Amphiphilic Eu complex and its preparation method and application in detecting tetracycline

Abstract

The invention provides an amphiphilic Eu complex and a preparation method thereof and an application thereof in detecting tetracycline, and relates to the field of biomedicine. The europium in the amphiphilic Eu complex of the invention can electrostatically interact with tetracycline hydrochloride, and the fluorescence at 590 nm, 615 nm, 650 nm and 705 nm is enhanced, thereby achieving qualitative and quantitative detection of tetracycline hydrochloride, and the selectivity for tetracycline hydrochloride is high, the sensitivity is high, the detection limit is low, and the complex has strong stability.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to an amphiphilic Eu complex and a preparation method thereof and an application thereof in detecting tetracycline. Background Art

[0002] Tetracycline hydrochloride (TC) antibiotics are a class of broad-spectrum drugs that are widely used in the livestock industry due to their low toxicity and low price. However, if livestock and poultry do not strictly abide by the regulations during the use of tetracycline veterinary drugs, or if they are not adequately cleaned and handled after use, the residues may enter the human food chain, posing a potential threat to human health. Long-term excessive consumption of foods containing tetracycline veterinary drug residues may cause damage to internal organs such as the liver and kidneys, further causing headaches, dizziness, nausea, vomiting and other discomfort symptoms. In severe cases, it may damage the central nervous system and hematopoietic function, and even affect the normal development and function of the reproductive system.

[0003] To ensure food safety, the state has clearly defined the standards for veterinary drug residues in food, among which the restrictions on tetracycline veterinary drug residues are very strict. According to the "Maximum Residue Limits of Veterinary Drugs in Food" (GB 31650-2019), the maximum limit standards for tetracycline veterinary drug residues in food are as follows:

[0004] The tetracycline veterinary drug residue in pork, beef, mutton, chicken and eggs shall not exceed 0.1 mg / kg;

[0005] The residue of tetracycline veterinary drugs in milk and other dairy products shall not exceed 0.05 mg / kg.

[0006] At present, tetracycline hydrochloride is mainly detected by traditional liquid chromatography analysis, and there are few reagents for rapid detection. Compared with instrumental detection methods, fluorescence detection methods have the advantages of rapidity, sensitivity, high selectivity, and portability, and have a lower limit of detection (LOD). Due to the unique photophysical properties of lanthanide ions, such as large Stokes shift, narrow ff transition spectral band, and long fluorescence lifetime, the interference of background fluorescence can be effectively avoided, and the signal-to-noise ratio and detection sensitivity can be improved. These superior photophysical properties have made lanthanide ions widely studied in the fields of fluorescence detection, fluorescence imaging, and time-resolved imaging. In summary, it is of great significance to invent a probe for rapid detection of tetracycline hydrochloride using lanthanide ions as the luminescence center. Summary of the invention

[0007] The present invention aims to provide an amphiphilic Eu complex and a preparation method thereof and an application thereof in detecting tetracycline. The amphiphilic Eu complex can specifically respond to tetracycline hydrochloride, can perform qualitative and quantitative detection of tetracycline hydrochloride, and has a low detection limit, high sensitivity, and certain stability.

[0008] In the first aspect, the present invention provides an amphiphilic Eu complex, the structure of which is shown in Formula I:

[0009] .

[0010] The amphiphilic Eu complex provided by the present invention has hydrophilicity and lipophilicity, and contains a nanoparticle structure. When a sample to be tested contains tetracycline hydrochloride, the tetracycline hydrochloride can generate an electrostatic interaction with europium in the amphiphilic Eu complex, and emission peaks can be detected at 590 nm, 615 nm, 650 nm and 705 nm during fluorescence measurement, thereby achieving qualitative and quantitative detection of tetracycline hydrochloride.

[0011] In a second aspect, the present invention provides a method for preparing an amphiphilic Eu complex, comprising the following steps:

[0012] In a solvent, 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester is alkanoamidated and then reduced, and then coordinated with europium salt to obtain an amphiphilic Eu complex.

[0013] Optionally, in a dichloromethane solvent, 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester is subjected to an alkanoyl amidation reaction with dodecanoyl chloride to generate 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester-10-carbon dodecanoyl amide.

[0014] Optionally, 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester is reacted with dodecanoyl chloride at 0° C. for 10 h-12 h to separate 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester-10-carbon dodecaneamide.

[0015] Optionally, the molar ratio of 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester to dodecanoyl chloride is 1:(0.8-2), preferably 1:1.5.

[0016] Alternatively, 1,4,7,10-tetraazacyclododecane-1,4,7-triacetate tri-tert-butyl ester is reacted with dodecanoyl chloride to introduce an alkyl chain.

[0017] Alternatively, trifluoroacetic acid is added to 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester-10-carbon dodecaneamide, and the mixture is reduced at 25° C. for 10 h-12 h to obtain the ligand.

[0018] Alternatively, the tert-butyl ester group can be reduced to a carboxyl group by reacting 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester-10-carbon dodecaneamide with trifluoroacetic acid.

[0019] Optionally, the molar ratio of 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester-10-carbon dodecaneamide to trifluoroacetic acid is 1:(3.2-4.5), preferably 1:4.

[0020] Optionally, in tetrahydrofuran solvent, the ligand is reacted with europium salt at 50° C. for 10 h-12 h to obtain an amphiphilic Eu complex.

[0021] Optionally, the europium salt includes any one of europium nitrate and europium chloride.

[0022] Optionally, the molar ratio of the ligand to the europium salt is 1:(2.5-4.5), preferably 1:3.

[0023] In a third aspect, the present invention provides the use of the amphiphilic Eu complex prepared by any of the above optional preparation methods in the detection of tetracycline hydrochloride.

