Tetracycline In Milk Samples Research Articles

Strip biosensors based on broad-spectrum aptamers and cationic polymers for the on-site rapid detection of tetracycline antibiotics residues in milk

A lateral flow chromatography strip (LFS) is a chromatography-based biosensor with advantages of convenient carrying, simple operation and rapid detection. In this study, a novel rapid detection technique of aptamer-based chromatography strip was developed and used for the first time for the residue detection of tetracycline antibiotics (TCs) in various milk samples. In this method, gold nanoparticles (AuNPs) modified by TCs specific aptamers were used as probes, and cationic polymers as a capture molecule in a test line (T-line). Meanwhile, the analysis of the gray value of the T-line enabled a linear detection range of 1–300 nM and a limit of detection (LOD) of 0.33 nM for quantitative analysis. The biosensor demonstrated specificity, stability, and successfully detected tetracyclines in milk samples with recovery between 93.60 %–106.20 %. This method sets a basis for multi-residue antibiotics detection in diverse samples, showing potential for on-site applications.

Synthesis of SnC@Au@Apta by electrospinning as an electrochemical sensor for detection of tetracycline

A highly efficient electrochemical aptamer sensor for the detection of tetracycline (TC) was prepared by using SnC@Au@Apta. Metal tin has good electrochemical activity and high conductivity. It is often used as an electrochemical sensing material. The nanofibers prepared by electrospinning machine make the metal distribution more uniform, not easy to agglomerate, and have a certain porosity, which can improve the sensitivity of sensor detection. Carbonization further enhances conductivity. The gold nanoparticles (AuNPs) on the surface of SnC nanomaterials improve the electrochemical detection performance, and also act as the binding site of the TC aptamer, which is stably combined with the thiol group at the end of the TC aptamer. The TC aptamer specifically binds to TC to detect TC in the sample. The electrochemical performance of SnC@Au@Apta was evaluated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Under the optimal conditions, the detection range of SnC@Au@Apta is wide (0.001–100 μM), the detection limit is low (0.83 nM), and it has excellent selectivity, stability and reproducibility. In addition, SnC@Au@Apta can be used to detect TC in milk samples.

Three in one: coordination-induced emission for inherent fluorescent Al-MOF synthesis combined with inner filter effect@aggregation-induced emission mechanisms for designing color tonality and ratiometric sensing platforms.

Three phenomena, namely coordination-induced emission (CIE), aggregation-induced emission (AIE), and inner filter effect (IFE), were incorporated into the design of a ratiometric and color tonality-based biosensor. Blue fluorescent Al-based metal-organic frameworks (FMIL-96) were prepared from non-emissive ligand and aluminum ions via CIE. Interestingly, the addition of tetracycline (TC) led to ratiometric detection and color tonality, as the blue emission at 380nm was quenched (when excited at 350nm) due to IFE, while the green-yellowish emission at 525nm was enhanced due to AIE. Based on that, an ultra-sensitive visual-based color tonality mode with smartphone assistance was developed for detection of TC. The sensor exhibited a linear relationship within a broad range of 2.0 to 85.0μMTC with a detection limit of68.0nM. TC in milk samples was quantified with high accuracy and precision. This integration of smartphone and visual fluorescence in solution isaccurate, reliable, cost-effective, and time-saving, providing an alternative strategy for the semi-quantitative determination of TC on-site.

Rapid detection and differentiation of chlortetracycline and tetracycline by N,P-Ti3C2 QDs

Ti3C2 QDs with good water solubility and photoluminescence properties was prepared by nitrogen and phosphorus doping (N,P-Ti3C2 QDs). The probe has a fluorescence emission peak at 420 nm and emits bright blue fluorescence, which can distinguish and detect two antibiotics with similar structures, chlortetracycline (CTC) and tetracycline (TC). The fluorescence enhancement effect can be realized by the interaction with CTC. The linear range is 2–22 μM, and the detection limit is as low as 0.53 μM. And the change of fluorescent color from bright blue to bright purple can be observed with the naked eye. At the same time, with the addition of TC, the fluorescence of N,P-Ti3C2 QDs can be quenched by static quenching effect, and the fluorescence color changes from bright blue to yellowish green. The detection range is 4.1–20.5 μM, and the detection limit is as low as 0.16 μM.Therefore, on this basis, a strategy for simultaneous detection and recognition of CTC and TC is proposed. The method has been successfully applied to the determination of CTC and TC in milk samples. This paper provides a new detection method for the identification of tetracycline antibiotics and taps the application potential of Ti3C2 QDs in food detection.

