A dual lanthanide MOF hydrogel system and its application in detecting enrofloxacin

Abstract

This invention provides a lanthanide-based MOF hydrogel system and its application in the detection of enrofloxacin, belonging to the technical field of detection kits. The lanthanide-based MOF hydrogel system provided by this invention is prepared by embedding lanthanide TbEu-MOF nanomaterials in an agarose hydrogel matrix. Under 254 nm excitation, the composite material exhibits orange-red fluorescence; upon contact with enrofloxacin, the fluorescence color gradually changes from orange-red to orange-pink, which can be identified by the naked eye. This invention requires no large-scale precision instruments, is simple and portable to operate, and is suitable for rapid screening in livestock fields, possessing advantages such as ease of operation, low cost, and visualization.

Description

Technical Field

[0001] This invention relates to the field of detection kit technology, and in particular to a bis-lanthanide MOF hydrogel system and its application in the detection of enrofloxacin. Background Technology

[0002] Fluoroquinolone antibiotics have become widespread environmental pollutants due to their extensive use in veterinary medicine and agriculture. As broad-spectrum antimicrobial agents, approximately 70% of fluoroquinolones are excreted into the environment in a nearly unchanged form after administration, leading to their persistent accumulation in soil and water bodies. Their long-term presence not only promotes the emergence and spread of antibiotic-resistant bacteria but also poses significant ecotoxicological risks to plant, animal, and even human health. Therefore, developing reliable and efficient strategies for monitoring fluoroquinolone residues in environmental and agricultural substrates is crucial. Enrofloxacin (ENX) is a typical fluoroquinolone antibiotic, widely used in livestock farming to treat urinary, respiratory, gastrointestinal, and skin bacterial infections due to its potent antimicrobial activity, favorable pharmacokinetic properties, and relatively low side effects. However, its extensive use has raised growing concerns about ENX residues in animal feces, which could become a significant source of environmental pollution. Furthermore, compared to other fluoroquinolones, ENX exhibits unique cytotoxicity and environmental behavior, highlighting the need to develop sensitive and selective detection methods.

[0003] Currently, routine analytical techniques for ENX detection mainly rely on high-performance liquid chromatography (HPLC), micellar liquid chromatography, and HPLC-MS / MS. Although these methods offer high accuracy and sensitivity, they have inherent limitations such as expensive equipment, complex and time-consuming sample pretreatment, and the need for professional operators. Furthermore, matrix interference from complex biological samples such as animal feces can affect analytical performance, limiting their application in rapid on-site monitoring.

[0004] Fluorescence sensing technology has emerged as a promising alternative due to its high sensitivity, ease of operation, low cost, and potential for real-time in-situ analysis. Metal-organic frameworks (MOFs)—crystalline porous materials composed of metal nodes and organic ligands—have attracted considerable attention as luminescent sensing platforms. Lanthanide MOFs, in particular, possess excellent photophysical properties such as high color purity, narrow emission bands, large Stokes shifts, and long fluorescence lifetimes, making them especially suitable for the design of advanced optical sensors. However, when pristine MOFs are exposed to complex biological matrices such as animal feces, they often exhibit aggregation, instability, or pore blockage, hindering mass transfer, reducing the accessibility of active sites, and ultimately weakening sensing performance. Introducing MOFs into functional matrices is an effective strategy to address this challenge. Agarose hydrogels, natural polysaccharide-based materials, possess a highly porous three-dimensional network, excellent biocompatibility, and environmental friendliness. They can physically encapsulate MOF nanoparticles, preventing aggregation and enhancing dispersibility, stability, and resistance to matrix interference. Summary of the Invention

[0005] The purpose of this invention is to provide a lanthanide MOF hydrogel system and its application in the detection of enrofloxacin.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a lanthanide MOF hydrogel system, which consists of a luminescent active component TbEu-MOF and a carrier matrix agarose hydrogel; The TbEu-MOF is composed of Tb 3+ Eu 3+ The TbEu-MOF was obtained by self-assembly of bislanthanide metal ions and the organic ligand benzene-1,3,5-tricarboxylic acid; the TbEu-MOF was embedded in an agarose hydrogel.

[0007] Preferably, the concentration of TbEu-MOF in the agarose hydrogel is 0.05~1.0 mg / mL.

