A method for detecting tetracycline antibiotics

Abstract

The application discloses a detection method of tetracycline antibiotics, and belongs to the technical field of organic chemical analysis, and comprises the following steps: S1, preparing a three-dimensional layered double hydroxide fluorescent sensor array; S2, measuring sample data of a series of concentrations; and S3, measuring the type of tetracycline antibiotics in an unknown sample. The application has the advantages of simple detection process, fast detection speed, short time consumption, simultaneous detection of multiple objects, and great application prospect.

Description

Technical Field

[0001] This invention belongs to the field of organic chemical detection technology, and specifically relates to a method for detecting tetracycline antibiotics. Background Technology

[0002] As broad-spectrum third-generation tetracycline antibiotics, tetracyclines (TCs) have been used to treat various bacterial infections in humans and animals due to their inhibitory effects on both Gram-negative and Gram-positive bacteria, with particularly high demand in the livestock and poultry farming industry and the veterinary drug sector. Currently, there are eight tetracyclines on the market, among which tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), and doxycycline (DOX) are the four most commonly used prescription antibiotics in veterinary medicine. However, because animals have limited metabolism of tetracyclines, residues can remain in animal-derived foods, potentially harming human health through the food chain. Therefore, it is necessary to develop an effective and convenient method for detecting tetracycline residues in animal-derived foods. Currently, methods such as chromatography, enzyme-linked immunosorbent assay (ELISA), electrochemical analysis, and microbiological analysis have been developed for the detection of tetracycline residues in food; however, these methods either only detect a single substance or are time-consuming and require complex sample pretreatment.

[0003] Fluorescence analysis methods are simple to operate, have a rapid response, and are highly selective, making them widely used in the detection of tetracycline residues in animal-derived foods. In particular, fluorescence sensor arrays offer advantages such as high sensitivity, low cost, simplicity, speed, and multiplex identification, making this method extremely effective for detecting multiple structurally similar substances in complex environments. Examples include constructing a dual-mode fluorescence sensing platform based on nitrogen-doped biomass-derived carbon dots, or a colorimetric fluorescence dual-channel sensor array based on the EriochromeBlack T / Eu complex, for the detection of tetracycline antibiotics in biological and food samples.

[0004] Quantum dots (QDs), also known as semiconductor nanocrystals (NCs), are quasi-zero-dimensional semiconductor nanomaterials with unique optical properties. The diameter of quantum dot molecules is generally between 1 and 10 nm, and they are usually composed of elements from groups II-VI, III-V, and IV. Common quantum dots include cadmium sulfide quantum dots (CdS QDs), cadmium selenide quantum dots (CdSe QDs), cadmium telluride quantum dots (CdTe QDs), zinc sulfide quantum dots (ZnS QDs), and zinc selenide quantum dots (ZnSe QDs).

[0005] Compared to conventional organic dyes or fluorescent proteins, quantum dots have been widely used in chemical / biological sensor arrays due to their advantages such as high fluorescence quantum yield, narrow emission band, and high resistance to photobleaching. Among them, CdTe quantum dots, as a type of semiconductor quantum dot, are an ideal multicolor luminescent material for sensor array technology because their particle size and emission wavelength can be easily adjusted by controlling the reaction time.

[0006] Layered double hydroxides (LDHs) are a class of versatile metal-organic framework derivatives, widely used in catalysis, electrochemistry, water remediation, and pharmaceuticals due to their layered structure and efficient modification of chemical components. Three-dimensional layered double hydroxides (3D LDHs) can be formed by stacking two-dimensional LDH crystals together in a unique flower-like structure. Combinations of LDHs with different geometries, ranging from roses to dahlias, from peonies to dandelions, have been obtained through specific methods. Compared to ordinary LDH materials, these flower-like 3D LDHs exhibit good chemical stability and multifunctionality, with higher surface area and larger pore volume. This material can serve as an ideal carrier for nano-quantum dot materials, preventing quantum dot aggregation and improving stability. Summary of the Invention

[0007] In view of this, the present invention provides a method for detecting tetracycline antibiotics. This method is simple in procedure, highly sensitive, and can rapidly detect a variety of different tetracycline antibiotics and their contents, and has great application prospects.

