SPR signals from a sequential two-step sandwich assay and preincubation one-step sandwich assay were 7.5 times and 14.0 times higher than the direct assay. The detection limits of the assay were 4.7 log cfu/mL in the buffer and 5.2 log cfu/g in romaine lettuce samples.

Keywords: Salmonella Typhimurium; biosensor; flagellin; magnetic nanoparticle; monoclonal antibody; surface plasmon resonance.

Figure 1

Illustrations of three different SPR assay formats: (a) direct assay—sample preparation is injected directly, (b) sequential two-step sandwich assay—MAb 1C8-coupled magnetic nanoparticle is injected following sample injection, and (c) preincubation one-step sandwich assay—sample preparation is preincubated with MAb 1C8-coupled magnetic nanoparticle before injection.

Figure 2

(a) SPR sensorgrams of the sequential two-step sandwich assay showing the interaction of MAb 1C8-coupled magnetic nanoparticles at various concentrations when the flagellin is captured on the MAb 1E10-immobilized sensor surface: (b) SPR responses at the peak of association phase, the vertical line in (a), showing a linear relation to the concentrations of MAb 1C8-coupled magnetic nanoparticles.

Figure 3

SPR responses at the peak of association phase of the three assay formats—direct assay, sequential two-step sandwich assay and preincubation one-step sandwich assay; same concentrations of flagellin extractions (9.2 µg/mL) from S. Typhmurium were analyzed. Same concentration of MNPs (41.6 µg/mL) was used in the sequential two-step sandwich assay and preincubation one-step sandwich assay. Two experiments were performed on different days, each with three replicates.

Figure 4

SPR responses of S. Typhimurium at various concentrations (A) 5.0 log cfu/mL, (B) 5.2 log cfu/mL, (C) 5.6 log cfu/mL and (D) 5.9 log cfu/mL, analyzed by the preincubation one-step sandwich assay. Two experiments were performed in different days each with three replicates.

Figure 5

SPR responses of the romaine lettuce samples contaminated with different levels of S. Typhimurium (A) 5.5 log cfu/g, (B) 6.0 log cfu/g and (C) 6.5 log cfu/g, analyzed by the preincubation one-step sandwich assay. Two experiments were performed on different days, each with two replicates.