A method for synthesizing a terbium complex fluorescent probe and its application in the detection of metronidazole.

Abstract

This invention discloses a method for synthesizing a terbium complex fluorescent probe and its application in the detection of metronidazole, belonging to the field of food safety technology. This invention utilizes 2,6-difluorobenzoic acid and 1,10-phenanthroline as ligands to synthesize a luminescent rare earth complex [Tb2(2,6-dfba)6(phen)2] with Tb(NO3)3·6H2O. The crystal structure shows that it is a binuclear terbium complex, which exhibits strong Tb2 fluorescence at 489, 545, 584, and 621 nm. 3+ The complex exhibits characteristic emission peaks and good time stability of its fluorescence intensity. It specifically and sensitively recognizes metronidazole, demonstrating rapid response (<1 min), a low limit of detection (43.7 nM), and a wide linear detection range (0–100 μM). It can be used for the quantitative detection of metronidazole in animal feed.

Description

Technical Field

[0001] This invention belongs to the field of food safety technology and relates to the detection of veterinary drug residues, specifically to a method for synthesizing a terbium complex fluorescent probe and its application in the detection of metronidazole. Background Technology

[0002] Due to its broad-spectrum anti-anaerobic and antiprotozoal activity, good therapeutic effects, and low price, metronidazole has been widely used in veterinary clinical practice since its introduction. More seriously, it has been indiscriminately added to animal feed for disease prevention and growth promotion in farmed animals. Metronidazole and its metabolites entering livestock and poultry not only cause environmental pollution through excretion in feces via bile, but also remain in animal products such as milk and eggs through lactation and egg production, posing food safety risks. Reports indicate that metronidazole metabolites retain imidazole rings with similar toxicity to the original drug, and these metabolites persist in animals for a longer period than the original drug, potentially causing serious diseases such as cancer and deformities in mammals during growth, and even exhibiting genotoxicity. Therefore, its use has been banned in many countries and regions. To prevent the overuse of metronidazole in animal husbandry and improve the rationality of veterinary drug clinical applications, ensuring environmental and food safety, it is essential to strengthen the monitoring of metronidazole in animal feed. Therefore, developing a stable, effective, and rapid method for detecting metronidazole in animal feed is of great practical significance.

[0003] Currently, metronidazole detection techniques include titration, thin-layer chromatography, enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and gas chromatography (GC). However, these conventional detection methods often suffer from drawbacks such as low sensitivity, large errors, susceptibility to interference, complex pretreatment, and high equipment costs. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings and deficiencies of the existing technology and to provide a method for synthesizing a terbium complex fluorescent probe and its application in the detection of metronidazole.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] The terbium complex fluorescent probe of this invention has the molecular formula [Tb2(2,6-dfba)6(phen)2], and its crystal structure shows that [Tb2(2,6-dfba)6(phen)2] is a binuclear terbium complex with strong Tb2 at 489, 545, 584, and 621 nm. 3+ It exhibits characteristic emission peaks, and its fluorescence intensity shows good time stability.

[0007] The synthesis method of the terbium complex fluorescent probe includes the following steps: dissolving rare earth ions Tb(III), 2,6-difluorobenzoate, NaHCO3, and 1,10-phenanthroline in a solvent, filtering to remove the precipitate, and allowing the colorless clear liquid to stand until colorless blocky crystals precipitate, thereby obtaining the terbium complex fluorescent probe. The solvent is preferably a water / ethanol mixture.

[0008] In some implementations, the rare earth ion Tb(III) is derived from terbium nitrate hexahydrate.

[0009] In some implementations, terbium nitrate hexahydrate, 2,6-difluorobenzoate, NaHCO3, and 1,10-phenanthroline are added in a molar ratio of 17:50:50:16.

[0010] Furthermore, the synthesis method of the terbium complex fluorescent probe includes the following steps: terbium nitrate hexahydrate is dissolved in ethanol, 2,6-difluorobenzoic acid and NaHCO3 are dissolved in a water / ethanol mixed solvent, and 1,10-phenanthroline is dissolved in ethanol. The above three solutions are mixed, and the resulting precipitate is removed by filtration. The colorless clear liquid is allowed to evaporate naturally at 20-30 °C. After two weeks, colorless blocky crystals precipitate, thus obtaining the terbium complex fluorescent probe.

