Citric acid coordinated europium-based fluorescent probe, fluorescent hydrogel and application in detection of tetracycline

Abstract

The invention discloses a europium-based fluorescent probe synergistically coordinated with citric acid, fluorescent hydrogel and application of the europium-based fluorescent probe and the fluorescent hydrogel in tetracycline detection, and belongs to the technical field of tetracycline detection. The preparation method of the europium-based fluorescent probe with citric acid synergistic coordination comprises the following steps: mixing and dissolving 2, 5-dihydroxyterephthalic acid, citric acid and a europium-based compound to form a precursor solution, reacting at 40-45 DEG C, after the reaction is finished, naturally cooling, centrifuging, washing and drying to obtain the europium-based fluorescent probe with citric acid synergistic coordination. And the citric acid synergistic coordination europium-based fluorescent probe and agarose act to generate the citric acid synergistic coordination fluorescent hydrogel. The europium-based fluorescent probe synergistically coordinated with citric acid has excellent water dispersibility and chemical stability, shows excellent luminescence characteristics, and has excellent sensing performance; and effective distinguishing of tetracycline antibiotics is realized through a principal component analysis technology.

Description

Technical Field

[0001] This invention belongs to the field of tetracycline detection technology, specifically relating to europium-based fluorescent probes with citric acid co-coordination, fluorescent hydrogels, and their application in the detection of tetracycline. Background Technology

[0002] Tetracycline (TC) antibiotics are a class of broad-spectrum antibiotics that play an important role in clinical medicine, animal husbandry, and aquatic disease control due to their high antibacterial activity and bioavailability. However, their overuse can lead to environmental residues that can accumulate in the food chain, threatening human health and inducing allergic reactions, liver and kidney damage, and intestinal flora imbalance. Monitoring data in my country show that tetracycline residues in livestock and poultry products and freshwater fish in some regions exceed the EU's maximum limits, highlighting the urgency of pollution control and accurate detection. Therefore, developing green analytical technologies that combine high sensitivity, rapid response, and environmental compatibility, and constructing a real-time detection platform for tetracycline antibiotics, is of great significance for ensuring food safety, curbing the spread of drug-resistant genes, and optimizing drug use regulation.

[0003] Currently, many methods for detecting tetracycline antibiotics have been explored, including high-performance liquid chromatography-mass spectrometry, capillary electrophoresis, and electrochemical analysis. In contrast, fluorescence methods have attracted widespread attention due to their high sensitivity, simple operation, fast response, and good visual effects. Traditional fluorescence sensing relies on changes in fluorescence intensity at a single wavelength. This signal mode is highly susceptible to significant interference from non-target factors such as uneven probe concentration distribution, instrument parameter fluctuations, and environmental factors, leading to low reliability and accuracy of detection results, and lacking an inherent self-calibration mechanism to eliminate systematic errors. Ratio fluorescence sensing technology, by measuring the ratio of fluorescence intensities at two different emission wavelengths as the output signal, effectively overcomes the shortcomings of a single intensity signal, providing inherent self-calibration capabilities, higher anti-interference ability, reliability, and potential advantages in visual visualization. Europium ions (Eu) 3+ Europium complexes, due to their unique long fluorescence lifetime, large Stokes shift, characteristic sharp emission peaks, and excellent photophysical properties such as sensitization through the "antenna effect," are considered ideal platforms for constructing fluorescent probes, and have been used in the detection of TCs. 2,5-Dihydroxyterephthalic acid (H2DHT), as a dicarboxylic acid bisphenol hydroxyl ligand, maintains stable yellow-green fluorescence during detection, forming a self-calibrating dual-signal system. This study aims to design and synthesize novel europium-based complex probes that produce significant and stable changes in the dual emission peak ratio in the presence of TCs, thereby achieving rapid detection of tetracycline antibiotics with high sensitivity, high selectivity, high reliability, and interference resistance, meeting the needs of complex practical sample analysis.