A facile nickel nanocluster-based fluorescent ‘‘turn-off” sensor to detect tetracycline antibiotics

Developing an effective method for monitoring of tetracycline (TC) concentrations is extremely crucial due to its adverse effects on both ecosystems and human health. In this study, we developed a novel glutathione-protected nickel nanoclusters (GSHNiNCs) fluorescent (FL) sensing platform to detect TC. The GSHNi NCs were synthesized by a facile one-pot route with GSH as a stabilizer and ascorbic acid (AA) as reducing agent. The experimental results confirmed that the FL of GSHNiNCs was quenched in the presence of TC based on both of the inner-filter effect (IFE) and photoinduced electron transfer (PET) mechanisms to achieve high sensitivity. Under the optimal conditions, excellent linear relationships existed between the FL intensities of GSHNiNCs and the logarithmic TC concentrations in the range of 0.5–120.0 and 120.0–300.0 µM. Even in the absence of surface modification or any complex signal amplification techniques, the proposed FL sensing platform for TC detection showed a low limit of detection of 58 nM (3σ/k). Thus, this approach provides considerable simplicity, low-cost, timesaving and flexibility for the construction of a TC sensor. Most remarkably, we could selectively analyze TC in milk samples without the need for tedious or time-consuming pretreatment processes. This work expands the FL analysis platform for TC detection, and facilitates the design and development of NiNC-based FL sensors for the monitoring of antibiotics.

A Sensitive Fluorescence Sensor for Tetracycline Determination Based on Adenine Thymine-Rich Single-Stranded DNA-Templated Copper Nanoclusters.

A sensitive fluorescent sensor has been developed for the determination of tetracycline (TC) using adenine thymine (AT)-rich single-stranded DNA (ssDNA) templated copper nanoclusters (CuNCs) as a fluorescent probe. Fluorescent ssDNA-CuNCs were synthesized by employing AT-rich ssDNA as templates and ascorbic acid as reducing agents through a facile one-step method. The as-prepared ssDNA-CuNCs exhibited strong fluorescence with a large Stokes shift (240 nm) and stable fluorescence emission. In the presence of TC, the fluorescent intensity of ssDNA-CuNCs was obviously decreased through the inner filter effect, due to the spectral overlapping between ssDNA-CuNCs and TC. Under the optimal conditions, the strategy exhibited sensitive detection of TC with a linear range from 2 nM to 30 μM and with a limit of detection of 0.5 nM. Furthermore, the sensor was successfully applied for the detection of TC in milk samples. Therefore, it provided a simple, rapid, and label-free fluorescent method for TC detection.

Towards direct detection of tetracycline residues in milk with a gold nanostructured electrode.

Tetracycline antibiotics are used extensively in veterinary medicine, but the majority of the administrated dose is eliminated unmodified from the animal through various excretion routes including urine, faeces and milk. In dairy animals, limits on residues secreted in milk are strictly controlled by legislation. Tetracyclines (TCs) have metal chelation properties and form strong complexes with iron ions under acidic conditions. In this study, we exploit this property as a strategy for low cost, rapid electrochemical detection of TC residues. TC-Fe(III) complexes in a ratio of 2:1 were created in acidic conditions (pH 2.0) and electrochemically measured on plasma-treated gold electrodes modified with electrodeposited gold nanostructures. DPV measurements showed a reduction peak for the TC-Fe(III) complex that was observed at 50 mV (vs. Ag/AgCl QRE). The limit of detection in buffer media was calculated to be 345 nM and was responsive to increasing TC concentrations up to 2 mM, added to 1 mM FeCl3. Whole milk samples were processed to remove proteins and then spiked with tetracycline and Fe(III) to explore the specificity and sensitivity in a complex matrix with minimal sample preparation, under these conditions the LoD was 931 nM. These results demonstrate a route towards an easy-to-use sensor system for identification of TC in milk samples taking advantage of the metal chelating properties of this antibiotic class.