[0011] The application of the terbium complex fluorescent probe in the detection of metronidazole. The terbium complex fluorescent probe can specifically detect metronidazole without interference from other veterinary drugs.

[0012] A method for detecting metronidazole using the aforementioned terbium complex fluorescent probe includes the following steps: adding the test sample to a solution containing the terbium complex fluorescent probe, detecting the change in fluorescence intensity at 545 nm, and detecting metronidazole based on the fluorescence quenching effect. The solution containing the terbium complex fluorescent probe is a DMF solution containing the terbium complex fluorescent probe.

[0013] In some embodiments, the test sample is animal feed. Further, the animal feed is processed by a method comprising the following steps: fully dissolving the animal feed in ethanol, filtering, removing the solvent ethanol from the filtrate under reduced pressure, and drying to obtain an extract. The dried extract is then added to a solution containing a terbium complex fluorescent probe for detection.

[0014] Advantages and beneficial effects of this invention: This invention utilizes 2,6-difluorobenzoic acid and 1,10-phenanthroline as ligands to synthesize a luminescent rare earth complex [Tb2(2,6-dfba)6(phen)2] with Tb(NO3)3·6H2O, which can specifically and sensitively recognize metronidazole. [Tb2(2,6-dfba)6(phen)2] exhibits rapid response to metronidazole (<1 min), a low detection limit (43.7 nM), and a wide linear detection range (0-100 µM). When [Tb2(2,6-dfba)6(phen)2] is used for the determination of metronidazole content in animal feed, the recovery rate is high. [Tb2(2,6-dfba)6(phen)2] can be used for the quantitative detection of metronidazole in animal feed. Attached Figure Description

[0015] Figure 1 It has the crystal structure of [Tb2(2,6-dfba)6(phen)2].

[0016] Figure 2 This is the infrared spectrum of [Tb2(2,6-dfba)6(phen)2].

[0017] Figure 3 This is the PXRD pattern of [Tb2(2,6-dfba)6(phen)2].

[0018] Figure 4 It is a thermogravimetric diagram of [Tb2(2,6-dfba)6(phen)2].

[0019] Figure 5 This is the fluorescence spectrum of [Tb2(2,6-dfba)6(phen)2].

[0020] Figure 6 This is the seven-day fluorescence spectrum of [Tb2(2,6-dfba)6(phen)2] in DMF solution.

[0021] Figure 7 It represents the fluorescence intensity of [Tb2(2,6-dfba)6(phen)2] before and after the addition of each analyte.

[0022] Figure 8 The fluorescence intensity of [Tb2(2,6-dfba)6(phen)2] before and after the addition of metronidazole is compared under different interfering conditions.

[0023] Figure 9 These are the fluorescence spectra of [Tb2(2,6-dfba)6(phen)2] in the presence of different concentrations of metronidazole.

[0024] Figure 10The expression represents the linear relationship between the fluorescence intensity I0 / I of [Tb2(2,6-dfba)6(phen)2] before and after the addition of metronidazole and the concentration of metronidazole added.

[0025] Figure 11 This is the time response plot of [Tb2(2,6-dfba)6(phen)2]metronidazole.

[0026] Figure 12 The image shows the UV spectra of [Tb2(2,6-dfba)6(phen)2] with metronidazole and terbium nitrate. Detailed Implementation

[0027] The following embodiments are used to further illustrate the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0028] Some of the materials used in the following examples: Terbium nitrate hexahydrate (analytical grade) was purchased from Shandong Henghua Reagent Co., Ltd., China. 2,6-Difluorobenzoic acid (analytical grade), 1,10-phenanthroline (analytical grade), and metronidazole (analytical grade) were purchased from Aladdin Reagent (Shanghai) Co., Ltd. N,N-Dimethylformamide was purchased from Xilong Chemical Co., Ltd., China. Lincomycin sulfate, kanamycin sulfate, streptomycin sulfate, dihydrostreptomycin sulfate, thiamphenicol, florfenicol, bisphenol A, erythromycin, dimethylpyridine, andrographolide, benzimidazole, chloral hydrate, cyanamide, ivermectin, amikacin sulfate, chlorpheniramine, abramycin, neomycin sulfate, baclofen, zithromycin hydrochloride, and other analytical grade reagents were purchased from Shanghai Maclean's Biochemical Technology Co., Ltd. All reagents were used directly without further purification.

