Upconversion nanoparticle-based aptasensor for rapid and ultrasensitive detection of Staphylococcus aureus by low-speed centrifugation

Abstract

Opportunistic foodborne pathogens such as Staphylococcus aureus (S. aureus) can cause a wide variety of threats to public health. There is an urgent clinical need for a fast, simple, low-cost, and sensitive method. Here, we designed a fluorescence-based aptamer biosensor (aptasensor) for S. aureus detection using core-shell structured upconversion nanoparticles (CS-UCNPs) as a beacon. A S. aureus-specific aptamer was modified on the surface of CS-UCNPs for binding pathogens. The S. aureus bound to CS-UCNPs can then be isolated from the detection system by simple low-speed centrifugation. Thus, an aptasensor was successfully established for the detection of S. aureus. The fluorescence intensity of CS-UCNPs correlated with the concentration of S. aureus within the range of 6.36 × 10² to 6.36 × 10⁸ CFU mL^-1, resulting in the detected limit of S. aureus being 60 CFU mL^-1. The aptasensor performed well in real food samples (milk) with a detection limit of 146 CFU mL^-1 for S. aureus. Furthermore, we applied our aptasensor in chicken muscles for S. aureus detection, and compared it with the plate count gold standard method. There was no significant difference between our aptasensor and the plate count method within the detected limit, while the time for the aptasensor (0.58 h) was shorter than that of the plate count method (3-4 d). Therefore, we succeeded in the design of a simple, sensitive and fast CS-UCNPs aptasensor for S. aureus detection. This aptasensor system would have the potential for the detection of a wide range of bacterial species by switching the corresponding aptamer.

Fig. 1. Schematic illustration of the CS-UCNPs based aptasensor and the detection of S. aureus. First, the core UCNPs were synthesized, then the UCNPs were coated with a NaYF₄ shell to form CS-UCNPs, and then the aptamer sensor for S. aureus detection was prepared by grafting aptamers. The aptamer sensor can bind specifically to S. aureus when S. aureus is present. After centrifugation at low speed (3000 g, 5 min) and separation precipitation (Staphylococcus aureus and bound aptasenors), the fluorescence of the solution was significantly reduced.

Fig. 2. Characterization of UCNPs and CS-UCNPs. (a and b) TEM images of the core-UCNPs (a) and CS-UCNPs (b). (c) Photographs of core-UCNPs (left) and CS-UCNPs (right) in the cyclohexane solution under the same 980 nm laser excitation. (d) Luminescence properties of core-UCNPs (red line) and CS-UCNPs (black line). (e) High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) image and its corresponding elemental contents of CS-UCNPs.

Fig. 3. Modification of CS-UCNPs. (a) FTIR spectra of prepared CS-UCNPs (red line) and OA-removed CS-UCNPs (blue line). (b) TEM image of water-soluble CS-UCNPs. (c) UV-vis absorption spectra of CS-UCNPs and aptamer-modified CS-UCNPs. (d) Normalized fluorescence spectra of CS-UCNPs and aptamer-CS-UCNPs conjugates.

Fig. 4. Sensitivity of CS-UCNPs based aptasensor for S. aureus detection. (a and b) Recording output for the detection of different concentrations of S. aureus by the CS-UCNPs-based aptasensor. (c) Standard curve of the decreased luminescence intensity (ΔI = I₀ − I) versus the concentrations of S. aureus.

Fig. 5. Specificity of CS-UCNP-based aptasensor for S. aureus detection. (a) Normalized fluorescence spectra of the CS-UCNP-based aptasensor in the presence of different bacteria. (b) Normalized decreased upconversion luminescence intensity (ΔI = I₀ − I) of the aptasensor for different bacteria detection (the concentration of all bacteria is 10³ CFU mL⁻¹). (c and d) SEM images of aptasensor-S. aureus conjugates, while the CS-UCNPs are colored in red (d). (f and g) TEM images of aptasensor-S. aureus conjugates. (e and h) Elemental contents of aptasensor-S. aureus conjugates, which confirmed the nanoparticles were CS-UCNPs.

Fig. 6. Detection of S. aureus in the milk sample. (a) Normalized fluorescence spectra of the simultaneous detection of different concentrations of S. aureus (2.1 × 10²–2.1 × 10⁷ CFU mL⁻¹) in milk sample by the CS-UCNP aptasensor. (b) The linear relationship between the decreased fluorescence relative intensity and the logarithm of S. aureus concentration with the range of 2.1 × 10²–2.1 × 10⁷ CFU mL⁻¹.