[0004] Composites of nanoparticles with substrates such as membranes, paper, or hydrogels can effectively suppress leaching losses of nanomaterials, significantly improve processing applicability, and expand application scenarios. Among these, hydrogel sensing materials stand out in the field of fluorescence sensing due to their portability, reusability, and long-term stability: this composite material not only inherits the high specific surface area, hierarchical porous structure, and excellent luminescence properties of the complex, but also integrates the high flexibility, strong processability, and chemical stability of the polymer matrix, forming a synergistic enhancement effect.

[0005] Tetracycline antibiotics (such as oxytetracycline, tetracycline, chlortetracycline, and doxycycline) have highly similar structures. Their structural homology leads to their extremely similar physicochemical properties, making it difficult for traditional sensing methods to achieve accurate identification based on a single response signal. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a citric acid-coordinated europium-based fluorescent probe, a fluorescent hydrogel, and its application in the detection of tetracycline. The preparation method and application of the ratiometric fluorescent probe and fluorescent hydrogel with a dual emission strategy are not only simple to prepare, but also have the characteristics of good selectivity, low cost, and visual detection. This invention can be used for the specific detection of tetracycline.

[0007] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a citric acid co-coordinated europium-based fluorescent probe, wherein the preparation method of the citric acid co-coordinated europium-based fluorescent probe is as follows: 2,5-dihydroxyterephthalic acid, citric acid and europium compound are mixed and dissolved to form a precursor solution, which is reacted at 40~45°C. After the reaction is completed, the solution is naturally cooled, and after centrifugation, washing and drying, the citric acid co-coordinated europium-based fluorescent probe is obtained.

[0008] Furthermore, the europium compound is europium chloride; the molar ratio of the europium compound, 2,5-dihydroxyterephthalic acid and citric acid is 3:1~3:1~2; and the reaction time is 2~5 hours.

[0009] Furthermore, the molar ratio of the europium compound, 2,5-dihydroxyterephthalic acid, and citric acid is 3:2:1.5.

[0010] In a second aspect, the present invention provides a citric acid-coordinated fluorescent hydrogel prepared from the citric acid-coordinated europium fluorescent probe. The citric acid-coordinated europium fluorescent probe solution is added to an agarose solution, stirred evenly at room temperature, and then placed in a mold and allowed to cool naturally to obtain the citric acid-coordinated fluorescent hydrogel.

[0011] Furthermore, the concentration of the citric acid co-coordinated europium fluorescent probe solution is 0.005~0.2 g / mL; the concentration of the agarose gel solution is 200~500 μg / mL; and the volume ratio of the citric acid co-coordinated europium fluorescent probe solution to the agarose gel solution is 1:40.

[0012] In a third aspect, the present invention provides the application of the citric acid co-coordinated europium-based fluorescent probe or the citric acid co-coordinated fluorescent hydrogel in the detection of tetracycline antibiotics.

[0013] Furthermore, the tetracycline antibiotic is one or more of oxytetracycline, tetracycline, chlortetracycline, and doxycycline.

[0014] In a fourth aspect, this invention provides the application of the citric acid co-coordinated europium-based fluorescent probe or the citric acid co-coordinated fluorescent hydrogel in distinguishing tetracycline antibiotics. This invention innovatively introduces principal component analysis (PCA), which analyzes the projection distribution patterns of different tetracycline samples in a low-dimensional feature space constructed from the first two or three principal components. This enables clear and effective visualization and classification of the four tetracycline antibiotics, ultimately achieving high-precision pattern recognition and differentiation of the four tetracycline antibiotics.

[0015] Furthermore, the tetracycline antibiotic is one or more of oxytetracycline, tetracycline, chlortetracycline, and doxycycline.

[0016] This invention is based on the β-diketone group in tetracycline molecules and its relation to Eu. 3+ The antenna effect sensitization effect, at an excitation wavelength of 280 nm, allows tetracycline to selectively enhance Eu through the ligand-metal energy transfer principle. 3+ The characteristic red light emission at 625 nm of H2DHT is stable, while the intrinsic fluorescence at 542 nm remains stable. Furthermore, the fluorescence intensity ratio at the emission peaks of 625 nm and 542 nm (F1) is significantly higher. 625 / F 542 The concentration of tetracycline increases with increasing tetracycline concentration.

