ABSTRACT
Tylosin (TYL) is a kind of antibiotics which promotes the development of animal husbandry, but it poses potential threats to food safety and human health. Therefore, it is necessary to establish a highly sensitive method for the detection of TYL. Here, an enzyme-linked immunosorbent assay (ELISA) combined with biotin–streptavidin (Bio-SA) system and hybridization chain reaction (HCR) technique (Bio-SA-HCR-ELISA) was developed. In the absence of the TYL, a strong fluorescence was generated through double signal amplification as signal output, while in the presence of TYL, the fluorescence intensity decreased. The results showed that the limit of detection for TYL was 0.39 ng/mL, and this method had a good specificity for 9 common antiboics. Compared to the traditional indirect competitive ELISA, the sensitivity of this method was 6.8 times higher. Additionally, the established double signal amplification Bio-SA-HCR-ELISA was used for detecting TYL in milk and honey, the LOD of this method were 4.9 and 2.5 ng/mL, respectively.
Introduction
Antibiotics are widely used as growth promoters and antibacterial drugs for disease prevention and treatment (Arsic et al., Citation2018). The emergence of antibiotics has greatly accelerated the development of animal husbandry and produced economic benefits. However, the large consumption has also caused threats to food safety and human health. Tylosin (TYL) is a macrolide antibiotic produced by Streptomyces fradiae and is mainly used for the treatment of infections caused by Gram-positive and certain Gram-negative bacteria (Gao et al., Citation2021). The use of veterinary drugs in food-producing animals has the potential to generate residues in animal-derived products (meat, milk, eggs, and honey) and poses a health hazard to the consumer (Gao et al., Citation2021; Liu et al., Citation2021). The improper use of TYL in food-producing animals caused residues in animal-derived products (meat, milk, eggs, and honey) transferred to the human body through the food chain and caused many adverse effects. One of the most important consequences is that the emergence of microbial resistance may lead to increased morbidity or mortality of bacterial infections (Lan et al., Citation2019; Mason et al., Citation2018). To address the problem and ensure food safety, maximum residue limits (MRLs) of TYL for each target tissue of domestic species are established by the European Union (EU), the fixed MRL is 100 μg/kg (Prats et al., Citation2002). The concentration of TYL residues in milk was higher than in tissue, the MRL established for TYL in milk is 50 μg/kg (Avci & Elmas, Citation2014). Therefore, it is necessary to establish a highly sensitive detection method for TYL residues in food.
Several methods have been developed to monitor TYL residues in foods. Microbiological inhibition methods are widely used in the screening stage with a broad spectrum, but this method is laborious, time-consuming, and with a high false-positive rate (Q. Wu et al., Citation2021). Methods based on instruments, such as liquid chromatography (Bernal et al., Citation2011; Prats et al., Citation2002) (LC), and mass spectrometry (Chopra et al., Citation2013; Lin et al., Citation2019) are the most common and sensitive techniques used for the determination of TYL. However, those high-cost methods need sample pretreatment which involves extraction or pre-concentration. Immunological methods for the detection of antibiotics based on highly specific antigen–antibody were developed (Taranova et al., Citation2015; Yang et al., Citation2022; Zeng et al., Citation2022). Traditional indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) is one of the most popular methods for the detection of antibiotic residues with high specificity (Peng et al., Citation2012), high throughput, and low cost, but the detection sensitivity is not high enough. Many researchers are devoted to improving the sensitivity of ELISA using new signal output materials (Zhao et al., Citation2021), improving biomolecular affinity (Hu et al., Citation2020), and other signal amplification systems. Biotin–streptavidin (Bio-SA) system (Guo et al., Citation2016; Yang et al., Citation2021) has been effectively used to improve the detection sensitivity by capturing more signal molecules due to the strong affinity caused by the extremely low dissociation constant between biotin and streptavidin (SA). Besides, a series of signal amplification tools based on nucleic acid amplification has also been developed (Guo et al., Citation2016; Pang et al., Citation2020). Hybridization chain reaction (HCR) can proceed under the condition of constant temperature without the participation of enzymes, which as an efficient amplification technique is widely used in the fields of bioimaging, biomedicine, and detection (Lv et al., Citation2019; J. Wu et al., Citation2021).
