Rapid detection of Vibrio parahaemolyticus using magnetic nanobead-based immunoseparation and quantum dot-based immunofluorescence

Abstract

In recent years, the scale of population exposure and food poisoning caused by Vibrio parahaemolyticus (V. parahaemolyticus) has shown a significant upward trend, becoming one of the primary food-borne pathogens. Herein, we developed a rapid and sensitive detection of V. parahaemolyticus by integrating the technology of magnetic nanobeads (MBs) based immunoseparation (IMS) with quantum dots (QDs) based immunofluorescence. Firstly, specific rabbit polyclone IgG antibodies (IgG) and chicken egg yolk antibodies (IgY) of V. parahaemolyticus were prepared. Then two sizes of MBs (1 μm; 180 nm) were coupled with IgG to form immuno-MB (IMB) capture probes for evaluating the effect of different sizes on the detection efficiency. For QDs, they were conjugated with IgY to form fluorescent reporting probes. In the process of detection, IMB probes were used to separate V. parahaemolyticus and then these complexes were labeled by QD probes on the principle of double antibody sandwich. The fluorescence intensity of the IMB-V. parahaemolyticus-QD complexes was measured by a fluorescence spectrophotometer. The detection method takes 150 min with a detection limit of 10² cfu mL^-1 ranging from 10² to 10⁶ cfu mL^-1 and it has been shown to work satisfactorily in real food samples.

Fig. 1. The technology road mapping. V. parahaemolyticus was separated by IMBs probes, then QDs probes were used as fluorescent label to measure the content of bacteria.

Fig. 2. Assessment of the purity of IgG (A) and IgY (B). Lane M represented the molecular weight marker. Assessment of the antibody titre of IgG (C) and IgY (D) produced by immunizing rabbits and hens. The titer was presented by dilution times. The optimization of 1 μm IMBs-probes (E), 180 nm IMBs-probes (F) and QDs probes (G). For IMBs probes, A280 nm of the IgG solution was determined before and after conjugation. The amount of conjugated IgG of IMBs was calculated to evaluate the most efficient probes. For QDs probes, the fluorescence intensity of the complex was increased with the increase of the amount of IgY. The fluorescence intensity reached the highest level and then stepped down.

Fig. 3. Fluorescence intensity was determined to optimized conditions of 1 μm IMBs-QDs group from the IMBs probes amount (A), QDs probes amount (B), labeling time (C) and enriching time (D). Fluorescence intensity was also determined to optimized conditions of 180 nm IMBs-QDs from the IMBs probes amount (E), QDs probes amount (F), labelling time (G) and enriching time (H).

Fig. 4. The sensitivity of 1 μm IMBs-QDs group method of V. parahaemolyticus and their plotted linear relationship (P/N ≥ 2.1 indicates positive and the LOD is 10² CFU mL⁻¹) (A). The specificity of 1 μm IMBs-QDs group method (B). The sensitivity of 180 nm IMBs-QDs group method of V. parahaemolyticus and their plotted linear relationship (P/N ≥ 2.1 indicates positive and the LOD is 10² CFU mL⁻¹) (C). The specificity of 1 μm IMBs-QDs group method (D).

Fig. 5. Fluorescence intensity of various concentration of V. parahaemolyticus detection in the simulation sample for 1 μm IMBs-QDs group (A) and 180 nm IMBs-QDs group (B). The LOD could both reach 10² CFU mL⁻¹.