[0008] This solution is achieved through the following technical means:

[0009] A method for detecting tetracycline antibiotics, comprising the following steps:

[0010] S1: Fabrication of a dual-channel fluorescence sensor array based on cadmium telluride quantum dots and three-dimensional layered double hydroxides;

[0011] S2: Determination of data from a series of concentration samples: Prepare a series of gradient concentration standard solutions of one or more tetracycline antibiotics, and then mix the dual-channel fluorescence sensor array obtained in step S1 with a trihydroxyaminomethane-hydrochloric acid buffer solution at pH 7.4 to obtain a mixed solution. Add each standard solution to the above mixed solution, shake well, and measure the fluorescence intensity value. Each sample is tested in parallel 4 times to obtain the detection and differentiation data of tetracycline antibiotics.

[0012] S3: Determination of the type and content range of tetracycline antibiotics in unknown samples: The dual-channel fluorescence sensor array obtained in step S1 is mixed with a trihydroxyaminomethane-hydrochloric acid buffer solution at pH 7.4 to prepare the test solution for the unknown sample. The test solution is added to the above mixed solution and shaken to mix. The fluorescence intensity value is measured. Each test sample is tested in parallel 4 times. The obtained data and the detection differentiation data of tetracycline antibiotics obtained in step S2 are plotted simultaneously to form an LDA diagram. The type and content range of tetracycline antibiotics in the unknown sample are determined by the overlap or positional relationship of the data points.

[0013] Compared to existing technologies, this invention constructs a fluorescence sensor array with dual fluorescence channels and uses this array to detect tetracycline antibiotic standard solutions, thereby obtaining detection discrimination data and ranges for tetracycline antibiotics. Then, the same fluorescence sensor array is used to detect unknown samples, collecting fluorescence intensity change data. Linear discriminant analysis is then employed to achieve simultaneous differentiation and detection of tetracycline antibiotics with similar structures. The detection method of this invention is simple, fast, and time-efficient, and can simultaneously differentiate and detect multiple targets, showing promising application prospects.

[0014] Preferably, step S1 includes the following steps:

[0015] S11: Synthesis of red-light CdTe quantum dots and yellow-light CdTe quantum dots;

[0016] S12: Preparation of three-dimensional layered double hydroxides;

[0017] S13: 50 mg of three-dimensional layered double hydroxide was dispersed in 10 mL of 0.05 M trihydroxyaminomethane-hydrochloric acid buffer solution and ultrasonically dispersed. 1.2 mL of yellow CdTe quantum dot solution (1 g / L) was added dropwise and stirred in a water bath at 20 °C for 30 min. 0.6 mL of red CdTe quantum dot solution (1 g / L) was added dropwise and stirred for 3 h. After stopping stirring, the mixture was centrifuged at 8000 rpm and washed three times with deionized water. It was then vacuum dried at 45 °C for 8 h. The dried powder was redispersed in deionized water to obtain a dual-channel fluorescence sensor array (1 g / L).

[0018] Preferably, CdTe quantum dots are synthesized in step S11 using the following steps: Weigh an appropriate amount of sodium citrate into a container and add deionized water. After complete dissolution, add CdCl2·2.5H2O and stir until homogeneous. Immediately add mercaptopropionic acid. Add 1M NaOH solution dropwise to the mixed solution to adjust the pH to 10.5. Add Na2TeO3 and NaBH4 under vigorous stirring. Heat at 100°C for 40-60 minutes under reflux. After stopping heating, add an equal amount of ethanol to precipitate the quantum dots. Centrifuge, wash, and redisperse with deionized water to prepare a solution.

[0019] Preferably, step S12 involves the following steps to prepare 3D LDH: Mg(NO3)2·6H2O, Al(NO3)3·9H2O and urea are dissolved in deionized water to obtain solution A; SDS is dissolved in deionized water to form a clear solution B at 60°C; solution B is added to solution A to obtain a mixture; the mixture suspension is stirred for 30 min, transferred to an autoclave, and subjected to hydrothermal reaction at 160°C for 6 h; the mixture is centrifuged and washed with deionized water and ethanol, and dried at 70°C to obtain 3D LDH.

[0020] Preferably, in step S11, the ratio of sodium citrate, CdCl2·2.5H2O, Na2TeO3 and NaBH4 is 0.22g:0.11g:0.02g:0.05g.

[0021] Preferably, in step S12, the ratio of Mg(NO3)2·6H2O, Al(NO3)3·9H2O, urea and SDS is 0.3g:0.48g:0.03g.