To achieve a more sensitive detection for TYL, we attempted to establish an ELISA assay based on Bio-SA system combined with HCR technology (Bio-SA-HCR-ELISA) to achieve double amplification of the signal. Gold nanoparticles (AuNPs) were used to label monoclonal antibodies (mAb) and SA as bifunctional probes (mAb-Au-SA) of immune recognition and binding to biomolecules. The biotinylated initiator (bio-initiator) was used to conjugate SA. In the absence of TYL, more mAb-Au-SA could be captured on the plate due to the immunological reaction between mAb and coated antigen (TYL-CMO-OVA) and more biotin-initiator can be captured owing to the strong interaction between SA and biotin. The biotinylated initiator hybridized with the hairpins modified with the fluorescent group, and the fluorescence generated was used for the subsequent signal output. In the presence of TYL, the higher the concentration of TYL is in the sample, the less mAb labelled on the probe binds to the coated antigen in the well, and the lower the final fluorescence intensity was obtained. Here, the approach of combining ELISA with the Bio-SA system and HCR technique was established with both specificity and sensitivity.
Experimental
Materials and apparatus
TYL, erythromycin, roxithromycin, tiamulin, spiramycin, azithromycin, abamectin, tilmicosin, natamycin, acetylisovaleryltylosin were purchased from Solarbio Science Technology Co., Ltd. (Beijing, China). Hydrogen tetrachloroaurate trihydrate (HAuCl4·3H2O), trisodium citrate (C6H5Na3O7·2H2O), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, Mo, USA). Dimethyl formamide (DMF) and tween-20 were purchased from Tianjin Yongda chemical reagent Co., Ltd (Tianjin, China). Enzyme-immunoassay-grade horseradish peroxidase-labelled goat anti-mouse immunoglobulin (HRP-IgG) was purchased from Jackon Immuno Research Laboratories, Inc. (Shanghai, China), and 96-well microplates were purchased from Shanxi Defcred Biotech Co., Ltd. (Shanxi, China), and Black 96-well microplates were purchased from Corning Co. (Corning, NY, USA). The 100 bp DNA ladder and 10× loading buffer were purchased from Takara Biotech Co., Ltd. (Beijing, China). Agarose was purchased from Bio-Rad Laboratories Co., Ltd., (Hercules, CA, USA). The 1× TAE buffer (pH 8.3) was from Solarbio Life Sciences (Beijing, China). All other chemicals and solvents were of analytical reagent grade. Ultrapure water was generated by Milli-Q Century instrument from USA (< 18.2 MΩ.cm).
Phosphate-buffered solution (0.01 mol/L PBS), prepared as follows: add 0.24 g KH2PO4, 3.62 g Na2HPO4·12H2O, and 0.2 g KCl, 8.0 g NaCl in 1 L ultrapure water. Add 1.59 g Na2CO3 and 2.93 g NaHCO3 in 1 L ultrapure water to prepare 0.05 mol/L carbonate-buffered solution (CBS). Tetramethylbenzidine (TMB) substrate solution contains solution A and solution B with the ratio of 5: 1. Solution A: 180 µL 30% H2O2 was added into 1 L 0.01 mol/L PBS, solution B: 300 mg TMB was dissolved in 500 mL ethylene glycol.
Electrophoresis equipment was obtained from Beijing Liuyi Biotechnology Co., Ltd. (Beijing, China). The gel imaging system (Gel Doc XR) was purchased from Bio-Rad Laboratories Co., Ltd., (Hercules, CA, USA). The SpectraMax i3x multimode microplate reader was purchased from Molecular Devices Corporation (San Jose, CA, USA) and used to measure the fluorescence intensity.