[0029] Example 1: Synthesis of terbium complex [Tb2(2,6-dfba)6(phen)2]

[0030] (1) Synthesis of terbium complex [Tb2(2,6-dfba)6(phen)2]

[0031] Tb(NO3)3·6H2O (780 mg, 1.7 mmol) was dissolved in 64 mL of ethanol, and 2,6-difluorobenzoic acid (750 mg, 5 mmol) and NaHCO3 (400 mg, 5 mmol) were dissolved in 96 mL of a water / ethanol mixture (V... H2O V EtOHIn a 5:1 ratio, 1,10-phenanthroline (315 mg, 1.6 mmol) was dissolved in 40 mL of ethanol. The three solutions were then mixed, and a small amount of white precipitate was removed by filtration. The colorless clear liquid was left to evaporate naturally at room temperature, and after two weeks, colorless blocky crystals [Tb2(2,6-dfba)6(phen)2] precipitated.

[0032] (2) Determination of crystal structure

[0033] Select dimensions of 0.150 × 0.1 × 0.08 mm. 3 The single-crystal data were measured using an Agilent Gemini CCD diffractometer (Xcalibur, Eos, Gemini) with a monochromatic graphite radiation source, Mo Kα radiation (λ = 0.71073 nm). The single-crystal structure was resolved using the Patterson method with SHELXTL-2008 software. The crystal data are listed in Table 1, and some bond lengths are listed in Table 2.

[0034] Table 1. Crystal structure data of [Tb2(2,6-dfba)6(phen)2]

[0035]

[0036] Table 2. Bond lengths [Å] and bond angles [Å] of [Tb2(2,6-dfba)6(phen)2]. 。 ]

[0037]

[0038] like Figure 1 As shown, the asymmetric unit of [Tb2(2,6-dfba)6(phen)2] is composed of a Tb 3+ ions, three 2,6-difluorobenzoate anions (dfba) - It consists of a 1,10-phenanthroline molecule and a Tb group. Each 8-coordinated Tb group 3+ Ions from 3 dfba - The six oxygen atoms at the ligand center and the two nitrogen atoms at the center of the phen molecule coordinate to form a deformed tetragonal antiprism configuration. Among them, O3 and O4... i O5, O6 i Four atoms are located on the base of the square antiprism, and atoms N1, N2, O1, and O2 are located on the top surface of the square antiprism. Each dfba - Ligands have two different coordination modes, such as Figure 1As shown, the carboxyl group O1-C13-O2 adopts a chelate coordination mode, while the carboxyl groups O3-C20-O4 and O5-C27-O6 adopt a monodentate bridging coordination mode, connecting two adjacent Tb groups. 3+ This forms a dual-core structure, in which Tb ... The distance to Tb is 4.325 Å, and each phen molecule uses two N atoms to interact with Tb. 3+ Coordination.

[0039] The infrared spectrum of the complex [Tb2(2,6-dfba)6(phen)2] is as follows: Figure 2 As shown, the complexes at 1624 and 1416 cm⁻¹ -1 The strong peak at that location belongs to -COO - The antisymmetric and symmetric stretching vibrations further prove dfba - It exists in coordination compounds. Located at 1235 cm. -1 The strong peak at 1578 cm⁻¹ represents the stretching vibration of the CF bond. -1 Stretching vibrations of CN were observed at [location missing], indicating that phen is contained in the complex. Furthermore, [details missing] were observed at 860–729 cm⁻¹. -1 The peaks within the range are attributed to the stretching vibrations of CH on the aromatic ring. Therefore, the infrared results indicate that the complex contains two ligands: 2,6-dfba and phen.

