Detection Of Tetracycline In Milk Research Articles

Emerging Electrochemical Sensors for Real-Time Detection of Tetracyclines in Milk.

Antimicrobial drug residues in food are strictly controlled and monitored by national laws in most territories. Tetracyclines are a major broad-spectrum antibiotic class, active against a wide range of Gram-positive and Gram-negative bacteria, and they are the leading choice for the treatment of many conditions in veterinary medicine in recent years. In dairy farms, milk from cows being treated with antibiotic drugs, such as tetracyclines, is considered unfit for human consumption. Contamination of the farm bulk tank with milk containing these residues presents a threat to confidence of supply and results in financial losses to farmers and dairy. Real-time monitoring of milk production for antimicrobial residues could reduce this risk and help to minimise the release of residues into the environment where they can cause reservoirs of antimicrobial resistance. In this article, we review the existing literature for the detection of tetracyclines in cow’s milk. Firstly, the complex nature of the milk matrix is described, and the test strategies in commercial use are outlined. Following this, emerging biosensors in the low-cost biosensors field are contrasted against each other, focusing upon electrochemical biosensors. Existing commercial tests that identify antimicrobial residues within milk are largely limited to beta-lactam detection, or non-specific detection of microbial inhibition, with tests specific to tetracycline residues less prevalent. Herein, we review a number of emerging electrochemical biosensor detection strategies for tetracyclines, which have the potential to close this gap and address the industry challenges associated with existing tests.

Paper-based analytical device for high-throughput monitoring tetracycline residue in milk

A low-cost and portable paper-based analytical device has been developed for high throughput and on-site monitoring TC residue in milk through visualized colorimetric reaction. The filtration and concentration effect induced by the porous nature of paper contribute to strengthen the color intensity, leading to quantitative and sensitive detection of tetracycline reaching 1 ppm detection limit, with the linear range of 1–100 ppm both in water and milk samples. The applicability was demonstrated by detection of TC in 18 different types of real milk samples with good recovery ranging from 88% to 113%. Furthermore, the dynamic degradation behavior of tetracycline was monitored through the device. To the best of our knowledge, this is the first report of colorimetric detection of tetracycline in milk using the paper-based device. This simple, fast, cost-effective (~$0.50 per device) and equipment-free paper-based platform provides a promising tool for future application in food and environmental safety.

A portable test strip based on fluorescent europium-based metal–organic framework for rapid and visual detection of tetracycline in food samples

Residual tetracycline (TC) in animal food caused by abuse of antibiotics leads to many chronic diseases in the human body. The development of a simple and on-site visualization method for TC detection is need of the hour. Herein, a fluorescent europium-based metal–organic framework (Eu-MOF) sensor for visual and rapid detection of TC was developed. Eu-MOF displays a red emission being excited at 260 nm. Upon exposure to TC, significant fluorescence quenching was observed due to the inner filter effect and photoinduced electron transfer. Moreover, the developed sensor was applied for the detection of TC in milk and beef samples with recoveries of 96.1% to 106.3%, respectively. More importantly, a portable test strip based on Eu-MOF was manufactured. It is a highly selective and sensitive portable device for TC detection. The results can be distinguished immediately by naked eyes, making it become an excellent choice to detect TC in real-time application.

A Sensitive Surface-Enhanced Raman Spectroscopy Method for Detecting Tetracycline in Milk.

Tetracycline, an animal antibiotic, may remain in milk to cause harm to human health. For economic reasons, the abuse of antibiotics is becoming more and more common. Therefore, the abuse of tetracycline has alarmed the dairy industry and many countries such as New Zealand, China, and the USA have proposed strict standards. Surface-enhanced Raman scattering (SERS) is an emerging detection method which has been applied in food detection with the advantages of no complex pretreatment, fast detection, and weak water environment interference. Considering the abuse of antibiotics in dairy industry, we used polydimethylsiloxane (PDMS) plasma cavity as SERS substrate to detect tetracycline in milk. We found that the enhancement ability of PDMS substrate is affected by addition of 4-amino-1-butanol and complex interplay in the milk--tetracycline system. The modified PDMS plasma cavity has high SERS sensitivity that allows us to achieve low detection limit of 0.28 μg/L. The correlation coefficient was 0.987. The detection of tetracycline in milk using PDMS substrate is quick (within 10 min) and it provides a possible method for in-site detection of tetracycline.