Design and fluorescence response experiments of oligonucleotide sequences
The oligonucleotide sequences were designed and synthesized by Nucleic Acid Package (NUPACK; http://www.nupack.org/) according to the previous report with slight modification (Lv et al., Citation2019) and included bio-initiator, H1, and H2, as listed in Table S1. The initiator was modified with biotin, and H1 was internally modified with two groups, TAMRA and Black Hole Quencher 2 (BHQ2). The fluorophores (TAMRA) were efficiently quenched by the adjacent quenching group (BHQ2) when H1 was present as a stem-loop structure, and the fluorescence was not able to be measured until HCR occurred. Oligonucleotides were dissolved in ultrapure water and stored at 20°C. Before use, all hairpins were heated to 95°C for 5 min and slowly cooled to room temperature to form a hairpin structure. Fluorescence measurements were obtained using the SpectraMax i3x multimode microplate reader to measure the fluorescence intensity of H1 lablled with TAMRA with excitation and emission wave length at 557 and 583 nm, respectively.
Verification of oligonucleotide sequences
NUPACK was used to imitate the secondary structure of oligonucleotide sequences (NUPACK; http://www.nupack.org/). Agarose gel electrophoresis analysis was performed to determine the occurrence of HCR. An aliquot of 4 g agarose was added into 10 mL 1×TAE buffer, and then heat the solution to boiling. Cool and then add 2 μL gel red. The mixture solution was poured into the electrophoresis tank. After setting, the agarose gel was immersed into 500 mL 1×TAE buffer and taken away from the tooth comb. An aliquot of 10 μL DNA hybridization buffer which contained 1μmol/L DNA and hairpins (1 μmol/L) was performed at 37°C for 60 min, after that the 10 μL HCR products were mixed with 1 μL loading buffer. After 10 min, the mixture was added into the wells of the agarose gel. The electrophoresis was performed at 85 V for 45 min.
Preparation of mAb-AuNP-SA probes
Synthesis of AuNPs with an average diameter of 18 nm was carried out according to the trisodium citrate method. Briefly, 1 mL of 1% HAuCl4 was added to 99 mL of ultrapure water and was heated to boil under constant stirring in a three-neck round flask. Subsequently, 1.8 mL of 1% trisodium citrate was rapidly added. During 15 min heating, the hue of the mixture solution changed from light yellow to red. The mAb-AuNP-SA probes were prepared by the electrostatic adsorption between AuNPs and protein (anti-TYL mAb and SA). With constant gentle stirring, 0.2 mol/L K2CO3 was added into 1 mL of AuNPs suspension to adjust the pH to 7.5, and then anti-TYL mAb and SA were mixed and dissolved in 100 μL ultrapure water and slowly added to AuNPs suspension. After stirring at room temperature for 60 min, the complex (mAb-AuNP-SA) was formed, and 100 μL of 1% PEG 20000 was added to the complex solution for 30 min to stabilize the complex. Then 100 μL of 10% BSA was added for another 60 min to block the mAb-AuNP-SA probs. The complex was separated by centrifugation at 10,000 rpm for 30 min and then resuspended in 200 μL of 0.01 mol/L PBS. The mAb-Au-SA probes were stored at 4°C.
The process of indirect competitive ELISA (ic-ELISA)
The process of ic-ELISA was performed as described in the literature to detect TYL. Briefly, 96-well microplates were coated with TYL-CMO-OVA dissolved in 0.05 mol/L CBS (2 μg/mL, 100 μL), incubated at 37°C for 2 h, and washed three times with 300 μL PBS containing 0.05% Tween 20 (PBST). Then the plates were blocked with 300 μL of CBS containing 0.02% gelatin for 2 h at 37°C. After washing with PBST, 50 μL of the anti-TYL mAb and 50 μL of varying concentrations of standard analyte were added to each well, and incubated at 37°C for 0.5 h. After washing with PBST, 100 μL HRP-IgG with 3k-fold dilution was added to the wells, and incubated at 37°C for 0.5 h. After washing with PBST, TMB substrate was added to the wells and incubated at 37°C for 15 min in the dark. Finally, the reaction was terminated followed by the addition of 50 μL H2SO4 (2 mol/L). The absorbance at 450 nm was measured using a microplate reader.