[0040] like Figure 3 As shown, the X-ray powder diffraction pattern of the complex [Tb2(2,6-dfba)6(phen)2] is compared with the simulated XRD pattern of single-crystal diffraction. Figure 1 The consistency indicates that the complex sample is a single, pure sample and has the same structure as the single crystal.

[0041] The thermogravimetric spectrum of the complex [Tb2(2,6-dfba)6(phen)2] is as follows: Figure 4 As shown, the complex exhibits good thermal stability with no significant weight loss before 280 °C. The complex [Tb2(2,6-dfba)6(phen)2] shows its first weight loss in the range of 280–310 °C, with a weight loss rate of 21%; as the temperature increases, the complex continues to decompose slowly.

[0042] Example 2: Fluorescence properties and detection performance of terbium complex [Tb2(2,6-dfba)6(phen)2]

[0043] 10.0 mg of terbium complex [Tb2(2,6-dfba)6(phen)2] crystals were dissolved in 50 mL of DMF to prepare a probe stock solution. Then, various analytes (such as lincomycin sulfate, kanamycin sulfate, streptomycin sulfate, dihydrostreptomycin sulfate, thiamphenicol, florfenicol, bisphenol A, erythromycin, dimethylpyridine, andrographolide, benzimidazole, chloral hydrate, cyanamide, ivermectin, amikacin sulfate, chlorpheniramine, abramycin, neomycin sulfate, baclofen, zizomycin hydrochloride, and metronidazole, etc.) were dissolved in DMF to prepare analyte stock solutions. Each analyte was then added to 2 mL of the probe [Tb2(2,6-dfba)6(phen)2] stock solution to obtain a final analyte concentration of 0.3 mM. Fluorescence data were recorded using a fluorescence spectrophotometer.

[0044] The fluorescence spectrum of [Tb2(2,6-dfba)6(phen)2] in DMF is as follows: Figure 5 As shown. When excited at an excitation wavelength of 298 nm, [Tb2(2,6-dfba)6(phen)2] exhibits Tb 3+ Characteristic emission peaks of the ion: The main emission peaks at 489, 545, 584, and 621 nm can be attributed to Tb. 3+ Ionic 5 D4→ 7 F J (J=6, 5, 4, 3) transitions. The main peak of the complex [Tb2(2,6-dfba)6(phen)2] at 545 nm is... 5 D4→ 7 The F5 transition is the main reason for its bright green fluorescence emission. Furthermore, the fluorescence spectrum of [Tb2(2,6-dfba)6(phen)2] in DMF showed almost no significant change after 7 days, indicating its excellent fluorescence stability. Figure 6 Therefore, [Tb2(2,6-dfba)6(phen)2] has the potential to be used as a fluorescent probe.

[0045] To confirm the specificity of the fluorescent probe [Tb2(2,6-dfba)6(phen)2] for the detection of metronidazole, this invention selected some veterinary drugs and chemicals that may be used in animal feed, including lincomycin sulfate, kanamycin sulfate, streptomycin sulfate, dihydrostreptomycin sulfate, thiamphenicol, florfenicol, bisphenol A, erythromycin, dimethylpyridine, andrographolide, benzimidazole, chloral hydrate, melamine, ivermectin, amikacin sulfate, chlorpheniramine, abramycin, neomycin sulfate, baclofen, zizomycin hydrochloride, and metronidazole, as analytes, and studied their effects on the fluorescence properties of the fluorescent probe [Tb2(2,6-dfba)6(phen)2]. Figure 7As shown, only lincomycin slightly enhanced the fluorescence intensity of [Tb2(2,6-dfba)6(phen)2] at 545 nm. Lincomycin sulfate, clindamycin, dihydrostreptomycin sulfate, thiamphenicol, florfenicol, erythromycin, bisphenol A, dimethylpyridine, andrographolide, benzimidazole, chloral hydrate, cyanamide, ivermectin, chlorpheniramine, abramycin, neomycin sulfate, and baclofen slightly weakened the fluorescence intensity of the probe [Tb2(2,6-dfba)6(phen)2] at 545 nm; kanamycin sulfate, streptomycin sulfate, amikacin sulfate, and zizomycin hydrochloride had almost no effect on the fluorescence intensity of the probe [Tb2(2,6-dfba)6(phen)2]. Only metronidazole almost completely quenched the fluorescence of [Tb2(2,6-dfba)6(phen)2]. Therefore, [Tb2(2,6-dfba)6(phen)2] exhibits good selectivity for metronidazole.