[0017] Compared with the prior art, the beneficial effects achieved by this application are as follows: This invention prepares a citric acid-coordinated europium-based fluorescent probe by a low-temperature stirring method. The preparation method is simple, the materials are easy to obtain, and the prepared citric acid-coordinated europium-based fluorescent probe has excellent water dispersibility and chemical stability, exhibits excellent luminescence characteristics, and has excellent sensing performance. This invention utilizes europium-based fluorescent probes with citric acid co-coordination to develop a visual hydrogel sensor for detecting tetracyclines, and achieves effective differentiation of four tetracycline antibiotics through principal component analysis. Attached Figure Description

[0018] Figure 1 Transmission electron microscopy image of the citric acid co-coordinated europium-based fluorescent probe prepared in Example 1; Figure 2 Scanning electron microscope image of the citric acid co-coordinated europium-based fluorescent probe prepared in Example 1; Figure 3 Fourier transform infrared (FT-IR) spectrum of europium-based fluorescent probes co-coordinated with H2DHT, Cit and citric acid; Figure 4 The citric acid-coordinated europium-based fluorescent probe prepared in Example 1 was excited at 270-330 nm. 625 / F 542 Intensity comparison chart; Figure 5 The figure shows the stability test results of the europium-based fluorescent probe with citric acid co-coordination prepared in Example 1. Figure 6 Europium-based fluorescent probe solutions co-coordinated with citric acid and their fluorescence lifetimes after the addition of tetracycline; Figure 7 Fluorescence response diagrams of europium-based fluorescent probes co-coordinated with citric acid to tetracycline and different metal ions; Figure 8 X-ray photoelectron spectra of europium complexes before and after the addition of tetracycline to the europium fluorescent probe solution co-coordinated with citric acid; Figure 9 X-ray photoelectron spectrum of europium-based fluorescent probe O 1s co-coordinated with citric acid; Figure 10 The O 1s X-ray photoelectron spectrum of the europium-based fluorescent probe solution with citric acid co-coordination after the addition of tetracycline. Figure 11 X-ray photoelectron spectrum of C1s of europium-based fluorescent probes co-coordinated with citric acid; Figure 12 This is the C1s X-ray photoelectron spectrum of a europium-based fluorescent probe co-coordinated with citric acid after the addition of tetracycline. Figure 13 X-ray photoelectron spectrum of N 1s, a europium-based fluorescent probe co-coordinated with citric acid. Figure 14 The fluorescence response diagrams of the europium-based fluorescent probes co-coordinated with citric acid prepared in Examples 1-5 before and after the addition of tetracycline are shown. Figure 15 The fluorescence intensity ratio (F) of europium-based fluorescent probe solutions before and after tetracycline addition with co-coordination of citrate under different pH conditions. 625 / F 542 ); Figure 16 The fluorescence intensity ratio (F) before and after adding tetracycline to europium-based fluorescent probe solutions with different concentrations of citric acid co-coordination. 625 / F 542 ); Figure 17 The fluorescence intensity ratio (F) of europium-based fluorescent probe solutions co-coordinated with citric acid after adding tetracycline at different incubation times 625 / F 542 ); Figure 18 Response curves of europium-based fluorescent probe solutions with citric acid co-coordination to different concentrations of tetracycline; Figure 19 The fluorescence intensity ratio (F) of europium-based fluorescent probe solutions co-coordinated with citric acid for different concentrations of tetracycline 625 / F 542 Linear curve; Figure 20 Two-dimensional score graphs of recognition of four tetracycline antibiotics by europium-based fluorescent probes with citric acid co-coordination; Figure 21 Two-dimensional score diagram of the recognition of bicyclic tetracycline antibiotics by europium-based fluorescent probes with citric acid co-coordination; Figure 22 The fluorescence color (365nm) of europium-based fluorescent hydrogels with co-coordination of citric acid under different concentrations of tetracycline is shown. Figure 23 The graph shows the linear relationship between the concentration of tetracycline and the co-coordination of citric acid in europium-based fluorescent hydrogels. Detailed Implementation

[0019] To make the objectives and technical solutions of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions; unless otherwise specified, the reagents and materials are commercially available.