Ratiometric Sensing for Ultratrace Tetracycline Using Electrochemically Active Metal-Organic Frameworks as Response Signals.

A novel ratiometric sensor using an electrochemically active metal-organic framework of Mo@MOF-808 and NH2-UiO-66 as response signals was developed to detect tetracycline (TET) in ultratrace quantities. To achieve the dual-response strategy, Mo@MOF-808, with a reduction peak at -1.06 V, and NH2-UiO-66, with an oxidation peak at 0.724 V, were used as signal probes directly. Concretely, Mo@MOF-808, single-stranded DNA (ssDNA), and complex system (Apt@NH2-UiO-66) of aptamer (Apt) and NH2-UiO-66 were sequentially immobilized on the electrode. With the addition of TET, Apt was hybridized with TET and Apt@NH2-UiO-66 was detached from the electrode, resulting in an increase in the current at -1.06 V and a decrease in the current at 0.724 V. Through this strategy, the sensor achieved a wide linear range (0.1-10000 nM) and a low limit of detection (0.009792 nM) for TET. Moreover, the ratiometric sensor exhibited better sensitivity, reproducibility, and stability than a single-signal sensor. Furthermore, the constructed sensor was successfully applied to detect TET in milk samples, suggesting excellent application prospects.

Electrochemical sensing of tetracycline based on Au NPs@MoS2/Ch hybrid structures

• For the first time, Au NPs@MoS 2 is used to detect tetracycline. • The Au NPs and MoS 2 form a novel complex. • We have constructed a new type of electrochemical platform based on AuNPs@MoS2, which is based on the synergy between AuNPs and MoS2 for simple, rapid, ultra-sensitive and quantitative detection of tetracycline. As a broad-spectrum antibiotic, tetracycline (TC) has a wide range in the treatment of diseases and feed additives. Molybdenum disulfide (MoS 2 ) is becoming more and more popular in terms of electrochemical sensing. So as to improve the sensitivity, AuNPs@MoS 2 is coated on the electrode surface by combining with chitosan (Ch) to improve the conductivity. Under optimized conditions, this assay has high sensitivity and excellent linear response for detection TC. That covers the 1–1000 μM and 5–1100 μM tetracycline (TC) concentration range with a 0.41 μM limit of detection. In addition, the sensor may become a promising new strategy for detecting tetracycline in milk samples. This study develop an unusual analytical chemistry method for the development of various challenging electrochemical sensors.

A novel probe for tetracyclines detection and its applications in cell imaging based on fluorescent WS2 quantum dots

In this study, a novel fluorescence sensor for tetracyclines (TCs) detection was designed using WS2 quantum dots (WS2 QDs). WS2 QDs could be quenched by TCs through the inner filter effect (IFE). The limit of detection of this proprosed method is 39 nM, 52 nM, and 28 nM for tetracycline (TC), doxycycline (DC), and oxytetracycline (OTC), respectively. The as-proposed strategy was successfully applied to detect TC in milk samples and human serum samples. The WS2 QDs were highly biocompatible and showed lower toxicity. Moreover, the WS2 QDs was successfully applied to imaging TC in HeLa cells owing to its excellent optical performance and great biocompatibility.

Cucurbit[8]uril-Based Potentiometric Sensor Coupled to HPLC for Determination of Tetracycline Residues in Milk Samples

The determination of chlortetracycline, doxycycline, oxytetracycline, and tetracycline in milk samples by HPLC coupled to a cucurbit[8]uril-based potentiometric sensor is herein presented. The new tetracycline-selective electrode is based on a polymeric membrane incorporating cucurbit[8]uril as a macrocyclic host, potassium tetrakis(p-chlorophenyl) borate as an ionic additive, 2-fluorophenyl 2-nitrophenyl ether as a plasticizer, and multi-walled carbon nanotubes as nanostructured materials. A microfluidic wall-jet flow-cell is implemented as a potentiometric detector after chromatographic separation by a C8 column using a gradient mobile phase of sulphuric acid and acetonitrile. The proposed methodology was validated following International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) and European Union (EU) guidelines. Linear regression models provided R2 in the range from 0.9973 ± 0.0026 to 0.9987 ± 0.0012 for all tetracycline antibiotics. The limits of detection and quantification ranged from 13.3 to 46.0 μg L−1 and 44.4 to 92.1 μg L−1, respectively. Precision intra-day, inter-day, and inter-electrode showed relative standard deviation values lower than 12.5%, 13.5%, and 12.9%, respectively. Accuracy was assessed by analysis of spiked milk samples around the maximum residue limit, yielding recovery values in the range from 81.3 to 108.5%. The simple, sensitive, cost-effective, and reliable HPLC-ion-selective electrode method justifies its use as a competitive alternative for the analysis of tetracycline residues in the food quality control sector.

