Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Dec 20:9:793355.
doi: 10.3389/fchem.2021.793355. eCollection 2021.

Europium Fluorescent Nanoparticles-Based Multiplex Lateral Flow Immunoassay for Simultaneous Detection of Three Antibiotic Families Residue

Yaping Wang  1 , Biao Ma  1 , Miaomiao Liu  1 , Erjing Chen  1 , Ying Xu  1 , Mingzhou Zhang  1
Affiliations

Europium Fluorescent Nanoparticles-Based Multiplex Lateral Flow Immunoassay for Simultaneous Detection of Three Antibiotic Families Residue

Yaping Wang et al. Front Chem. .

Abstract

A fluorescent immunoassay based on europium nanoparticles (EuNPs-FIA) was developed for the simultaneous detection of antibiotic residues, solving the problems of single target detection and low sensitivity of traditional immunoassay methods. In the EuNPs-FIA, EuNPs were used as indictive probes by binding to anti-tetracyclines monoclonal antibodies (anti-TCs mAb), anti-sulphonamides monoclonal antibodies (anti-SAs mAb) and anti-fluoroquinolones monoclonal antibodies (anti-FQs mAb), respectively. Different artificial antigens were assigned to different regions of the nitrocellulose membrane as capture reagents. The EuNPs-FIA allowed for the simultaneous detection of three classes of antibiotics (tetracyclines, fluoroquinolones and sulphonamides) within 15 min. It enabled both the qualitative determination with the naked eye under UV light and the quantitative detection of target antibiotics by scanning the fluorescence intensity of the detection probes on the corresponding detection lines. For qualitative analysis, the cut-off values for tetracyclines (TCs), fluoroquinolones (FQs) and sulphonamides (SAs) were 3.2 ng/ml, 2.4 ng/ml and 4.0 ng/ml, respectively, which were much lower than the maximum residue limit in food. For quantitative analysis, these ranged from 0.06 to 6.85 ng/ml for TCs, 0.03-5.14 ng/ml for FQs, and 0.04-4.40 ng/ml for SAs. The linear correlation coefficients were higher than 0.97. The mean spiked recoveries ranged from 92.1 to 106.2% with relative standard deviations less than 8.75%. Among them, the three monoclonal antibodies could recognize four types of TCs, seven types of FQs and 13 types of SAs, respectively, and the detection range could cover 24 antibiotic residues with different structural formulations. The results of the detection of antibiotic residues in real samples using this method were highly correlated with those of high performance liquid chromatography (R 2 > 0.98). The accuracy and precision of the EuNPs-FIA also met the requirements for quantitative analysis. These results suggested that this multiplex immunoassay method was a promising method for rapid screening of three families of antibiotic residues.

Keywords: europium nanoparticle fluorescence immunochromatography; fluoroquinolone antibiotics; multi-residue detection; sulfonamide antibiotics; tetracycline antibiotics.

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
The EuNPs-FIA detection principle. (A) The cycle of antibiotics between man and nature. (B) Principle of assemble EuNPs-FIA test strips. (C) Test results of the EuNPs-FIA reader test results. (D) The actual testing process and test results of the EuNPs-FIA reader test results. (E) Diagram of the test results. (F) Number of detectable target species.
FIGURE 2
FIGURE 2
Optimisation of EuNPs-antibody conjugates. (A) Effect of the amount of FQs-mAb labelling on the detection capability of the probe. (B) Effect of the amount of SAs-mAb labelling on the detection capability of the probe. (C) Effect of the amount of TCs-mAb labelling on the detection capability of the probe. (D) Effect of probe incorporation on the detection capacity of single-EuNPs-FIA.
FIGURE 3
FIGURE 3
Parameter optimisation of single-EuNPs-FIA. (A) Effect of encapsulation concentrations of FQs-BSA and GAM-IgG on the detection capacity of single-EuNPs-FIA. (B) Effect of encapsulation concentrations of SAs-BSA and GAM-IgG on the detection capacity of single-EuNPs-FIA. (C) Effect of encapsulation concentrations of TCs-BSA and GAM-IgG on the detection capacity of single-EuNPs-FIA. (D) Three standard curves for single-EuNPs-FIA.
FIGURE 4
FIGURE 4
Parameter optimisation of three-multiple-EuNPs-FIA. (A) Effect of probe mixing ratio on the detection capability of three-multiple-EuNPs-FIA. (B) Effect of reaction time duration on the detection capacity of three-multiple-EuNPs-FIA.
FIGURE 5
FIGURE 5
Sensitivity and specificity analysis of three-multiple-EuNPs-FIA. (A) Simultaneous sensitivity analysis of SAs, FQs and TCs by three-multiple-EuNPs-FIA. (B) The standard curve for SAs, FQs and TCs by three-multiple-EuNPs-FIA. (C) Specific analysis of three-multiple-EuNPs-FIA.
FIGURE 6
FIGURE 6
Comparison of EuNPs-FIA and HPLC. (A) Comparative method scatter diagram of EuNPs-FIA and HPLC. (B) Passing-Bablok regression.
See this image and copyright information in PMC

References

    1. Abafe O. A., Gatyeni P., Matika L. (2020). A Multi-Class Multi-Residue Method for the Analysis of Polyether Ionophores, Tetracyclines and Sulfonamides in Multi-Matrices of Animal and Aquaculture Fish Tissues by Ultra-high Performance Liquid Chromatography Tandem Mass Spectrometry. Food Additives & Contaminants: A 37, 438–450. 10.1080/19440049.2019.1705399 - DOI - PubMed
    1. Ashuo A., Zou W., Fu J., Yang T., Yu L., Liu W., et al. (2020). High Throughput Detection of Antibiotic Residues in Milk by Time-Resolved Fluorescence Immunochromatography Based on QR Code. Food Additives & Contaminants: Part A 37, 1481–1490. 10.1080/19440049.2020.1778192 - DOI - PubMed
    1. Bartosh A. V., Sotnikov D. V., Hendrickson O. D., Zherdev A. V., Dzantiev B. B. (2020). Design of Multiplex Lateral Flow Tests: A Case Study for Simultaneous Detection of Three Antibiotics. Biosensors 10, 17. 10.3390/bios10030017 - DOI - PMC - PubMed
    1. Ben Y., Hu M., Zhang X., Wu S., Wong M. H., Wang M., et al. (2020). Efficient Detection and Assessment of Human Exposure to Trace Antibiotic Residues in Drinking Water. Water Res. 175, 115699. 10.1016/j.watres.2020.115699 - DOI - PubMed
    1. Booth A., Aga D. S., Wester A. L. (2020). Retrospective Analysis of the Global Antibiotic Residues that Exceed the Predicted No Effect Concentration for Antimicrobial Resistance in Various Environmental Matrices. Environ. Int. 141, 105796. 10.1016/j.envint.2020.105796 - DOI - PubMed