[0020] Example 1 Preparation of europium-based fluorescent probes with citric acid co-coordination: (1) At room temperature, 33.02 mg of 2,5-dihydroxyterephthalic acid (H2DHT, 0.167 mmol) and 26.25 mg of citric acid monohydrate (Cit, 0.125 mmol) solid powder were dissolved in 15 mL of ethanol solution and 139 μL of triethylamine solution was added. (2) Dissolve 91.5 mg europium chloride hexahydrate (0.25 mmol) in 5 mL of ultrapure water and sonicate for 5 min until fully dissolved; (3) The solutions from steps (1) and (2) were mixed and heated and stirred in a water bath at 40 °C for 2 h. The resulting suspension was centrifuged and washed. The precipitate obtained was dried in a vacuum drying oven at 40 °C for 12 h to obtain a europium-based fluorescent probe with citric acid co-coordination.

[0021] Transmission electron microscopy (TEM) image of the citric acid-coordinated europium-based fluorescent probe prepared in Example 1 is shown below. Figure 1 As shown in the figure, the europium-based fluorescent probe with citric acid co-coordination prepared in Example 1 exhibits an irregular shape.

[0022] The scanning electron microscope image of the citric acid-coordinated europium-based fluorescent probe prepared in Example 1 is shown below. Figure 2 As shown, by Figure 2 It is known that the europium-based fluorescent probe with citric acid co-coordination exhibits a network structure with obvious pores and an average size of about 50 nm.

[0023] Figure 3 The Fourier transform infrared (FT-IR) spectrum of the europium-based fluorescent probe co-coordinated with H2DHT, Cit, and citrate is shown in the figure. H2DHT is visible at 3084 cm⁻¹. -1 An OH bond stretching vibration peak appears at 3416 cm⁻¹, while the OH bond stretching vibration peak of the europium-based fluorescent probe with citric acid co-coordination redshifts to 3416 cm⁻¹. -1 Location; Cit at 1746cm -1 and 1708cm -1 The characteristic peak at 1650 cm⁻¹ is caused by the stretching vibration of the C=O bond in the carboxyl group. Meanwhile, H₂DHT at 1650 cm⁻¹... -1 and 1290 cm -1 The strong peaks at 1650 cm⁻¹ are attributed to the stretching vibrations of the C=O and CO bonds in the carboxyl group, respectively, while the europium-based fluorescent probe with citric acid co-coordination at 1650 cm⁻¹... -1 The stretching vibration of the C=O bond at that point disappears, and the stretching vibration of the C-OH group decreases from 1290 cm⁻¹. -1 Displaced to 1240 cm -1 This indicates that H2DHT, Cit, and Eu 3+ Matching successful.

[0024] The citric acid-coordinated europium-based fluorescent probe prepared in Example 1 was excited at a wavelength of 270-330 nm. 625 / F 542 Intensity comparison chart as follows Figure 4 As shown in the figure, when the excitation wavelength is 280 nm, F625 / F 542 The ratio is the largest.

[0025] The citric acid-coordinated europium-based fluorescent probe prepared in Example 1 was dissolved in HEPES buffer solution and stored at room temperature for five days. The F-value was measured at an excitation wavelength of 280 nm. 625 / F 542 Stability testing was conducted, and the test results are as follows: Figure 5 As shown; by Figure 5 It can be seen that the fluorescence intensity ratio of the europium-based fluorescent probe with citric acid co-coordination is (F... 625 / F 542 The value remains basically unchanged, proving its good stability.