Homogeneous Photoelectrochemical Aptasensors for Tetracycline Based on Sulfur-Doped g-C3N4/n-GaN Heterostructures Formed through Self-Assembly.

The complex synthesis of photoelectric materials and the difficulty of fixing the identification elements on the photoelectrode are long-standing problems in the field of photoelectrochemical (PEC) biosensing. In this work, a simple PEC aptasensor construction strategy based on a sulfur-doped g-C3N4 (SCN)/n-GaN heterostructure photoelectrode was proposed. The SCN/n-GaN heterostructure can be formed through self-assembly in solution since SCN can be uniformly dispersed in solution. In addition, as a dual-function mediate, an aptamer can be fixed on an SCN substrate automatically because of the good adsorption performance of SCN. Therefore, tedious steps of PEC electrode preparation and the fixing of recognition elements were both avoided. Compared with the traditional ones, the construction difficulty and time cost of the prepared PEC aptasensors are greatly reduced. The simplified experimental process improves the stability and reproducibility of the aptasensor. Finally, tetracycline (TET) was used as a model target to verify the sensing performance of the proposed PEC strategy. TET can consume the photogenerated holes of the SCN/n-GaN heterostructure, promote carrier migration, and result in the change in the photocurrent. The linear relationship between the change in the photocurrent intensity and the TET concentration can be used to detect TET. The aptasensor has a linear range of 0.10-10.0 nmol L-1 and the detection limit is 0.030 nmol L-1 (3S/N). The aptasensor was applied to the detection of TET in milk samples with satisfactory results.

Bioinspired Carbon Dots as an Effective Fluorescent Sensing Platform for Tetracycline Detection and Bioimaging

AbstractBioinspired carbon dots (CDs) with excellent biocompatibility were prepared from egg white as precursors through a simple hydrothermal method at 200 °C for 4 hours. The obtained CDs exhibited bright blue fluorescence, excellent optical stability, and sensitive response to tetracycline. Under the optimized synthetic conditions, the fluorescence quantum yield is 17.6 %. The fluorescence intensity of CDs at 440 nm could be quenched linearly with the addition of tetracycline in the range of 0.1–200 μmol L−1 due to the internal filtering effect. The proposed detection platform has been applied in the determination of tetracycline in milk samples with good results. The limit of detection is 32.3 nmol L−1, and the spiked recoveries are in the range of 93–103 %. Additionally, the CDs could also be applied to cell imaging, implying good biocompatibility and low toxicity. As a type of green and effective multi‐functional fluorescence materials, the CDs have potential applications in biochemical sensing and biomedical imaging.

Nitrogen-doped cyan-emissive carbon quantum dots for fluorescence tetracycline detection and lysosome imaging.

Tetracyclines (TCs) prevent the growth of peptide chains and the synthesis of proteins, and they are widely used to inhibit Gram-positive and -negative bacteria. For the detection of tetracyclines in cell and in vitro, a convenient and simple detection system based on nitrogen-doped cyan carbon quantum dots (C-CQDs) was developed. C-CQDs have excellent excitation-independent properties, the best optimal excitation peak is 360 nm and the best emission peak is 480 nm. Based on the inner filter effect (IFE), the fluorescence intensity of C-CQDs in solution decreases with the increase of tetracyclines. In the range of 0-100 μM, C-CQDs present a good linear relationship with three tetracyclines (CTC, TET, OCT), with R 2 all greater than 0.999. C-CQDs can detect tetracycline in milk samples with recovery in the range of 98.2-103.6%, which demonstrates their potential and broad application in real samples. Furthermore, C-CQDs exhibit excellent lysosomal targeting, as indicated by a Pearson's coefficient of 0.914 and an overlap of 0.985. The internalisation of C-CQDs was mainly affected by lipid raft-mediated endocytosis in endocytic pathway experiments. These experiments indicate that C-CQDs can be effectively used to detect TC content and target lysosomes as an alternative to commercial dyes.