The process of ELISA-assisted biotin–streptavidin system combined with HCR (ELISA-Bio-SA-HCR)
TYL-CMO-OVA was coated in the black 96-well microplates, incubated at 37°C for 2 h, and washed with PBST. After blocking, 50 μL of standard analyte and 10 μL probe mAb-Au-SA were added to the ELISA wells and incubated at 37°C for 0.5 h. After washing with PBST, 50 μL of biotin-initiator was added to the wells, and incubated at 37°C for 0.5 h. Finally, hairpins (0.15 nmol/mL) were added to each well and then incubated at 37°C for 1 h. Through the specific recognition between SA and biotin molecules, the probe mAb-Au-SA was combined with biotin-initiators. HCR could occur when the detection system contains biotin-initiators by adding matching hairpins. Once the stem-loop structure of H1 was opened, the fluorescent signal could be measured by a multimode microplate reader at excitation at 557 nm and emission at 583 nm.
Specificity test
To evaluate the specificity of the proposed method, several commonly used macrolide antibiotics including erythromycin, roxithromycin, tiamulin, spiramycin, azithromycin, abamectin, tilmicosin, natamycin, acetylisovaleryltylosin were analysed at 20 ng/mL.
Detection of TYL in food samples
Pasteurized skim milk and honey purchased from a local supermarket were used to investigate the application of this method. Before the test, a simple treatment was performed to reduce the effect of the matrix. Firstly, skim milk and honey were diluted 10-fold with 0.01 mol/L PBS. And various concentrations of TYL standard analyte were added to the dilution samples make the final concentration of TYL 100, 50, 25, 12.5, 6.25, 3.125, 1.5615, and 0 ng/mL. And then the samples were detected by ELISA-Bio-SA-HCR as above procedures.
Results and discussion
Experimental principle
The principle is shown in Scheme 1. This method was mainly based on immunological reaction, and the biotin-SA system was combined with HCR to achieve double signal amplification (Scheme 1(A)). The mAb-AuNP-SA probes which were prepared simply by modifying AuNPs with mAb and SA could recognize the target analytes and bind the initiator efficiently. In the absence of the target, the TYL-CMO-OVA-mAb-AuNPs-SA complex was formed through an immunological reaction between mAb and TYL, and SA in the complex was combined with bio-initiators. After adding hairpins, bio-initiators caused hairpins open alternately for hybridization. The fluorescence of TAMRA modified on H1 was recovered as the signal output. As we know, AuNPs with the diameter of 18 nm has low ability of fluorescence quenching. The absorbance curve of AuNPs (18 nm) solution with a peak at 520 nm (Figure S1). And the excitation and emission wave length of TAMRA was at 557 and 583 nm. So the effect of AuNPs on the fluorescence intensity of TAMRA is negligible. In the presence of the target, the immobilized TYL-CMO-OVA and the TYL in the sample compete for the limited number of binding sites on mAb labelled on the probes, and the fluorescence intensity decreased.
Scheme 1. Schematic illustration of HCR combined with biotin-SA signal amplification method for the detection of TYL. (A) The biotin-SA system was combined with HCR to achieve double signal amplification. (B) HCR combined with biotin-SA signal amplification method for the detection of TYL.