Functional Up-Conversion Nanoparticle-Based Immunochromatography Assay for Simultaneous and Sensitive Detection of Residues of Four Tetracycline Antibiotics in Milk.

An ultrahigh-sensitivity lateral flow immunochromatography (LFIC) assay based on up-converting nanoparticles (UCNPs) was developed to carry out a multi-residue detection of tetracycline in milk. The sensitivity of the immunoassay was greatly improved by the use of a broad-spectrum monoclonal antibody attached to UCNPs to form a signal probe. Under the optimal conditions, the UCNP-LFIC assay enabled sensitive detection of tetracycline (TC) as well as of oxytetracycline (OTC), chlortetracycline (CTC), and doxycycline (DOX) within 10 min, with IC50 values of 0.32, 0.32, 0.26, 0.22 ng/mL, respectively. There was no cross-reactivity with ten other antibiotics. Similarly, we evaluated the experimental results for matrix effects. Experiments involving spiking showed the four tetracycline antibiotics displaying mean recoveries ranging from 93.95 to 111.90% with relative standard deviations (RSDs) of < 9.95%. The detection results of actual samples using the developed method showed a good correlation (R2 ≥ 0.98) with the results using high-performance liquid chromatography (HPLC). Thus, the assay can achieve an ultrahighly sensitive detection of antibiotics in milk, and can hence promote human health and provides promising applications in the bio-detection field.

Development of an electrochemical biosensor for impedimetric detection of tetracycline in milk.

This study dealt with the fabrication of an impedimetric biosensor based on nanomaterial modified with pencil graphite electrode for the detection oftetracycline (TET) in milk samples. For response of the impedimetric aptasensor to be improved, the influence of different parameters (immobilization time of reduced grapheme oxide, time of aptamer, and TET binding, and concentration of aptamer) was optimized. In optimum conditions,the aptasensor provided a concentration range within 1 × 10-16 - 1 × 10-6M and with a limit of detection of 3 × 10-17M TET. The proposed impedimetric aptasensor was then used in milk samples analysis, and the acceptable recovery was achieved ranging from 92.8 to 102.1%. According to this study, the combination of an aptamer and electrochemical impedance spectroscopy is a promising method for detection of TET in milk samples with high reproducibility and stability.

Size-dependent modulation of CoOOH nanoflakes light scattering for rapid and selective detection of tetracycline in milk

As an antibiotic, tetracycline has been widely used against bacterial infection or as feed additives for growth promotion, which may lead to the enrichment of antibiotic residues in animals or their products. Therefore, the detection of tetracycline is closely related to human health. The morphological transformation of CoOOH nanoflakes induced by tetracycline was monitored by scanning electron microscope images, dark-field light scattering images and optical spectra, which found that tetracycline could decompose CoOOH nanoflakes into small nanoparticles with low light scattering signal. As a result, the reduced light scattering of CoOOH nanoflakes was proportional to the increased concentration of tetracycline in the range of 1.0–250 μmol/L, and the limit of determination (LOD) was 0.23 μmol/L (3σ/k). Most foreign substances did not interfere in the analysis of tetracycline. Furthermore, the concentration of tetracycline in different milk samples detected with the standard addition method was so low that it accorded with the safety regulation, which was similar to the detection result with high-performance liquid chromatography (HPLC).

Detection of Tetracycline in Milk using NIR Spectroscopy and Partial Least Squares

The feasibility of measuring tetracycline in milk was investigated by near infrared (NIR) spectroscopic technique combined with partial least squares (PLS) method. The NIR transmittance spectra of 40 pure milk samples and 40 tetracycline adulterated milk samples with different concentrations (from 0.005 to 40 mg/L) were obtained. The pure milk and tetracycline adulterated milk samples were properly assigned to the categories with 100% accuracy in the calibration set, and the rate of correct classification of 96.3% was obtained in the prediction set. For the quantitation of tetracycline in adulterated milk, the root mean squares errors for calibration and prediction models were 0.61 mg/L and 4.22 mg/L, respectively. The PLS model had good fitting effect in calibration set, however its predictive ability was limited, especially for low tetracycline concentration samples. Totally, this approach can be considered as a promising tool for discrimination of tetracycline adulterated milk, as a supplement to high performance liquid chromatography.