Theoretical design and experimental study of new aptamers with the enhanced binding affinity relying on colorimetric assay for tetracycline detection

In this research, a screening procedure was applied for designing new aptamers with the enhanced binding affinity for tetracycline detection through a common theoretical and experimental study. In the first step, among the specific aptamers introduced from the experimental studies, the 8-mer aptamer was chosen toward tetracycline with a low limit of detection. The different mutants were designed by changing the nucleotides of the native sequence and, their MD simulations were performed in the presence of tetracycline. The conformational factor (Pi) was computed as a parameter that indicates the affinity of each aptamer nucleotides to target. To calculate the ΔGbind as a suitable factor for the specificity of aptamer toward the target, the umbrella sampling simulation was applied. Theoretical results proved that some of the mutants, such as the M-5T and M-5C mutants possess enhanced binding affinity toward tetracycline compared to the native aptamer. In the next step, the enhanced selective detection of designed mutants toward tetracycline was investigated by utilizing an easy colorimetric test relying on the gold nanoparticles (AuNPs). Based on the experimental outcomes, the limit of detection values of 159, 1.2 and, 0.1 pM were determined for the native, M-5T and, M-5C aptamers, respectively. The results of colorimetric showed that the designed mutants have good selectivity to tetracycline compared to other antibiotics. Besides, the designed mutants were successfully applied to detect tetracycline in milk samples. The agreement among the theoretical and experimental results proves the ability of theoretical approaches for designing the new aptamers with enhanced binding affinity to identify different targets.

Using magnetic nanoparticles/MIP-based electrochemical sensor for quantification of tetracycline in milk samples

This paper reports the development of a biomimetic sensor and its application for the determination of tetracycline antibiotics. Tetracycline is an extremely vital antibiotic usually used for the treatment of people and livestock with bacterial disorder and respiratory issues. This antibiotic is widely used in poultry farms for the prevention of bacterial infection and the treatment of advanced bacterial growth. The sensor was constructed using magnetic nanoparticles (mag) and molecularly imprinted polymer (MIP). Based on the application of the co-precipitation approach, FeCl3·6H2O and FeCl2·4H2O salts were used to obtain [email protected] magnetic nanoparticles. Prior to the synthesis of the selective MIP material, the density functional theory (DFT) was used to select the best functional monomer for the analyte investigated. After choosing the best monomer, the modified magnetic nanoparticles were used for the synthesis of the MIP in order to enhance the detection capability of the electrochemical sensor. The imprinted polymer was synthesized using optimized amounts of acrylic acid (functional monomer), ethylene–glycol-dimethacrylate (cross-linker), and 2,2 azobisisobutyronitrile (radical initiator). The control material – denoted by mag-NIP (magnetic non-molecularly imprinted polymer), which was used for comparison purposes, was synthesized based on the same procedure applied for the synthesis of the MIP but in the absence of the analyte (tetracycline). The analysis of surface morphology and the identification of the functional groups used in the synthesized magnetic materials were performed by fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscopy (FESEM), and high-resolution electron microscopy. The detection of tetracycline was performed under optimized conditions using square wave voltammetry, 15 mg of mag-MIP (on carbon paste), and 80 s of pre-concentration. Through the application of this analysis, well-defined peaks ranging from 0.5 to 1.0 V vs. Ag/AgCl (KClsat) were obtained. Analytical curves were constructed using the second peak at 0.83 V, and the following results were obtained: linear range of 5.0 × 10−7 to 4.0 × 10−5 mol L−1 (R2 of 0.9993), limit of detection (LOD) of 1.5 × 10−7 mol L−1 and repeatability of 2.82% (in terms of relative standard deviation) for n = 10. The results obtained from the analysis of selectivity demonstrated that the proposed electrochemical sensor is highly efficient for tetracycline determination. The sensor was successfully applied in commercial and raw milk samples for tetracycline detection, and recovery rates ranging from 93 − 103% were obtained. The results obtained show that the proposed electrochemical technique is suitable for application toward the determination of tetracycline.