[0022] The present invention has the following technical effects:

[0023] This invention fully utilizes the advantages of CdTe QDs' multicolor luminescence and the porous structure and large specific surface area of ​​3D LDH to successfully synthesize two types of CdTe QDs emitting orange-yellow and red light. These two QDs are then loaded onto 3D LDH via electrostatic adsorption to prepare a single-particle dual-emission three-dimensional luminescent material (3D LDH-CdTe QDs). A fluorescence sensor array with two fluorescence channels is constructed to distinguish and detect four tetracycline antibiotics with similar structures. The four TCs interact with CdTe QDs, causing varying degrees of fluorescence quenching. The fluorescence sensor array is applied to distinguish TCs at the same concentration, to differentiate and quantify TCs of different concentrations, and to detect and differentiate TCs with different compositions. It is also used for the identification and semi-quantitative detection of TCs in milk samples, demonstrating good differentiation and identification performance, capable of distinguishing four TCs down to 0.1 μM. The invention features a simple detection process, fast detection speed, short processing time, and the ability to simultaneously distinguish and detect multiple objects, showing promising application prospects. Attached Figure Description

[0024] Figure 1 The column fingerprints of the four analytes at 0.1 μM, 0.3 μM, 1 μM and 5 μM are shown.

[0025] Figure 2 Fluorescence spectra, two-dimensional LDA plots, and linear relationship plots of different concentrations of TC (A1, B1, C1), DOX (A2, B2, C2), CTC (A3, B3, C3), and OTC (A4, B4, C4).

[0026] Figure 3 LDA plots for different TC / DOX mixing ratios (10:0, 8:2, 6:4, 5:5, 3:7, 1:9, 0:10).

[0027] Figure 4 Two-dimensional LDA scatter plots of TC, DOX, CTC and OTC in a series of mixed proportions.

[0028] Figure 5 Two-dimensional LDA scatter plot of milk with a spiked mixture concentration ratio of TC / DOX = 4:6.

[0029] Figure 6 Two-dimensional LDA scatter plots of milk spiked with 2 μM and 10 μM TC and a series of TC concentration standards. Detailed Implementation

[0030] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0031] The experimental instruments and reagents used in the following examples are shown in Tables 1 and 2 below;

[0032] Table 1 Experimental Instruments

[0033]

[0034] Table 2 Experimental Reagents

[0035]

[0036]

[0037] Example 1: Detection and differentiation of four tetracycline antibiotics at the same concentration

[0038] This embodiment uses the following detection method to distinguish four antibiotics—tetracycline (TC), oxytetracycline (OTC), chlortetracycline (DOC), and doxycycline (CTC)—at the same concentration. The detection method includes the following steps:

[0039] S1: Constructing 3D LDH-CdTe QDs: This includes the following steps:

[0040] S11: Preparation of orange-yellow and red-light CdTe quantum dots

[0041] This embodiment uses two types of CdTe quantum dots, namely two stable CdTe quantum dots (CdTe QDs) with emission wavelengths of 550nm (yellow) and 630nm (red).

[0042] The preparation process of orange-yellow CdTe quantum dots (o-CdTe QDs) is as follows: Weigh 0.22 g of sodium citrate into a 100 mL round-bottom flask and add 50 mL of deionized water. After complete dissolution, add 0.11 g of CdCl2·2.5H2O, stir well, and immediately add 52 μL of MPa. Add 1 M NaOH solution dropwise to the mixed solution to adjust the pH to 10.5. Add 0.02 g of Na2TeO3 and 0.05 g of NaBH4 under vigorous stirring, and heat at 100 °C for 40 minutes under reflux. After stopping heating and cooling, add an equal amount of ethanol to precipitate the quantum dots, centrifuge, wash, and redisperse with deionized water to prepare a 1 g / L o-CdTe QDs solution, which is stored at 4 °C in the dark for later use.

[0043] The preparation process of red CdTe quantum dots (r-CdTe QDs) is the same as that of orange-yellow CdTe quantum dots, but the heating time is 60 min, and an r-CdTe QDs solution is prepared.

