Detection of Vibrio parahaemolyticus Based on Magnetic and Upconversion Nanoparticles Combined with Aptamers

Abstract

Vibrio parahaemolyticus is a halophilic and heat-labile gram-negative bacterium and is the most prevalent foodborne bacterium in seafood. In order to develop a rapid and sensitive method for detecting the foodborne pathogenic bacterium Vibrio parahaemolyticus, an aptamer-modified magnetic nanoparticle and an aptamer-modified upconversion nanoparticle were synthesised and used as a capture probe and a signal probe, respectively. The aptamer-modified magnetic nanoparticle, V. parahaemolyticus cell, and aptamer-modified upconversion nanoparticle formed a sandwich-like complex, which was rapidly separated from a complex matrix using a magnetic force, and the bacterial concentration was determined by fluorescence intensity analysis. The results showed that the fluorescence intensity signal correlated positively with the concentration of V. parahaemolyticus in the range of 3.2 × 10² to 3.2 × 10⁵ CFU/mL, with a linear equation of y = 296.40x - 217.67 and a correlation coefficient of R² = 0.9610. The detection limit of the developed method was 4.4 CFU/mL. There was no cross-reactivity with other tested foodborne pathogens. This method is highly specific and sensitive for the detection of V. parahaemolyticus, and can achieve the qualitative detection of this bacterium in a complex matrix.

Keywords: Vibrio parahaemolyticus; aptamer; detection; magnetic nanoparticles; upconversion nanoparticles.

Figure 1

A schematic description of the proposed fluorescence detection platform.

Figure 2

Characterisation of Fe₃O₄ magnetic nanoparticles modified with amino groups (TEM image of MNPs (A), XRD patterns of MNPs (B), FTIR spectra of MNPs (C), and VSM magnetisation curves of MNPs (D)).

Figure 3

Characterisation of UCNPs and UCNPs@SiO₂ (TEM image of UCNPs (A), TEM image of UCNPs@SiO₂ (B), XRD patterns of UCNPs (C), FTIR spectra of UCNPs and UCNPs@SiO₂ (D), and fluorescence spectrum of UCNPs, and UCNPs@SiO₂ (E)).

Figure 4

Confirmation of aptamer immobilisation on the Fe₃O₄ magnetic nanoparticles (A) and UCNPs (B) using UV-vis spectra.

Figure 5

The fluorescence spectra of different additions of aptamer-MNPs (A), absorption peaks at 542 nm of different additions of aptamer-MNPs (B), the fluorescence spectra of different additions of aptamer-UCNPs (C), absorption peaks at 542 nm of different additions of aptamer-UCNPs (D).

Figure 6

Fluorescence spectra of compounds in the presence of different V. parahaemolyticus concentrations (A), the linear relationship between the fluorescence intensity and concentration of V. parahaemolyticus (B), where F is the sample fluorescence intensity and F₀ is the blank control.

Figure 7

The specificity of the developed method with other bacteria.