Determination of seven tetracyclines in milk by dissolvable layered double hydroxide-based solid-phase extraction coupled with high-performance liquid chromatography

A dissolvable layered double hydroxide-based solid-phase extraction combined with high-performance liquid chromatography was developed for the analysis of minocycline, oxytetracycline, tetracycline, demeclocycline, metacycline, chlortetracycline and doxycycline in milk samples. In situ formation of the layered double hydroxide was achieved by the addition of MgCl2-AlCl3 solution to alkaline deproteinized milk. The analytes were efficiently extracted by the Mg/Al layered double hydroxide. After centrifugation, the co-precipitates were dissolved in 0.1 mol L-1 Na2EDTA-McIlvaine buffer prior to HPLC analysis. Under optimized conditions, the method achieved low detection limits of 0.414-0.986 μg L-1 and quantification limits of 1.38-3.29 μg L-1, and good recoveries of 93.5-100% with intra- and inter-day RSDs of 0.498-4.08% and 1.23-10.0%, respectively. This method is convenient, accurate, sensitive, rapid, cost-effective, eco-friendly, and suitable for the determination of seven tetracycline antibiotics in milk samples.

Mixed-mode fabric phase sorptive extraction of multiple tetracycline residues from milk samples prior to high performance liquid chromatography-ultraviolet analysis

Herein, a new extraction mode of classical fabric phase sorptive extraction (FPSE), namely mixed-mode strong cation exchanging FPSE is presented and evaluated for the first time for the determination of tetracycline residues in milk, after protein precipitation. FPSE is a microextraction technique characterized by high chemically stable sorbent, higher analyte retention capacity and faster analyte diffusion capability, where the target analytes interact with the extraction sorbent during the pre-treatment process via a plethora of intermolecular/interionic interactions exerted by different functional moieties implanted on the sponge-like porous sol–gel sorbent network. Subsequently, the selectively retained analytes are eluted using a small volume of solvent system. A novel extraction membrane containing sol–gel C18/propyl sulfonic acid, a mixed mode sorbent consisting of neutral, C18 and strong cation exchanger, propyl sulfonic acid (C3H6SO3−) coated on 100% cotton cellulose fabric substrate was designed, synthesized, characterized and evaluated for the selective isolation of four tetracycline antibiotic residues from milk samples.For quantitative analysis, high performance liquid chromatography coupled with a UV detector (at 350 nm) was used. The method was validated according to the 2002/657/EC decision with regards to linearity, selectivity, stability, limits of detection and quantitation (LOD/LOQ), decision limit, detection capability, trueness, precision and ruggedness, according to the Youden’s test. Recoveries ranged between 88.9 and 122.4%, while the LOD and LOQ were obtained at 15 μg kg−1 and 50 μg kg−1, respectively. Finally, the composite analytical method was applied to monitor the presence of residual tetracyclines in milk samples purchased from a local Greek market.

Selective removal of tetracycline residue in milk samples using a molecularly imprinted polymer

In the present work, the synthesis and characterisation of a molecularly imprinted polymer (MIP) by precipitation polymerisation using tetracycline (TC) as a template molecule, methacrylic acid as a functional monomer, ethylene glycol dimethacrylate as a cross-linker, persulphate as an initiator, and methanol as a porogen solvent is described. The molecular recognition properties and selectivity of MIPs against four TCs [(TC), oxytetracycline (OT), chlortetracycline (CT), and doxycycline (DT)] were evaluated, and the results demonstrated high selectivity for four TCs in milk samples without sample treatment. Under the optimal synthesis conditions (TC/methacrylic acid/ethylene glycol dimethacrylate ratio of 1.0:7.0:50.0), the percent removal obtained was 81.83% for TC, 95.47% for OT, 96.44% for CT, and 93.25% for DT using 1.0 mL of the sample, 20 mg of the sorbent, a pH of 6.0, and 15 min of contact time. One of the tested samples was positive for the presence of OT, with a concentration of 19.83 μg L−1, ensuring the complete removal of TC using the proposed method. The method provided significant data regarding the development of efficient and selective materials in the removal of several residues and contaminants.

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.