[0044] S12: Synthesize 3D LDH

[0045] 0.44 g Mg(NO3)2·6H2O, 0.30 g Al(NO3)3·9H2O, and 0.48 g urea were dissolved in 50 mL of deionized water. Then, 0.03 g SDS was dissolved in 30 mL of deionized water, and a clear solution was formed at 60 °C. This solution was immediately added to the salt solution, and the mixture suspension was stirred for 30 min. The mixture was then transferred to a 100 mL autoclave and subjected to hydrothermal reaction at 160 °C for 6 hours. After centrifugation, the solution was washed with deionized water and ethanol, and dried at 70 °C to obtain 3D LDH.

[0046] S13: Constructing 3D LDH-CdTe QDs

[0047] 50 mg of 3D LDH was dispersed in 10 mL of 0.05 M Tris-HCl solution and sonicated. 1.2 mL of Lo-CdTe QDs (1 g / L) solution was added dropwise, and the mixture was stirred in a 20 °C water bath for 30 minutes. Then, 0.6 mL of r-CdTe QDs (1 g / L) solution was added dropwise, and stirring continued for 3 hours. After stirring was stopped, the mixture was centrifuged at 8000 rpm and washed three times with deionized water. The mixture was then vacuum dried at 45 °C to obtain 3D LDH-CdTe QDs.

[0048] S2: Measurement data of a series of concentration samples

[0049] Standard solutions of TC, OTC, CTC, and DOX with gradient concentrations of 0.1 μM, 0.3 μM, 1 μM, and 5 μM were prepared. These standard solutions were then rapidly added to a mixture of 0.5 mL of 3D LDH-CdTe QDs (final concentration 0.6 mg / mL) and 1.1 mL of Tris-HCl buffer solution (pH = 7.4, 0.05 M). After shaking thoroughly, the fluorescence intensity values ​​at emission wavelengths of 550 nm and 630 nm were immediately measured using a fluorescence spectrophotometer. Four independent parallel experiments were conducted for each standard solution (including blank samples), and linear discriminant analysis in SPSS was used to distinguish between the four antibiotics at the same concentration. The detection data for tetracycline (TC), oxytetracycline (OTC), chlortetracycline (DOC), and doxycycline (CTC) were obtained.

[0050] Example 2: Detection, differentiation, and semi-quantitative analysis of tetracycline antibiotics at different concentrations

[0051] A series of gradient standard solutions for TC, OTC, CTC, and DOX were prepared separately, and the detection was performed using the method and steps of Example 1. Taking TC as an example, 3D LDH-CdTe QDs (final concentration 0.6 mg / mL) and Tris-HCl buffer solution (pH = 7.4, 0.05 M) were mixed in a 2 mL centrifuge tube to obtain a mixed solution. Subsequently, the target analytes of different concentrations of TC standard solutions were rapidly added to the above mixed solution. Four independent parallel experiments were set up for each sample (including blank samples). After shaking and homogenization, the fluorescence intensity values ​​at emission wavelengths of 550 nm and 630 nm were immediately measured using a fluorescence spectrophotometer to obtain the detection differentiation data of TC at different concentrations. The same method was used to detect and differentiate the series of standard concentrations of OTC, CTC, and DOX to obtain the detection differentiation data of the four antibiotics TC, OTC, CTC, and DOX at different concentrations.

[0052] Example 3: Detection and differentiation of tetracycline antibiotics with different compositions

[0053] In this embodiment, solutions with TC / DOX ratios of 10:0, 8:2, 6:4, 5:5, 3:7, 1:9, and 0:10 were prepared, with a total concentration of 5 μM. The detection method of Example 1 was used to detect and distinguish the mixed concentration of TC and DOX. Four parallel samples were set for each analyte to obtain detection and differentiation data of TC and DOX dual components under different mixing ratios.

[0054] In this embodiment, solutions with the following ratios were also prepared: TC:CTC:OTC = 10:0:0, TC:CTC:OTC = 0:10:0, TC:CTC:OTC = 0:0:10, TC:CTC:OTC = 4:6:0, TC:CTC:OTC = 0:3:7, TC:CTC:OTC = 2:0:8, and TC:CTC:OTC = 3:3:4, with a total concentration of 5 μM. The detection method of Example 1 was used for detection and differentiation. Four parallel samples were set for each analyte to obtain detection and differentiation data of the three components of TC, CTC, and OTC under different mixing ratios.