[0008] The present invention also provides a method for preparing the lanthanide MOF hydrogel system according to claim 1, comprising the following steps: 1) Tb(NO3)3 6H2O, Eu(NO3)3 6H2O was dissolved in a solvent, and a solution of benzene-1,3,5-tricarboxylic acid ligand was added and stirred to react. After centrifugation, washing, and drying, TbEu-MOF solid powder was obtained. 2) Dissolve agarose in water by heating to prepare agarose sol; add TbEu-MOF dispersion and Tris-HCl buffer to the agarose sol and mix thoroughly to obtain a lanthanide MOF hydrogel system.

[0009] Preferably, the Tb(NO3)3 described in step 1) 6H2O and Eu(NO3)3 The molar ratio of 6H2O is 2~4:1~3.

[0010] Preferably, the stirring reaction in step 1) is carried out at 20~25℃ for 40~80 min; the centrifugation is carried out at 8000~12000 rpm for 2~4 min; and the drying is carried out at 55~65℃ for 5~7 h.

[0011] Preferably, the heating and melting temperature in step 1) is 75~85℃.

[0012] Preferably, the concentration of the agarose sol in step 2) is 0.4~0.6wt%; the TbEu-MOF dispersion is a mixture of TbEu-MOF solid powder and water, with a concentration of 0.2~0.4 mg / mL.

[0013] Preferably, the pH of the Tris-HCl buffer solution in step 2) is 4 to 6.

[0014] The present invention also provides a kit containing the aforementioned lanthanide MOF hydrogel system.

[0015] The present invention also provides an application of the aforementioned lanthanide MOF hydrogel system or the aforementioned kit in the rapid screening of enrofloxacin in livestock and poultry fecal samples.

[0016] Beneficial effects: This invention successfully constructed a TbEu-MOFs@hydrogel fluorescence sensing platform for the rapid detection of enrofloxacin (ENX) in livestock and poultry manure. By combining lanthanide luminescent MOFs with porous agarose sol, this composite sensor exhibits enhanced fluorescence stability, strong resistance to matrix interference, and reliable performance in complex biological samples. ENX alters the Tb / Eu emission balance, resulting in a visible color change from orange-red to orange-pink. Spectral overlap and fluorescence lifetime variations support an antenna-assisted response mechanism, but further evidence is needed to clarify the specific interaction pathway. Under selected conditions (pH 5, 25°C), the response time is 5 min, the linear range is 1–100 μM, and the detection limit is 0.155 μM. Integrating smartphone RGB analysis and CNN-assisted image recognition enables portable on-site screening of ENX contamination. This provides a practical and intelligent sensing strategy for monitoring veterinary antibiotic residues in livestock and poultry manure, with the potential for both on-site screening and, when necessary, chromatographic confirmation. Attached Figure Description

[0017] Figure 1 This provides structural and photophysical characterization of the TbEu-MOF in Example 1 and mechanistic evidence for its ENX response. Figure 2 The figures show the morphology and elemental characterization results of the TbEu-MOF in Example 1. Figure 3 This is a graph showing the optimized experimental conditions for ENX detection in Example 1; Figure 4 The graph shows the analytical response of TbEu-MOF@hydrogel to ENX in Example 1. Figure 5 The graph shows the selectivity, anti-interference properties, and storage stability of the TbEu-MOF@hydrogel in Example 1. Figure 6 This is the result of the CNN-based binary classification of smartphone fluorescence images in Example 2; Figure 7 Flowchart for the preparation of TbEu-MOF@hydrogel and the smartphone-assisted detection of ENX in livestock and poultry manure. Detailed Implementation

[0018] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0019] Example 1

[0020] 1. Preparation of TbEu-MOFs@hydrogel composite materials

[0021] 90.6 mg (0.203 mmol) of Eu(NO3)3·6H2O, 133.8 mg (0.295 mmol) of Tb(NO3)3·6H2O, and 123.1 mg (1.50 mmol) of anhydrous CH3COONa were dissolved in 10 mL of deionized water and stirred to prepare a metal precursor solution. Separately, 105 mg (0.500 mmol) of H3BTC was dissolved in 10 mL of anhydrous ethanol by sonication. The metal precursor solution was slowly added dropwise to the ligand solution and stirred at 25°C for 1 hour. The resulting white precipitate was centrifuged at 10,000 rpm for 3 minutes, washed three times with water and ethanol, and finally dried at 60°C for 6 hours to obtain a white powder of TbEu-MOFs (3:2). The nominal Tb:Eu precursor molar ratio is approximately 3:2; this value represents the feed ratio, not the composition as measured by ICP or XPS.

