Quantitative fluorescent detection of tetracycline in animal-derived foods using quantum dots

Abstract

Tetracycline (Tc) antibiotics, a class of synthetically produced broad-spectrum antimicrobial drugs, have been widely used in animal husbandry, leading to their widespread presence in animal-derived foods. However, misuse, overuse, and non-compliance with withdrawal periods in animal farming have resulted in excessive Tc residues in these foods, which can cause various adverse reactions in humans, induce bacterial resistance, and pose a significant threat to public health. Consequently, the detection of Tc antibiotic residues in animal-derived food has become a critical issue. This study aims to establish a novel method for quantifying Tc residues in animal-derived food using quantum dots (QDs) fluorescence immunoassay (FLISA). The developed method was optimized to achieve a detection limit of 0.69 ng/mL and a quantitative detection range of 1.30 ~ 59.22 ng/mL. The applicability of the method was demonstrated by successfully determining Tc residues in pork, chicken, fish, milk, eggs, and honey samples spiked with Tc standard solutions, yielding recoveries ranging from 94.01% to 110.19% and relative standard deviations between 1.10% and 11.39%. The significance of this study lies in its potential to provide a rapid and reliable approach for monitoring Tc residues in animal-derived food products, thereby contributing to the enhancement of food safety monitoring practices. KEY POINTS: • Screen out tetracycline-specific blocking monoclonal antibodies • The quantitative detection has high specificity and sensitivity • This method can be a useful tool for laboratories or testing facilities.

Keywords: Animal-derived foods; Fluorescence immunoassay; Quantum dots; Tetracycline.

Fig. 1

Schematic representation of the synthesis of Tc-BSA and Tc-OVA complete antigens

Fig. 2

Schematic representation of the amino group of anti-Tc-mAb coupled to the carboxyl group on the quantum dot

Fig. 3

Identification of Tc-BSA and Tc-OVA complete antigens. (a) Spectral analysis for Tc-BSA using UV absorption spectroscopy. (b) UV absorption spectroscopy analysis for Tc-OVA. (c) SDS-PAGE analysis of Tc-BSA: Lane 1 represents the protein BSA, Lane 2 is Tc-BSA, and Lane 3 is marker. (d) SDS-PAGE analysis of Tc-OVA: Lane 1 is the protein marker, Lane 2 is OVA, and Lane 3 is Tc-OVA

Fig. 4

Immunization procedures and evaluation of immunization in mice. (a) Tc-BSA was injected subcutaneously at multiple points on the back, with a total of four immunizations, each two weeks apart. (b) Following the four immunizations, mouse No. 1 exhibited a serum potency of 204.8K, while mouse No. 2 displayed a serum potency of 102.4K. (c) After undergoing four immunizations, mouse No. 1 exhibited a serum sensitivity IC50 of 439.64 ng/mL with a fitted curve described by the equation y = -1.66X—0.09(R² = 0.99). Similarly, mouse No. 2 displayed a serum sensitivity IC50 of 450.68 ng/mL with a regression model represented by the equation y = -1.77X—0.11(R.² = 0.99)

Fig. 5

Characterisation and analysis of fluorescent probes. (a) Fluorescence emission spectra of QDs and QDs-labeled antibodies. (b) Agarose gel electrophoresis: lane 1 indicates QDs, and lane 2 indicates anti-Tc-mAb-QDs. (c) SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis): lane 1 represents QDs, and lane 2 represents anti-Tc-mAb-QDs

Fig. 6

Schematic representation and standard curve of the competitive fluorescence-linked immunosorbent assay (FLISA) for target analyte detection. (a) Schematic representation of FLISA competitive detection. (b) FLISA standard curve (y = -0.36X—0.24, R.² = 0.99)

Fig. 7

Determination of optimal dilution of sample extracts. (a) The optimal dilution of the pork sample extract was tenfold. (b) Optimal dilution of chicken sample extract is tenfold. (c) The optimum dilution for fish sample extracts is tenfold. (d) The optimum dilution for milk sample extracts is eightfold. (e) The optimum dilution for egg sample extract is 12-fold. (f) The optimum dilution for honey sample extract is 13-fold