[0055] Example 4: Application and semi-quantitative analysis in actual samples

[0056] According to existing literature, tetracycline antibiotics are often added to animal feed for disease prevention, and there are reports of residues being detected in milk and eggs. In this embodiment, a commercially available brand of milk was selected as the actual sample. After removing protein and fat, high-performance liquid chromatography (HPLC) analysis showed no TCs detected. Therefore, a spiked recovery experiment was performed on the milk before testing. First, the milk sample was mixed with acetonitrile in an equal proportion and stirred for 5 minutes. The mixture was centrifuged at 10,000 rpm for 10 minutes. Before use, the supernatant was filtered twice through a 0.22 μm membrane to remove protein. The supernatant was diluted 50 times with Tris-HCl buffer (pH = 7.4, 0.05 M) for later use. TC and DOX standard solutions were added to the pretreated milk sample to prepare a series of test solutions of different concentrations. Then, the following steps were performed for detection:

[0057] S1: Construct 3D LDH-CdTe QDs. This step is the same as in Example 1 and will not be repeated here.

[0058] S2: Measurement data of a series of concentration samples: This refers to the detection and differentiation data of a series of concentration samples from Examples 1, 2 and 3, which will not be elaborated here;

[0059] S3: Determine the types and content ranges of tetracycline antibiotics in milk samples, specifically including the following steps:

[0060] S31: A certain amount of the test solution was added to a 0.05 M Tris-HCl buffer solution containing 0.6 mg / mL 3D LDH-CdTe QDs at pH 7.4, making the mixing ratio of TC:DOX = 4:6 and the total concentration 5 μM. This was recorded as the Spiked sample. The detection method of Example 1 was followed for differentiation. Each test was repeated four times, and the results, along with the mixed ratio of TC and DOX from Example 3, were further determined by LDA.

[0061] S32: Take a certain amount of the TC-spiked test solution and add it to a 0.05M Tris-HCl buffer solution with pH=7.4 containing 0.6mg / mL 3D LDH-CdTe QDs, so that the TC concentrations are 2.0μM and 10μM respectively. Detect and distinguish them according to the detection method in Example 1, and repeat the test 4 times.

[0062] Effect Experiment 1:

[0063] This experiment was conducted based on the data from Examples 1-3.

[0064] To differentiate four tetracycline antibiotics at the same concentration, the four antibiotics showed different responses in the two fluorescence emission channels of the 3DLDH-CdTe QDs. A fingerprint map was constructed using the variation data matrix. To investigate the ability of the 3DLDH-CdTe QDs fluorescence sensor array to distinguish different TCs at the same concentration, this experiment selected TC concentrations of 0.1 μM, 0.3 μM, 1 μM, and 5 μM as representatives. The results are as follows: Figure 1 As shown. Figure 1 The histogram of fingerprint (A) with the relative change of (F0-F) / F0 on the ordinate clearly shows that the two fluorescence emission channels at 550nm and 630nm respond differently to the four TCs at the same concentration. This indicates that the dual-channel fluorescence sensor constructed in Example 1 is highly feasible for distinguishing four TCs at the same concentration. Furthermore, linear discriminant analysis (LDA) was performed on 160 data matrices (2 sensing units × 4 analytes × 4 replicates × 4 concentrations) using SPSS software for statistical analysis. Figure 1 (B) The centroid function plots use dashed lines of different colors to represent the centroid regions of the four TCs at 0.1 μM, 0.3 μM, 1 μM and 5 μM. Each region does not overlap, which shows that the detection method of Example 1 has a strong ability to distinguish TCs. Although the constructed 3D LDH-CdTe QDs only has two sensing units, it can still successfully distinguish the four analytes.

[0065] The detection, differentiation, and semi-quantitative analysis of tetracycline antibiotics at different concentrations were performed, and the results are as follows: Figure 2 As shown. Figure 2 The LDA plot visually demonstrates the pattern of the scatter clusters arranged from left to right as the target concentration increases, with no overlap between them, achieving 100% complete differentiation. This sufficiently proves that the fluorescence sensor array constructed in the example has good application capabilities. The linear relationship is as follows: Figure 2 As shown in (C1-C4), Factor 1 exhibits correlation coefficients consistent with array data processing for different concentrations of TC, DOX, CTC, and OTC. The linear ranges for TC and DOX are 0.1 μM–50 μM, for CTC 0.1 μM–10 μM, and for OTC 0.1 μM–20 μM. The detection and differentiation results for different concentrations of tetracycline antibiotics demonstrate that the sensor array constructed in Example 1 can successfully distinguish between different concentrations of TCs.