[0022] Preparation of 0.5 wt% agarose sol: Dissolve 0.05 g agarose in 10 mL of ultrapure water at 80 °C. Add 100 μL of agarose sol and 100 μL of TbEu-MOFs dispersion (obtained by mixing white TbEu-MOFs powder with water, 0.3 mg·mL⁻¹). - ¹) and 150 μL of Tris–HCl buffer (pH 5) were mixed in a 96-well plate to form a TbEu-MOF@hydrogel composite material.

[0023] 2. Detection of ENX by TbEu-MOFs@hydrogel

[0024] First, a 96-well hydrogel detection plate containing the composite material was prepared, with equal amounts of the composite material and enrofloxacin solutions of different concentrations (0.01–200 μM) added to each well. The plate was then incubated at 25°C for 5 minutes. Next, the plate was transferred to a detection box with a pre-drilled imaging port, irradiated with 254 nm UV light, and images of different fluorescence intensities were captured using a mobile phone through the imaging port on the top of the detection box. Subsequently, quantitative analysis of the RGB channels of the images was performed using ImageJ software, selecting the green channel (G value) as the signal indicator. Finally, the experimental data were converted into a bar chart of green fluorescence intensity for visualization. The results are as follows: Figure 1 As shown.

[0025] Depend on Figure 1 It can be seen that the experimental PXRD pattern reproduced the main diffraction peaks of the simulated La(BTC)·6H2O reference calculated according to CCDC290771. Figure 1 a) This supports the formation of a closely related lanthanide-BTC framework. Minor peak shifts may originate from Tb³. + / Eu³ + With La³ + Differences in ionic radius, lanthanide contraction, hydration, microstrain, and preferred orientation; according to the Bragg equation, minute changes in lattice spacing manifest as 2θ shifts. The observed similarities suggest the existence of related structural units, but isomorphism needs to be confirmed through indexing or Rietveld refinement. FT-IR spectroscopy further confirms metal-carboxylate coordination (…). Figure 1 b). Non-ionized carboxyl groups in free H3BTC at 3112, 1717, and 538 cm⁻¹ - The absorption peak of ¹ disappears after MOF formation, while the symmetry of the coordinated carboxylate group (1437–1374 cm⁻¹) remains. - ¹) and asymmetry (1616–1561 cm) - ¹) The stretching and vibration bands become more pronounced. 534cm - ¹The absorption band near this point belongs to the lanthanide-oxygen vibration, 3443 cm⁻¹ - The broad peak ¹ indicates the presence of coordination water and lattice water. The MOF excitation spectrum overlaps with the ENX absorption band ( Figure 1 c) provides a possible pathway for perturbations in competitive excitation, internal filtering effects, and ligand antenna processes. Time-resolved emission spectra and smartphone photos show a visible color evolution from orange-red to orange-pink. Figure 1 d, f), excitation peak positions support 254nm excitation ( Figure 1 e).

[0026] It is evident that the TbEu-MOF@hydrogel composite material can both inhibit particle aggregation and allow small molecules to enter the luminescent phase. Under 254 nm excitation, Eu³ + and Tb³ + The center emits red and green emissions respectively, which mix to form an orange-red color. Upon addition of ENX, the relative equilibrium of Tb / Eu emission changes, and the complex color gradually changes from orange-red to orange-pink. This indicates that the PXRD pattern remains essentially unchanged after ENX exposure, suggesting that the framework is preserved during the optical response process. Spectral overlap, PXRD pattern retention, and lifetime changes indicate that ENX interferes with the sensitization process of the ligands to lanthanides, and also affects Tb³. + The impact of the center's contribution is more significant.