[0026] Example 2 Preparation of europium-based fluorescent probes with citric acid co-coordination: (1) At room temperature, 16.51 mg of 2,5-dihydroxyterephthalic acid (0.083 mmol) and 17.51 ​​mg of citric acid monohydrate (0.083 mmol) solid powder were dissolved in 15 mL of ethanol solution, and 139 μL of triethylamine solution was added. (2) Dissolve 91.5 mg europium chloride hexahydrate (0.25 mmol) in 5 mL of ultrapure water and sonicate for 5 min until fully dissolved; (3) The solutions from steps (1) and (2) were mixed and heated and stirred in a water bath at 40 °C for 2 h. The resulting suspension was centrifuged and washed, and the precipitate was dried in a vacuum drying oven at 40 °C for 12 h to obtain a europium-based fluorescent probe with citric acid co-coordination.

[0027] Example 3 Preparation of europium-based fluorescent probes with citric acid co-coordination: (1) At room temperature, 33.02 mg of 2,5-dihydroxyterephthalic acid (0.167 mmol) and 17.51 ​​mg of citric acid monohydrate (0.083 mmol) solid powder were dissolved in 15 mL of ethanol solution and 139 μL of triethylamine solution was added. (2) Dissolve 91.5 mg europium chloride hexahydrate (0.25 mmol) in 5 mL of ultrapure water and sonicate for 5 min until fully dissolved; (3) The solutions from steps (1) and (2) were mixed and heated and stirred in a water bath at 40 °C for 2 h. The resulting suspension was centrifuged and washed, and the precipitate was dried in a vacuum drying oven at 40 °C for 12 h to obtain a europium-based fluorescent probe with citric acid co-coordination.

[0028] Example 4 Preparation of europium-based fluorescent probes with citric acid co-coordination: (1) At room temperature, 33.02 mg of 2,5-dihydroxyterephthalic acid (0.167 mmol) and 35.02 mg of citric acid monohydrate (0.167 mmol) solid powder were dissolved in 15 mL of ethanol solution and 139 μL of triethylamine solution was added. (2) Dissolve 91.5 mg europium chloride hexahydrate (0.25 mmol) in 5 mL of ultrapure water and sonicate for 5 min until fully dissolved; (3) The solutions from steps (1) and (2) were mixed and heated and stirred in a water bath at 40 °C for 2 h. The resulting suspension was centrifuged and washed, and the precipitate was dried in a vacuum drying oven at 40 °C for 12 h to obtain a europium-based fluorescent probe with citric acid co-coordination.

[0029] Example 5 Preparation of europium-based fluorescent probes with citric acid co-coordination: (1) At room temperature, 49.53 mg of 2,5-dihydroxyterephthalic acid (0.25 mmol) and 17.51 ​​mg of citric acid monohydrate (0.083 mmol) solid powder were dissolved in 15 mL of ethanol solution and 139 μL of triethylamine solution was added. (2) Dissolve 91.5 mg europium chloride hexahydrate (0.25 mmol) in 5 mL of ultrapure water and sonicate for 5 min until fully dissolved; (3) The solutions from steps (1) and (2) were mixed and heated and stirred in a water bath at 40 °C for 2 h. The resulting suspension was centrifuged and washed, and the precipitate was dried in a vacuum drying oven at 40 °C for 12 h to obtain a europium-based fluorescent probe with citric acid co-coordination.

[0030] Example 6 Preparation of europium-based fluorescent hydrogels with citric acid co-coordination: (1) Add 1 g of agarose to 100 mL of pure water and heat to dissolve; (2) Add 2.5 mL of the citric acid co-coordinated europium fluorescent probe aqueous solution (400 μg / mL) from Example 1 to the agarose solution and stir until homogeneous; (3) Pour into a silicone mold and let cool naturally to obtain europium fluorescent hydrogel with citric acid co-coordination.

[0031] Detection of tetracycline compounds using europium-based fluorescent probes with citric acid co-coordination. (1) Mix 50 μL (400 μg / mL) of citric acid co-coordinated europium fluorescent probe with 950 μL of HEPES buffer solution to obtain citric acid co-coordinated europium fluorescent probe solution. Add 1000 μL of tetracycline (TC) of different concentrations to each solution, mix well and react at room temperature for 5 min. Record the fluorescence spectrum using an excitation wavelength of 280 nm.