[0066] To detect and differentiate tetracycline antibiotics with different components, Example 3 selected TC and DOX, which have similar structures, as representative target analytes to explore the ability of the sensor array constructed in Example 1 to distinguish the coexistence of different components. The results are as follows: Figure 3 As shown. Figure 3 LDA plots for different TC / DOX mixing ratios (10:0, 8:2, 6:4, 5:5, 3:7, 1:9, 0:10). Figure 3 The LDA plots show that the positions of each cluster are arranged from left to right according to different TC:DOX molar ratios (TC / DOX = 10:0, 8:2, 6:4, 5:5, 3:7, 1:9, 0:10, with a total molar concentration of 5 μM), without interference between them. Furthermore, the Factor 1 value gradually increases, and Factor 1 shows a good linear relationship with the mixed concentration of TC and DOX, indicating that this method can estimate the mixing ratio of TCs within a certain mixing concentration range. Secondly, in Example 3, tetracyclines and their derivatives TC, CTC, and OTC, which have very similar structures, were mixed in different proportions for detection and differentiation. The results are as follows... Figure 4 As shown. Figure 4 A two-dimensional LDA scatter plot of TC, DOX, CTC, and OTC in a series of mixed proportions. From Figure 4 It can be seen that, on the one hand, the clusters are well distinguishable and do not overlap. On the other hand, the mixture clusters exhibit certain distribution patterns. For example, the blue mixture cluster (TC:CTC = 4:6) is located between the TC and CTC single-component clusters; similarly, the purple mixture cluster (CTC:OTC = 3:7) and the blue mixture cluster (TC:OTC = 2:8) also exhibit similar patterns. Data analysis from Example 3 shows that the LDA distribution maps for both two-component and three-component mixtures confirm that the detection method of Example 1 has the ability to distinguish TC mixtures.

[0067] Effect Experiment 2

[0068] This experiment processed and analyzed the data obtained from Examples 1-4.

[0069] The detection data from Example 4 were combined with the data from Example 2, which had a mixing ratio of TC:DOX = 4:6, and linear discriminant analysis was performed. The results are as follows: Figure 5 As shown. Figure 5 A two-dimensional LDA scatter plot of spiked milk with a TC / DOX concentration ratio of 4:6. Figure 5 It can be seen that the scatter clusters of spiked samples fell into the predicted positions, namely between TC:DOX = 5:5 and TC:DOX = 3:7, without any overlap or interference. Since the spiked samples are distributed between 5:5 and 3:7, the TC concentration is between 1.5 μM and 2.5 μM, and the DOX concentration is between 2.5 μM and 3.5 μM, thus proving that this detection method can detect the concentration of TCs in milk samples.

[0070] Then, the Euclidean distances between the spiked samples and the corresponding series of standard aqueous solutions in the LDA plot were further calculated. Linear discriminant analysis was performed on the milk samples spiked with 10 μM TC from Example 4 together with aqueous solutions containing 0.1-50 μM TC. The results are as follows: Figure 6 As shown. Figure 6 Two-dimensional LDA scatter plots of milk spiked with 2 μM and 10 μM TC and a series of TC standards. From Figure 6 It can be seen that the 10 μM TC spiked sample is located between the clusters of 5 μM and 10 μM TC in the aqueous solution. Euclidean distance calculations were performed on the data of the three samples, and the results are shown in Table 3 below. The difference between the EDs of the spiked sample and the EDs of 5 μM TC in the water is d1 = 35.890 - 2.066 = 33.824, and the difference between the spiked sample and the 10 μM EDs in the water is d2 = 35.890 - 33.573 = 2.317. Clearly, d2 is closer, and the spiked sample and the 10 μM TC in the standard water sample can be grouped together, indicating that this method can semi-quantitatively analyze and detect TCs in actual samples.

[0071] Table 3 Summary of Euclidean distance calculations for spiked samples

[0072]

[0073]

[0074] In summary, the detection method of the present invention has good differentiation and identification effects for TCs at the same concentration, TCs at different concentrations, quantitative analysis, and detection and differentiation of TCs with different components, as well as for the identification and semi-quantitative detection of TCs in milk samples. It can distinguish four types of TCs as low as 0.1 μM.