Simulation and verification of oligonucleotide sequences
Secondary structures and assembly of initiator, H1, and H2 were simulated by NUPACK as shown in Figure S2. Bio-initiator was a single and non-folded strand, and the free energy was 0 kcal/mol. H1 and H2 were stem-loop structure hairpins, and the free energy was −22.78 and −23.41 kcal/mol, respectively. The bio-initiator can react with the sticky end of H1. The unpaired portion exposed by H1 can react with H2 after H1 was triggered by bio-initiators. A lot of H1 and H2 were hybridized alternately to form long double-strand DNA in the presence of the initiator, resulting in a large number of HCR products. The results showed that more stable products with lower free energy than the reactants were formed, which was beneficial to the process of HCR.
Agarose gel electrophoresis was performed to verify the practicality of the sequence. As shown in Figure S3, only in the presence of initiators and hairpins (H1 and H2) (lanes 7–11), was the HCR reaction conducted and a great deal of trailing production bands could be produced by HCR. The formed HCR products were short fragments when the initiator was in high concentration (lane 7). As the concentration of initiators decreased, longer fragment HCR products were generated (lanes 8–10). Few products were generated in lane 11 because the concentration of the initiator was too low to trigger HCR.
The results of fluorescence response experiments were shown in Figure S4, neither the lack of bio-initiator nor the mismatched initiator of will enable H1 and H2 to hybridize. Only the bio-initiator could make H1 and H2 hybridize and produce a strong fluorescent signal.
Exploration of the effect of probe parameters
The amounts of mAb and SA on the probe were the key factors to determine the sensitivity of the integrated double signal amplification system with ELISA assay. In the process of the preparation of the probe, the effects of various amounts of mAb (2, 4, 8, and 15 μg) and SA (2, 5, 10, and 15 μg) were analysed. As shown in (A), when the amount of mAb labelled on the probe was 2 μg, that was too low to produce significant inhibition due to the insufficient recognition of TYL. With the increase in the amounts of mAb, the fluorescence intensity increased in the absence of TYL. The fluorescence intensity was relatively high and the inhibition effect on the target was obvious when the amounts of mAb reached 8 μg. (B) showed that more addition of SA brought stronger fluorescence intensity in the absence of TYL. Nevertheless, in the presence of TYL, the fluorescence intensity was not significantly changed when different amounts of SA were applied. When the amounts of SA reached 10 μg, the fluorescence intensity was not significantly increased, and the inhibition effect was the best one. Therefore, 8 μg of mAb and 10 μg of SA were selected as the optimal label amount of the probe in the sequent experimental.
Figure 1. Optimization of the probe preparation parameters. (A) Amounts of mAb labelled on AuNPs (B) Amounts of SA labelled on AuNPs.
Optimization of HCR conditions
The addition of biotin-initiators and hairpins and the reaction time of HCR affect the sensitivity of the integrated double signal amplification system with ELISA assay. Various amounts of biotin-initiators and hairpins were added to the HCR reaction to explore the effect on fluorescent signals. As shown in (A,B), with the addition of biotin-initiators and hairpins increased, the fluorescence intensity significantly improved. When the addition of biotin-initiators reached 0.0125 nmol, the fluorescent signal was the strongest. However, the excess biotin-initiators could occupy all the recognition sites of SA, which caused incomplete steric hindrance for biotin-initiators and hairpins. More than 0.00375 nmol of hairpins was added to the reaction system, the fluorescence would no longer increase, due to the amount of biotin-initiator was limited. The effect of the HCR time was studied, with the increase in reaction time, the fluorescence intensity increased continuously and reached a steady state at 75 min ((C))
Figure 2. Optimization of HCR conditions. (A) Amounts of biotin-initiator (B) Amounts of hairpins (C) Time of HCR.