[0032] (2) The fluorescence lifetime of the citric acid-coordinated europium-based fluorescent probe (Eu-H2DHT / Cit) prepared in Example 1 after adding tetracycline (TC (100 μM)) is shown in the figure. Figure 6 As shown; by Figure 6 It can be seen that the fluorescence lifetime of the europium complex system is significantly enhanced after the addition of tetracycline.

[0033] (3) Tetracycline (TC) and different metal ions were added to a citric acid-coordinated europium-based fluorescent probe solution. The concentration of the metal ions was 100 μM, and the concentration of tetracycline was 10 μM. The response of the citric acid-coordinated europium-based fluorescent probe to different ions is shown in the figure. Figure 7 As shown, by Figure 7 It is known that the europium-based fluorescent probe with citric acid co-coordination has a significant response to tetracycline but a lower response to other ions. Therefore, this fluorescent probe can specifically detect tetracycline.

[0034] (4) X-ray photoelectron spectroscopy (XPS) was performed on the europium fluorescent probe solution with citric acid co-coordination before and after the addition of tetracycline (TC (100 μM)). Figure 8 X-ray photoelectron spectra of europium complexes before and after the addition of tetracycline to the europium fluorescent probe solution co-coordinated with citric acid; Figure 9 X-ray photoelectron spectrum of europium-based fluorescent probe O 1s co-coordinated with citric acid; Figure 10 The O 1s X-ray photoelectron spectrum of the europium-based fluorescent probe solution with citric acid co-coordination after the addition of tetracycline. Figure 11 X-ray photoelectron spectrum of C1s of europium-based fluorescent probes co-coordinated with citric acid; Figure 12 This is the C1s X-ray photoelectron spectrum of a europium-based fluorescent probe co-coordinated with citric acid after the addition of tetracycline. Figure 13 X-ray photoelectron spectrum of N 1s, a europium-based fluorescent probe co-coordinated with citric acid; by Figures 8-13It was found that after the addition of tetracycline (TC), the relative content of O-Eu bonds in the europium complex increased from 18% to 21%, indicating a chelation effect between the citric acid-coordinated europium fluorescent probe and tetracycline. Furthermore, after sensing tetracycline, the citric acid-coordinated europium fluorescent probe exhibited a CN characteristic peak at 286.1 eV, and N-(C)3, CN, NH, and NO bonds appeared at 402.6, 401, 400.1, and 399.5 eV, respectively, in the N 1s high-resolution XPS spectrum, indicating that the europium complex chelated with the β-diketone group in the tetracycline molecule.

[0035] (5) The fluorescence response diagrams of the europium-based fluorescent probes co-coordinated with citric acid prepared in Examples 1-5 before and after the addition of tetracycline (TC) are shown in the figure. Figure 14 As shown, by Figure 14 It can be seen that when the EuCl3:H2DHT:Cit ratio is 3:2:1.5, after adding tetracycline, the fluorescence intensity ratio of the europium complex is (F... 625 / F 542 The strongest response is achieved with a fixed EuCl3 content. Excessive H2DHT ratio reduces fluorescence response to TC, while insufficient ratio affects material yield; insufficient Cit ratio negatively impacts EuCl3 content. 3+ - Insufficient TC luminescence enhancement assist, excessive TC reduces Eu 3 + -TC complexation site.

[0036] (6) Adjust the pH of the citric acid co-coordinated europium-based fluorescent probe solution to 6-10, and compare the fluorescence intensity ratio (F) of the citric acid co-coordinated europium-based fluorescent probe solution before and after the addition of tetracycline (TC). 625 / F 542 The test was conducted, and the results are as follows: Figure 15 As shown, the fluorescence intensity ratio (F) of the europium-based fluorescent probe solution with citric acid co-coordination varies under different pH conditions. 625 / F 542 There was no significant change; after the addition of TC, its fluorescence intensity was lower than that of (F). 625 / F 542 The effect is strongest at pH=8, therefore pH 8 was chosen as the optimal pH for detecting tetracycline.