A novel M-shape electrochemical aptasensor for ultrasensitive detection of tetracyclines

Analytical techniques for detection and quantitation of tetracyclines in food products are greatly in demand. In this study, a novel electrochemical aptasensor was designed for ultrasensitive and selective detection of tetracyclines, based on M-shape structure of aptamer (Apt)-complementary strands of aptamer (CSs) complex, exonuclease I (Exo I) and gold electrode. The aptasensor was developed to make a noticeable electrochemical difference in the absence and presence of tetracycline. In the absence of tetracycline, the M-shape structure, which acts as a gate and barrier for the access of redox probe to the surface of gold electrode remains intact, leading to a weak electrochemical signal. Upon addition of tetracycline, Apt leaves CSs, resulting in disassembly of M-shape structure and following the addition of Exo I, a strong electrochemical signal was observed. The developed analytical assay indicated high selectivity toward tetracycline with a limit of detection (LOD) as low as 450 pM. Moreover, the designed aptasensor was effectively used for the detection of tetracycline in milk and serum samples with LODs of 740 and 710 pM, respectively.

Electrochemical Immunosensor Based on the Chitosan-Magnetic Nanoparticles for Detection of Tetracycline

In this paper, a low-cost, simple, and highly sensitive electrochemical immunosensor was fabricated for the tetracycline detection based on gold electrode-modified carboxyl-Fe3O4 nanoparticle (MNPs) by chitosan (CS) as linker. The anti-tetracycline monoclonal antibody (Ab) was immobilized on the modified electrode surface. The binding of tetracycline to Ab was analyzed by differential pulse voltammetry. Here, MNPs were used as the signal amplifier to improve the sensitivity of the immunosensor. The stepwise assembly process of the electrochemical immunosensor was characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Under the optimum operating conditions, the fabricated immunosensor showed a linear current response to the target concentration in the range from 0.08 to 1 ng/mL with a lower detection limit of 0.0321 ng/mL (S/N = 3). It was successfully applied to the detection of tetracycline in milk. Enzyme-linked immunoassay analysis was also conducted to detect tetracycline in the same samples for demonstrating the applicability of the electrochemical immunosensor.

Novel method for the determination of tetracycline antibiotics in bovine milk based on digital-image-based colorimetry

Solid-phase extraction was used to increase the intrinsic colour of tetracycline (TC), so that colorimetry could be employed as an analytical method for the detection of TC in milk. Digital-image-based colorimetry, a measurement based on capturing colour of solutions by a webcam camera, was coupled with an artificial neural network learning model for quantitative chemical analysis. The red, green, and blue values were used for data processing and were calibrated using values of standard solutions (from a database). The results were obtained directly as TC concentration. The colour intensity of the extracts was tested for accuracy using samples spiked with three TC concentrations. The average percentage recoveries were favourable in all samples and the relative accuracy of the method was between 91.9% and 105.0%. The method was validated by comparing the results with measurements performed using ultra-violet/visible spectrophotometry. There were no significant difference observed between the two methods.

A Colorimetric Aptamer Biosensor Based on Gold Nanoparticles for the Ultrasensitive and Specific Detection of Tetracycline in Milk

This paper proposes a sensing strategy which employs an aptamer, unmodified gold nanoparticles (AuNP), and hexadecyltrimethylammonium bromide (CTAB) to detect tetracycline (TET) in raw milk. The method is based on the colorimetric assay of aggregating AuNP. In the absence of TET, the CTAB and aptamer form a complex which allows the aggregation of AuNP. In the presence of TET, the TET aptamer is exhausted first due to the formation of aptamer-TET complexes, which prevents assembly of the CTAB–aptamer supramolecule, causing a colour change and no aggregation of AuNP. This mechanism for the detection of TET proved to be sensitive and convenient. The colorimetric assay has a detection limit of 122 nM TET. This sensor has great potential for the sensitive, colorimetric detection of a wide range of molecular analytes.