[0075] The above description is only a part of the embodiments of the present invention, and is not intended to limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the content of the present invention specification should be included within the protection scope of the present invention.

Claims

1. A method for detecting tetracycline antibiotics, characterized in that, Includes the following steps: S1: Fabrication of a dual-channel fluorescence sensor array based on cadmium telluride quantum dots and three-dimensional layered double hydroxides; S2: Determination of data from a series of concentration samples: Prepare a series of gradient concentration standard solutions of one or more tetracycline antibiotics, and then mix the three-dimensional layered double hydroxide fluorescence sensor array obtained in step S1 with a trihydroxyaminomethane-hydrochloric acid buffer solution at pH 7.4 to obtain a mixed solution. Add each standard solution to the above mixed solution, shake well, and measure the fluorescence intensity value. Each sample is tested in parallel 4 times to obtain the detection and differentiation data of tetracycline antibiotics. S3: Determination of the type of tetracycline antibiotics in unknown samples: The three-dimensional layered double hydroxide fluorescence sensor array obtained in step S1 is mixed with a trihydroxyaminomethane-hydrochloric acid buffer solution at pH 7.4 to prepare the test solution for the unknown sample. The test solution is added to the above mixed solution and shaken to mix. The fluorescence intensity value is measured. Each test sample is tested in parallel 4 times. The obtained data and the detection and differentiation data of tetracycline antibiotics obtained in step S2 are plotted simultaneously to form an LDA diagram. The type and content range of tetracycline antibiotics in the unknown sample are determined by the overlap or positional relationship of the data points. Step S1 includes the following steps: S11: Synthesis of red-light cadmium telluride quantum dots and orange-yellow-light cadmium telluride quantum dots; S12: Preparation of three-dimensional layered double hydroxides; S13: The three-dimensional layered double hydroxide was dispersed in 0.05 M trihydroxyaminomethane-hydrochloric acid buffer solution and ultrasonically dispersed. Orange-yellow cadmium telluride quantum dot solution was added dropwise, and the mixture was stirred in a water bath at 20°C for 30 min. Red cadmium telluride quantum dot solution was added dropwise, and the mixture was stirred for 3 h. After stirring was stopped, the mixture was centrifuged at 8000 rpm and washed three times with deionized water. The mixture was then vacuum dried at 45°C to prepare a dual-channel fluorescence sensor array. In step S11, CdTe quantum dots are synthesized using the following steps: Weigh an appropriate amount of sodium citrate and place it in a container, add deionized water, and after complete dissolution, add cadmium chloride hemipentahydrate, stir evenly, and immediately add mercaptopropionic acid; add 1 M sodium hydroxide solution dropwise to the mixed solution to adjust the pH of the solution to 10.5, add sodium tellurite and sodium borohydride under vigorous stirring, and heat at 100°C for 40-60 min under reflux; after stopping heating, add an equal amount of ethanol to precipitate the quantum dots, centrifuge, wash, and redisperse with deionized water to prepare a solution; In step S12, the three-dimensional layered double hydroxide is prepared using the following steps: magnesium nitrate hexahydrate, aluminum nitrate nonahydrate, and urea are dissolved in deionized water to obtain solution A; sodium dodecyl sulfate is dissolved in deionized water to form a clear solution B at 60°C; solution B is added to solution A to obtain a mixture; the mixture suspension is stirred for 30 min, transferred to an autoclave, and subjected to hydrothermal reaction at 160°C for 6 h; after centrifugation, the mixture is washed with deionized water and ethanol, and dried at 70°C to obtain the three-dimensional layered double hydroxide. The tetracycline antibiotics mentioned are tetracycline, oxytetracycline, chlortetracycline, and doxycycline.

2. The method for detecting tetracycline antibiotics according to claim 1, characterized in that, In step S11, the ratio of sodium citrate, cadmium chloride hemihydrate, sodium tellurite, and sodium borohydride is 0.22g:0.11g:0.02g:0.05g.

3. The method for detecting tetracycline antibiotics according to claim 1, characterized in that, In step S12, the ratio of magnesium nitrate hexahydrate, aluminum nitrate nonahydrate, urea, and sodium dodecyl sulfate is 0.44g:0.3g:0.48g:0.03g.

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