Detection of TYL by ELISA-Bio-SA-HCR
The high-sensitive detection of TYL was achieved by the Immuno-Biotin-HCR system. In (A), as the concentration of TYL increased, since TYL competed with TYL-CMO-OVA for mAb labelled on the probe, the complex (TYL-CMO-OVA-mAb-Au-biotin-initiator) formed decreased, and the fluorescence intensity eventually weakened. The linear range for TYL was 1.5625–50 ng/mL. The regression equation was y = –1.54248x + 2.99204 (R2 = 0.99126), where y represents the fluorescence intensity and x represents the concentration of TYL, the LOD was 0.39 ng/mL according to the definition of the mean value of the negative control minus three standard deviations (Shan et al., Citation2016). A calibration curve was established for the detection of TYL by traditional indirect competitive ELISA. As shown in (B), the regression equation was y = –0.62235x + 1.50469 (R2 = 0.9917), where y represents the fluorescence intensity and x represents the concentration of the target. The LOD was 2.67 ng/mL, which was calculated on the basis of the mean value of the negative control minus three standard deviations.
Figure 3. Standard curve of TYL in PBS by ELISA-Bio-SA-HCR (A) and traditional indirect competitive ELISA (B).
Compared with the traditional indirect competitive ELISA, the Immuno-Biotin-HCR signal amplification system achieved the double signal amplification which improves the detection sensitivity of TYL by 6.8-fold. That owes to SA is a tetramer that can be combined with four biotin molecules and HCR is a powerful tool of nucleic acid amplification. The sensitivity of the method combined with the signal amplification system was also compared to that of other methods in .
Table 1. Comparison to other methods.
Specificity analysis
As shown in , other common antibiotics (erythromycin, roxithromycin, tiamulin, spiramycin, azithromycin, abamectin, tilmicosin, natamycin, and acetylisovaleryltylosin) based on Immuno-Biotin-SA-HCR were detected. When other antibiotics were added to the detection system, there was no significant difference in their fluorescence intensity between the control. In the presence of TYL, the competitive inhibition effect of TYL and TYL-CMO-OVA with the probe reduced the fluorescence intensity. The fluorescence intensity of acetylisovaleryltylosin was also significantly reduced due to its similar structure to tylosin. Besides, this method had good specificity.
Figure 4. The evaluation of specificity.
Feasibility in food samples by ELISA-Bio-SA-HCR
In this study, skimmed milk and honey were tested to verify the feasibility of the method in food samples. As depicted in (A), a calibration curve for detecting TYL in spiked milk was established, the fluorescence intensity increased as concentrations of TYL in milk increased, and the linear regression equation was y = –0.95795x + 3.08295 (R2 = 0.93165), where y represents the fluorescence intensity and x represents the concentration of TYL in 10-fold diluted milk, so the LOD of this method in skim milk should be 4.9 ng/mL. As depicted in (B), a calibration curve for detecting TYL in spiked honey was established, the linear regression equation was y = –1.09798x + 3.50666 (R2 = 0.99321), where y represents the fluorescence intensity and x represents the concentration of TYL in 10-fold diluted honey, the LOD of this method in honey should be 2.5 ng/mL.
Figure 5. The detection of TYL in spiked samples. (A) Standard curve of TYL in honey (B) Standard curve of TYL in milk.
Conclusions
In order to realize a novel sensing platform for the high-sensitive detection of hazards in food samples, the approach of fusing ELISA, Bio-SA, and HCR into a comprehensive system with advanced sensitivity and specificity was developed. The linear range for TYL was 1.5625–50 ng/mL. The regression equation was y = –1.54248x + 2.99204 (R2 = 0.99126), where y represents the fluorescence intensity and x represents the concentration of TYL, and the LOD was 0.39 ng/mL. This method was also applied for detecting 9 common antiboics (erythromycin, roxithromycin, tiamulin, spiramycin, azithromycin, abamectin, tilmicosin, natamycin, and acetylisovaleryltylosin), the results showed it had a good specificity. Compared with the traditional indirect competitive ELISA, the Immuno-Biotin-HCR signal amplification system achieved the double signal amplification which improves the detection sensitivity of TYL by 6.8-fold. The combination of ELISA with Bio-SA and HCR was suggested to be potential for sensitive and specific detection of tylosin-related compounds.
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References
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