[0037] (7) Prepare europium-based fluorescent probe solutions with different concentrations of citric acid co-coordination, and compare the fluorescence intensity ratio (F) of the europium-based fluorescent probe solutions with citric acid co-coordination before and after the addition of tetracycline (TC). 625 / F 542 The test was conducted, and the results are as follows: Figure 16 The figure shows the fluorescence intensity (F) before and after the addition of tetracycline when the concentration of the europium-based fluorescent probe solution co-coordinated with citrate is 10 μg / mL.625 / F 542 The ratio change is most obvious, and the detection effect is the best.

[0038] (8) Tetracycline (TC) was added to the europium-based fluorescent probe solution with citric acid co-coordination, and its fluorescence intensity (F) at different incubation times was tested. 625 / F 542 The ratio, the result is as follows Figure 17 As shown in the figure; it can be seen from the figure that the fluorescence intensity (F) after adding tetracycline was [value missing] after 2 minutes. 625 / F 542 The ratio tends to stabilize, so an incubation time of 2 min was chosen as the optimal incubation time for detecting tetracycline.

[0039] (9) Different concentrations of tetracycline (TC) were added to the europium-based fluorescent probe solution with citric acid co-coordination, and the response curves are shown in the figure. Figure 18 As shown in the figure, the fluorescence intensity of the europium complex at 542 nm remains essentially unchanged with increasing TC concentration, while the fluorescence intensity at 625 nm gradually increases. Correspondingly, the fluorescence intensity (F...)... 625 / F 542 The ratio gradually increases with increasing TC concentration, indicating that the method of quantitatively detecting TC using fluorescence detection is feasible. When different concentrations of TC are added to a citric acid-coordinated europium-based fluorescent probe solution, the fluorescence intensity F... 625 / F 542 The linear curve of the ratio (fluorescence intensity at the emission peak of 635 nm to that at 542 nm) is shown below. Figure 19 As shown, this indicates that the fluorescent probe exhibits good linearity for TC, with a linear range of 0.1 to 15 μM. This confirms the reliability of the citrate-coordinated europium-based fluorescent probe in the quantitative detection of tetracycline.

[0040] (10) Principal component analysis was used to differentiate four tetracycline antibiotics, and the results are as follows: Figure 20 As shown, in the two-dimensional score map constructed based on principal components PC1 and PC2, the signals of tetracycline antibiotics (TC, CTC, OTC, DOX) at a concentration of 10 μM exhibit four spatially separated clusters. Each cluster is elliptically distributed with a 95% confidence interval, and the boundaries between clusters are clear and non-overlapping. This significant separation phenomenon confirms the specific recognition capability of the europium complex fluorescence sensing array for the target antibiotics.

[0041] (11) Principal component analysis was used to differentiate between binary mixtures of tetracycline antibiotics (TC, CTC, OTC, DOX), and the results are as follows: Figure 21 As shown, by Figure 21It can be seen that the response signals of the six target mixtures exhibit a clear and distinguishable separation in the feature space, without cross-over or non-specific interference, confirming that the europium complex fluorescence sensing array can realize reliable analysis of the complex tetracycline system and effectively eliminate background interference.

[0042] Performance testing of europium-based fluorescent hydrogels with citric acid co-coordination Different concentrations of tetracycline (TC: 0-10 μM) were added to the citric acid-coordinated europium-based fluorescent hydrogel prepared in Example 6. After reacting for 2 hours, the reaction was observed under a 365 nm UV lamp. The results are as follows: Figure 22 As shown, the fluorescence color of the europium-based fluorescent hydrogel with citric acid co-coordination gradually changes from yellow-green to orange-red as the tetracycline concentration increases.

[0043] The fluorescence and color development of citric acid-coordinated europium-based fluorescent hydrogels under UV light were analyzed using the ColorMax application under different concentrations of tetracycline. The results were plotted with tetracycline concentration on the x-axis and R / G value on the y-axis. Figure 23 As shown in the figure, the R / G value has a good linear relationship with the tetracycline concentration (0-10 μM), indicating that the hydrogel prepared by the europium-based fluorescent probe with citric acid co-coordination can be successfully used with a smartphone platform, transforming traditional fluorescence detection into portable real-time analysis and significantly improving detection efficiency.