Silver nanoparticle enhanced fluorescence of europium (III) for detection of tetracycline in milk

Abstract Metal-enhanced fluorescence (MEF) has shown a promising potential in fluorometric assays. In this work, the fluorescence of europium (Eu3+) was enhanced by silver nanoparticles (AgNPs) through MEF effect in solution phase, which was applied to the detection of tetracycline (Tc). Tc can coordinate with Eu3+ modified on the surface of AgNPs to form EuTc complex, which emits strong fluorescence due to an intramolecular energy transfer from Tc to Eu3+. The MEF effect of AgNPs further enhanced the emission of EuTc complex; the fluorescence of EuTc on the surface of AgNPs was 4 times higher than that of EuTc in a solution phase. The fluorescence intensity of this probe displayed a good linear response to Tc concentrations in the range of 10 nM to 10 μM with a detection limit of 4 nM, and was applied successfully to determine the levels of Tc in milk with a high selectivity. Compared with other fluorescent detection methods for Tc, the presented method allows simple, direct analysis for Tc without requiring fluorescence enhancement reagents or complicated prepreparation processes. This strategy of AgNP-enhanced fluorescence of lanthanide ions could be applicable to improving homogeneous assays of other molecules.

Direct screening of tetracyclines in water and bovine milk using room temperature phosphorescence detection

A fast and simple flow-through optosensor was designed and characterized for the direct screening of four tetracycline (TCC) antibiotics (tetracycline, oxytetracycline, chlortetracycline and doxycycline) in water and bovine milk samples. The proposed optosensor provides rapid binary yes/no overall responses, being appropriate for the screening of this family of antibiotics above or below a pre-set concentration threshold. The experimental set-up is based on a flow-injection manifold coupled on-line to a phosphorescence detector. Aliquots of the samples are pretreated with Eu(III) to form room temperature phosphorescent metal chelates and injected in the flow manifold. Those chelates are then on-line retained on a conventional flow-cell (packed with polymeric Amberlite XAD-4 particles) which is placed inside the cell holder of the phosphorimeter. After the emission is registered, the antibiotic–metal complexes are eluted from the packed resin with 1 M HCl (for milk samples a second regeneration step, using methanol, should be performed). A sample throughput of about 20 samples per hour was obtained. Optimum experimental conditions include a pH 9, a Eu(III) concentration of 2 × 10 −4 M and 8 mM sodium sulphite as chemical deoxygenant. The phosphorescence emitted by the europium–TCC complexes was measured at 394 and 617 nm for excitation and emission wavelengths, respectively. The unreliability region, given by the probability of false positives and false negatives, respectively (set at 5% in both cases) was in the range between 0.2 and 11.6 nМ for detection of tetracyclines in water samples (at a cut-off level of 4 nM) and in the range between 165 and 238 nM for detection of tetracyclines in milk (cut-off level fixed at the normative EU level of 200 nM). Finally, the applicability of the proposed screening optosensor was tested for the reliable control of tetracyclines in contaminated and uncontaminated water and milk samples.

Evaluation of a Modified Enzymatic Test for the Detection of Tetracyclines in Milk

The tetracycline galactosidase (TG) test, a new method for the detection of tetracycline residues in raw milk based on the inhibition of β-galactosidase biosynthesis in Escherichia coli, was previously validated with spiked milk samples. It has now been applied to milk from cows treated with oxytetracycline. In view of the occurrence of false positives, related to highly elevated somatic cell counts (>106/ml), the improved TG test was developed, in which a heating step (80°C, 15 min) preceded the original TG test protocol. A good agreement with other assays (Delvotest SP, the Bacillus cereus microtiter test, the LacTek tetracycline milk screening test, the Charm HVS-8100 tetracycline test) as well as with high-pressure liquid chromatography was obtained. No false negatives were observed with reference to the established maximum residue limit for tetracyclines of 100 μg/kg milk.