[0046] Anti-interference capability is another key factor in ensuring the performance of a sensing system. An excellent probe can effectively identify the analyte even when other interfering substances coexist. To understand the anti-interference capability of the fluorescent probe [Tb2(2,6-dfba)6(phen)2] in detecting metronidazole, the changes in fluorescence intensity before and after adding 0.3 mM metronidazole to probe solutions containing 0.3 mM of each potential interfering substance were tested. Figure 8 As can be seen, the presence of any potential interfering substances did not significantly affect the fluorescence intensity of the probe itself; however, after introducing metronidazole in the presence of interfering substances, the fluorescence of the probe was almost completely quenched, and the degree of quenching was almost equivalent to that in the absence of interfering substances. This means that the quenching effect of metronidazole on the fluorescence of [Tb2(2,6-dfba)6(phen)2] is not affected by the interfering substances. These results indicate that the probe [Tb2(2,6-dfba)6(phen)2] has good anti-interference ability for the detection of metronidazole.

[0047] Example 3 Sensitivity of terbium complex [Tb2(2,6-dfba)6(phen)2] for detecting metronidazole

[0048] A suitable amount of high-concentration (10 mmol / L) metronidazole DMF solution was gradually added dropwise to a 0.5 mg / mL probe [Tb2(2,6-dfba)6(phen)2] stock solution until the metronidazole concentration was 0, 1, 5, 10, 20, 40, 60, 80, and 100 μmol / L (since the added metronidazole DMF solution was of high concentration, the total amount was very small and its effect on the probe concentration was negligible; the probe [Tb2(2,6-dfba)6(phen)2] concentration was maintained at almost 0.5 mg / mL). The fluorescence intensity of the mixed solution at each concentration was recorded, and the quenching efficiency (I0 / I) versus metronidazole concentration curve was plotted. Figure 9 As shown, the fluorescence intensity of the probe [Tb2(2,6-dfba)6(phen)2] gradually decreased with increasing metronidazole concentration. When the metronidazole concentration was in the range of 0-100 µM, a good linear relationship was observed between the quenching efficiency (I0 / I) and the metronidazole concentration. Figure 10 ), I0 / I = 0.017C 甲硝唑 +1.019, correlation coefficient (R²) 2 The value was 0.998. The fluorescence intensity of the probe [Tb2(2,6-dfba)6(phen)2] stock solution at 545 nm was tested 11 times at a concentration of 0.5 mg / mL. Based on the signal-to-noise ratio (S / N = 3), the three-fold noise signal value was substituted into the linear relationship I0 / I = 0.017C. 甲硝唑 +1.019 yielded C 甲硝唑 The limit of detection (LOD) is 43.7 nM. Therefore, the complex [Tb2(2,6-dfba)6(phen)2] has very high sensitivity as a fluorescent probe for detecting metronidazole.

[0049] Example 4: Response rate of terbium complex [Tb2(2,6-dfba)6(phen)2] in detecting metronidazole

[0050] Metronidazole solution was added to the 0.5 mg / mL probe [Tb2(2,6-dfba)6(phen)2] stock solution to a concentration of 0.3 mmol / L, and the solution was quickly mixed. The fluorescence intensity of the solution at 545 nm was recorded on a fluorescence spectrophotometer at time points of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 10.0, 15.0, 20.0, and 25.0 min. The fluorescence intensity of the probe [Tb2(2,6-dfba)6(phen)2] stock solution before the addition of metronidazole was recorded as the fluorescence intensity at min 0.

[0051] Response speed is one of the important indicators for measuring the detection performance of a sensor. From Figure 11It can be seen that the fluorescence at 545 nm was almost completely quenched within 1 minute, indicating that the fluorescent probe [Tb2(2,6-dfba)6(phen)2] responds very quickly to metronidazole and can be used for real-time detection of metronidazole.