[0027] Further investigation was conducted using Fourier transform infrared spectroscopy (FTIR) to study the composition and elemental chemical state of the material. Figure 2 As can be seen, SEM images show that the particles have a uniform rod-shaped morphology. Figure 2 a and b), EDS and elemental distribution maps qualitatively confirm the coexistence of Tb and Eu ( Figure 2 Elements such as c~h), Eu, and Tb did not aggregate or precipitate; instead, they were very uniformly distributed within the carbon, nitrogen, and oxygen matrix. These elements are extremely uniform and highly dispersed within the material. This demonstrates the successful synthesis of MOF materials with bimetallic nodes.

[0028] 3. Optimization of experimental conditions

[0029] The effects of different TbEu-MOF concentrations, solution pH, reaction temperature, and reaction time were investigated, and the results are as follows: Figure 3 As shown.

[0030] Depend on Figure 3 It can be known that 0.3 mg / mL -1 At this point, the optimal concentration can achieve a balance between the number of emitted particles and self-absorption or aggregation under high load. Figure 3 a). The optimal response is observed at pH 5, reflecting a good trade-off between ENX protonation, lanthanide site interactions, and material stability. Figure 3 b). The highest response was observed at 25°C. Figure 3 c) This eliminates the need for additional heating during detection. The signal stabilizes within approximately 5 minutes. Figure 3 d). Therefore, 0.3 mg / mL was selected for subsequent experiments. -1 TbEu-MOF was reacted at pH 5 and 25℃ for 5 min.

[0031] 4. Detection performance against ENX

[0032] Under the aforementioned optimal reaction conditions, the TbEu-MOF@hydrogel composite material was further used to analyze ENX at different concentrations (0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200 μmol). The results are as follows: Figure 4 As shown.

[0033] The results showed that as the ENX concentration increased, the color gradually changed from orange-red to orange-pink, which was visible to the naked eye. Figure 4 a, b). The intensity of the green channel on the smartphone monotonically increases in the range of 0–200 μM, providing quantitative readings ( Figure 4 c, d). The fitted equation is Y = 35.29X + 82.52, which exhibits a log-linear relationship in the range of 1–100 μM (R² = 0.97); Figure 4d). The limit of detection (LOD) was calculated as 3σ / k (σ is the standard deviation of the blank signal, and k is the calibration slope), and the result was 0.155 μM. The error bar represents the standard deviation of three measurements. In addition to analytical sensitivity, this method also features a 5-minute room temperature response, a visually visible lanthanide color change, and smartphone-compatible reading functionality. This method is suitable for rapid screening of ENX in complex matrices, and positive or critical results can be validated using a confirmed chromatographic method.

[0034] 5. Selectivity, anti-interference and stability verification

[0035] Selectivity was assessed using eight common veterinary antibiotics, following the same procedure as ENX, and the results are as follows: Figure 5 As shown.

[0036] Depend on Figure 5 It can be seen that at the tested concentration (110 μM), the responses of norfloxacin (NDF), sulfamethoxazole (SMR), sulfadiazine (SMZ), sulfadiazine (SD), florfenicol (FF), chloramphenicol (CAP), erythromycin (ERY), and penicillin potassium (PEN) were all significantly lower than those of ENX. Figure 5 a) indicates that the method is selective for antibiotics under the stated conditions. When 100 μM of each potential interfering agent was added, only limited signal changes were observed at a detection rate of 100 μM MENX. Figure 5 (b) Supports tolerance to interference from the tested antibiotics, but does not cover all chemicals in feces; matrix specificity validation is required. Approximately 75% of the initial signal was retained after 20 days of storage at 4°C. Figure 5 c), supports refrigerated storage for screening purposes.

[0037] Example 2

[0038] Chicken and cow manure were collected from local farms, dried, extracted, centrifuged at 12,000 rpm for 10 min, filtered through a 0.22 μm filter membrane, and analyzed. The results are shown in Table 1.

[0039] Table 1. Recovery rate of ENX in chicken and cow manure (n=3)

[0040] As shown in Table 1, the recoveries ranged from 83.21% to 100.7% in five out of six tests, with RSDs ranging from 2.40% to 6.22%. The recovery rate in chicken manure at a 1 μM spike level was 123.4%, indicating a positive matrix effect at the lowest detection level. Therefore, this platform is more suitable for preliminary screening; positive or borderline results require confirmation by a validated LC-MS / MS method.