[0044] Recovery rate and relative standard deviation tests: (1) Milk sample preparation: Purchase fresh milk, mix 50 μL of trichloroacetic acid with 5 mL of milk, sonicate for 20 min to denature the protein, and then centrifuge the mixture at 10000 rpm for 15 min. Filter the obtained supernatant through a 0.22 μm membrane, dilute the filtrate with ultrapure water, and use it for subsequent analysis and detection; (2) Egg white sample preparation: Egg white was collected from commercially available eggs and homogenized using a glass rod. 5 g of the homogenized egg white was mixed with 20 mL of HEPES buffer, stirred thoroughly, and separated into layers. 5 mL of the resulting supernatant was mixed with an equal volume of HEPES buffer, centrifuged at 5000 rpm for 10 minutes, and filtered through a 0.22 μm membrane. The filtrate was diluted with ultrapure water before use. (3) The spiked milk and egg white samples containing concentrations of 2, 4, and 8 μM TC were analyzed by recording the fluorescence spectra under 280 nm excitation; the results are shown in Table 1 below. Table 1 Table 1 shows that the recoveries of tetracycline in milk and egg white samples ranged from 95% to 105%, with a relative standard deviation (RSD) of less than 4.7%, indicating that this ratio fluorescent probe has the potential to achieve quantitative detection of tetracycline in real samples.

[0045] Of course, the above are merely preferred embodiments of this method and are not intended to limit the method. Although the method has been described in detail with reference to the foregoing, those skilled in the art can still modify the technical solutions described above or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A citric acid-coordinated europium-based fluorescent probe, characterized in that, The preparation method of the citric acid co-coordinated europium-based fluorescent probe is as follows: 2,5-dihydroxyterephthalic acid, citric acid and europium compound are mixed and dissolved to form a precursor solution, which is reacted at 40~45℃. After the reaction is completed, the solution is naturally cooled, and after centrifugation, washing and drying, the citric acid co-coordinated europium-based fluorescent probe is obtained.

2. The citric acid-coordinated europium-based fluorescent probe according to claim 1, characterized in that, The europium-based compound is europium chloride; the molar ratio of the europium-based compound, 2,5-dihydroxyterephthalic acid, and citric acid is 3:1 to 3:1 to 2; the reaction time is 2 to 5 hours.

3. The citric acid-coordinated europium-based fluorescent probe according to claim 2, characterized in that, The molar ratio of the europium compound, 2,5-dihydroxyterephthalic acid and citric acid is 3:2:1.

5.

4. A citric acid-coordinated fluorescent hydrogel prepared using a europium-based fluorescent probe with citric acid co-coordination as described in any one of claims 1 to 3, characterized in that, A solution of europium-based fluorescent probes co-coordinated with citric acid was added to an agarose solution, stirred until homogeneous at room temperature, and then placed in a mold and allowed to cool naturally to obtain a fluorescent hydrogel co-coordinated with citric acid.

5. The fluorescent hydrogel with citric acid co-coordination according to claim 4, characterized in that, The concentration of the citric acid co-coordinated europium fluorescent probe solution is 0.005~0.2 g / mL; the concentration of the agarose gel solution is 200~500 μg / mL; and the volume ratio of the citric acid co-coordinated europium fluorescent probe solution to the agarose gel solution is 1:

40.

6. The use of a citric acid co-coordinated europium fluorescent probe according to any one of claims 1 to 3 or a citric acid co-coordinated fluorescent hydrogel according to any one of claims 4 to 5 in the detection of tetracycline antibiotics.

7. The application according to claim 6, characterized in that, The tetracycline antibiotics mentioned are one or more of oxytetracycline, tetracycline, chlortetracycline, and doxycycline.

8. The use of a citric acid co-coordinated europium fluorescent probe according to any one of claims 1 to 3 or a citric acid co-coordinated fluorescent hydrogel according to any one of claims 4 to 5 in distinguishing tetracycline antibiotics.

9. The application according to claim 8, characterized in that, The tetracycline antibiotics mentioned are one or more of oxytetracycline, tetracycline, chlortetracycline, and doxycycline.

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