[0052] Example 5: Fluorescence quenching mechanism of metronidazole on probe [Tb2(2,6-dfba)6(phen)2]

[0053] DMF solutions of metronidazole, 1,10-phenanthroline, terbium nitrate, and 2,6-difluorobenzoic acid molecules at certain concentrations were prepared, and the ultraviolet absorption spectra of the solutions were scanned using a UV-Vis spectrophotometer.

[0054] like Figure 12 As shown, the UV-Vis absorption spectra of 2,6-dfba, phen, and metronidazole almost overlap. Therefore, metronidazole competes for absorption with the ligands 2,6-dfba and phen, thereby reducing the energy released from the ligands to donate the rare earth ion Tb. 3+ The probability of this weakens the ligand's effect on Tb. 3+ The "antenna effect" of ions leads to the quenching of fluorescence in the complex [Tb2(2,6-dfba)6(phen)2].

[0055] Example 6: Application of probe [Tb2(2,6-dfba)6(phen)2] in the detection of metronidazole in feed samples

[0056] 1.0 g of rabbit feed was placed in a round-bottom flask, and an appropriate amount of metronidazole ethanol solution (0.3 mM) was added. The rabbit feed sample solution was diluted to 10 mL with ethanol and extracted by sonication for 30 min. The extract was filtered through a 0.22 µm Millipore membrane, and this extraction process was repeated 3 times. The collected filtrate was purified by removing the solvent ethanol under reduced pressure. The dried extract was diluted to 10 mL with 0.5 mg / mL probe [Tb2(2,6-dfba)6(phen)2] stock solution, and its fluorescence intensity at 545 nm was recorded.

[0057] As shown in Table 3, the recovery rate of metronidazole in rabbit feed ranged from 94.8% to 110.2%, and the RSD values ​​were all less than 2.0%. These results indicate that the probe has the potential to quantitatively detect metronidazole in actual animal feed samples.

[0058] Table 3. Application of metronidazole in actual samples.

[0059]

[0060] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A terbium complex fluorescent probe, characterized in that: The formula of the terbium complex fluorescent probe is [Tb2(2,6-dfba)6(phen)2], 2,6-dfba is 2,6-difluorobenzoate anion, and phen is 1,10-phenanthroline molecule.

2. The method for synthesizing the terbium complex fluorescent probe according to claim 1, characterized in that, The method comprises the following steps: dissolving terbium nitrate hexahydrate, 2,6-difluorobenzoate, NaHCO3 and 1,10-phenanthroline in a solvent, removing the precipitate by filtration, and standing the colorless and clear liquid to precipitate colorless block-shaped crystals to obtain the terbium complex fluorescent probe.

3. The method of synthesis of claim 2, wherein: The terbium nitrate hexahydrate, 2,6-difluorobenzoate, NaHCO3 and 1,10-phenanthroline are added in a mass ratio of 17:50:50:

16.

4. The method of synthesis of claim 2, wherein, The method comprises the following steps: dissolving terbium nitrate hexahydrate in ethanol, dissolving 2,6-difluorobenzoate and NaHCO3 in a water / ethanol mixed solvent, and dissolving 1,10-phenanthroline in ethanol; mixing the three solutions, removing the precipitate generated by filtration; and naturally evaporating the colorless and clear liquid to precipitate colorless block-shaped crystals to obtain the terbium complex fluorescent probe.

5. The terbium complex fluorescent probe of claim 1 is applied to the detection of metronidazole.

6. Use according to claim 5, characterized in that: The detection sample is animal feed.

7. A method for detecting metronidazole by using the terbium complex fluorescent probe of claim 1, comprising the following steps: adding a detection sample to a solution containing the terbium complex fluorescent probe, detecting the fluorescence intensity at 545 nm, and detecting metronidazole according to the fluorescence quenching effect.

8. The method of claim 7, wherein the solution containing the terbium complex fluorescent probe is a DMF solution containing the terbium complex fluorescent probe.

9. The method of claim 7, wherein the detection sample is animal feed.

Images