[0041] This sensor features a 5-minute response at 25°C, a visible lanthanide color change, smartphone-based quantitative analysis, and suitability for fecal samples. These complementary characteristics support its use in rapid on-site screening. This system also differs from previously reported Eu-MOF@hydrogel sensors for ciprofloxacin. Early platforms contained only Eu emitters, detecting chicken and duck droppings and producing a red to light blue color change; whereas in this invention, Tb³… + and Eu³ + Dual emission signals were provided for ENX in chicken and cow manure, exhibiting a color change from orange-red to orange-pink. Furthermore, CNN-based binary classification and a second-device image test were introduced, with results as follows... Figure 6 As shown.

[0042] Depend on Figure 6 It can be seen that, using a fixed imaging box and observation geometry, images acquired under 254nm illumination fall into two categories: both training and validation accuracies exceed 97%, and the loss value continuously decreases during training. Figure 6 a, b). The validation set image-level AUC is 0.97, and the confusion matrix shows that 66 images are correctly classified ( Figure 6 c, d). The AUC of the second device on the test set is 1.00, and there are no classification errors in the confusion matrix. Figure 6 (e, f). The above results validate the image classification performance of this method under controlled lighting conditions. Quantitative analysis employed green channel regression, and CNN was used for binary classification.

[0043] Because the images are acquired under controlled lighting, the CNN results can serve as image-level proof-of-concept. Evaluation using independent datasets from different farms, different mobile phones, and different lighting conditions will more effectively test the model's generalization ability. The CNN results can complement smartphone RGB calibration and enable rapid classification of ENX fluorescence images. This method, combining sensing and image analysis, is suitable for preliminary on-site screening of livestock manure.

[0044] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A dual lanthanide MOF hydrogel system, characterized in that, It consists of the luminescent active component TbEu-MOF and the carrier matrix agarose hydrogel; The TbEu-MOF is self-assembled from Tb 3+ , Eu 3+ double lanthanide ions and the organic ligand benzene-1,3,5-tricarboxylic acid; the TbEu-MOF is embedded in an agarose hydrogel.

2. The dual lanthanide MOF hydrogel system of claim 1, wherein, The concentration of TbEu-MOF in the agarose hydrogel was 0.05~1.0 mg / mL.

3. A method of preparing the dual lanthanide MOF hydrogel system of claim 1, characterized in that, Includes the following steps: 1) Tb (NO3)3 6H2O and Eu (NO3)3 6H2O were dissolved in a solvent, and then a solution of benzene-1, 3, 5-tricarboxylic acid ligand was added to stir for reaction, centrifugation, washing, and drying to obtain a TbEu-MOF solid powder; 2) Dissolve agarose in water by heating to prepare agarose sol; add TbEu-MOF dispersion and Tris-HCl buffer to the agarose sol and mix thoroughly to obtain a lanthanide MOF hydrogel system.

4. The preparation method according to claim 3, characterized in that, Step 1) said Tb (NO3)3 6H2O and Eu (NO3)3 in a molar ratio of 2-4: 1-3; said solvent is anhydrous CH3COONa.

5. The preparation method according to claim 3, characterized in that, Step 1) The stirring reaction is carried out at 20~25℃ for 40~80 min; the centrifugation is carried out at 8000~12000 rpm for 2~4 min; the drying is carried out at 55~65℃ for 5~7 h.

6. The preparation method according to claim 3, characterized in that, The heating and melting temperature in step 1) is 75~85℃.

7. The preparation method according to claim 3, characterized in that, Step 2) The concentration of the agarose sol is 0.4~0.6wt%; the TbEu-MOF dispersion is a mixture of TbEu-MOF solid powder and water, with a concentration of 0.2~0.4 mg / mL.

8. The preparation method according to claim 3, characterized in that, Step 2) The pH of the Tris-HCl buffer solution is 4-6.

9. A kit comprising the bis-lanthanide MOF hydrogel system of claim 1 or 2.

10. The application of the lanthanide MOF hydrogel system of claim 1 or 2 or the kit of claim 9 in the rapid screening of enrofloxacin in livestock and poultry manure samples.

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