[0024] When the amphiphilic Eu complex provided by the present invention is used for the detection of tetracycline hydrochloride, it can specifically respond to tetracycline hydrochloride, can perform qualitative and quantitative detection of tetracycline hydrochloride, and has a low detection limit, high sensitivity, and certain stability.

[0025] Optionally, applications include qualitative and quantitative detection.

[0026] Optionally, the qualitative detection of tetracycline hydrochloride includes: mixing the sample to be tested with the amphiphilic Eu complex and then performing fluorescence measurement, when emission peaks are detected at 590 nm, 615 nm, 650 nm and 705 nm, the sample to be tested contains tetracycline hydrochloride.

[0027] Optionally, the quantitative detection of tetracycline hydrochloride includes: mixing the sample to be tested with the amphiphilic Eu complex, measuring the fluorescence intensity at 590 nm, 615 nm, 650 nm and 705 nm, and calculating the concentration of tetracycline hydrochloride in the sample to be tested according to the fluorescence intensity and the titration curve; wherein: the titration curve is a relationship curve between the molar ratio of the pre-determined tetracycline hydrochloride standard solution and the amphiphilic Eu complex and the fluorescence ratio.

[0028] Optionally, the molar ratio of tetracycline hydrochloride to the amphiphilic Eu complex in the titration curve is 1:1.

[0029] Optionally, the linear indication range of tetracycline hydrochloride in the sample to be tested is 6 μmol / L-22 μmol / L, and when σ=0.2, the detection limit of tetracycline hydrochloride is 9.5×10 -3 μmol / L.

[0030] The amphiphilic Eu complex provided by the invention is used for the detection of tetracycline hydrochloride, has low raw material cost, high practical application value and simple preparation method, and is suitable for industrial batch production. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly introduce the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments.

[0032] Figure 1 The structural formula of the amphiphilic Eu complex I provided by the present invention;

[0033] Figure 2 The synthetic route of the amphiphilic Eu complex I provided by the present invention;

[0034] Figure 3 is the H NMR spectrum of the ligand in the amphiphilic Eu complex in Example 1 of the present invention;

[0035] Figure 4 is the mass spectrum of the amphiphilic Eu complex in Example 1 of the present invention;

[0036] Figure 5 It is a fluorescence spectrum diagram of the response of 20 μmol / L amphiphilic Eu complex in solution to 0 μmol / L-30 μmol / L tetracycline hydrochloride in the performance test of the present invention;

[0037] Figure 6 The detection limit calculation for the solution of 20 μmol / L amphiphilic Eu complex and 0 μmol / L-30 μmol / L tetracycline hydrochloride in the performance test of the present invention;

[0038] Figure 7 The selectivity test result of the amphiphilic Eu complex to tetracycline hydrochloride in the selectivity test of the present invention;

[0039] Figure 8 This is the test result of the time stability of the amphiphilic Eu complex in the stability test of the present invention. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described through the following embodiments in conjunction with the accompanying drawings. However, the following embodiments are illustrative and are only used to specifically describe the present invention rather than to limit the present invention.

[0041] The present invention provides an amphiphilic Eu complex, the structure of which is shown in Formula I:

[0042] .

[0043] See also Figure 2 The present invention also provides a method for preparing an amphiphilic Eu complex, comprising the following steps:

[0044] S1. In a dichloromethane solvent, 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester is subjected to an alkanoamidation reaction and then reduced to obtain a ligand;

[0045] S2. In tetrahydrofuran solvent at 50 °C, the ligand obtained in S1 coordinates with europium salt for 10 h-12 h to obtain an amphiphilic Eu complex.

[0046] In some embodiments, when executing step S1, the following sub-steps are included:

[0047] S1.1. At 0 °C, in dichloromethane solvent, react 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester with dodecanoyl chloride for 10 h-12 h to obtain 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester-10-carbon dodecaneamide;

[0048] S1.2. At 25 °C, in dichloromethane solvent, add trifluoroacetic acid to 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester-10-carbon dodecaneamide and react for 10 h-12 h to obtain the ligand.

[0049] In some embodiments, when performing step S1.1, the molar ratio of 1,4,7,10-tetraazacyclododecane-1,4,7-triacetate to dodecanoyl chloride is 1:(0.8-2), preferably 1:1.5.

[0050] In some embodiments, when performing step S1.2, the molar ratio of 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester-10-carbon dodecaneamide to trifluoroacetic acid is 1: (3.2-4.5), preferably 1:4.

[0051] In some embodiments, when performing step S2, the europium salt includes any one of europium nitrate and europium chloride.

[0052] In some embodiments, when performing step S2, the molar ratio of the ligand to the europium salt is 1: (2.5-4.5), preferably 1:3.

[0053] The present invention also provides the use of the amphiphilic Eu complex in the detection of tetracycline hydrochloride, including qualitative detection and quantitative detection:

[0054] (1) The qualitative detection of tetracycline hydrochloride includes: mixing the sample to be tested with the amphiphilic Eu complex and then performing fluorescence measurement. When emission peaks are detected at 590 nm, 615 nm, 650 nm and 705 nm, the sample to be tested contains tetracycline hydrochloride.

[0055] (2) The quantitative detection of tetracycline hydrochloride comprises: mixing the sample to be tested with the amphiphilic Eu complex, measuring the fluorescence intensity at 590 nm, 615 nm, 650 nm and 705 nm, and calculating the concentration of tetracycline hydrochloride in the sample to be tested according to the fluorescence and titration curve; wherein: the titration curve is a relationship curve between the molar ratio of the tetracycline hydrochloride standard solution and the amphiphilic Eu complex and the fluorescence ratio determined in advance.

[0056] In some embodiments, when performing application (2), the molar ratio of tetracycline hydrochloride to the amphiphilic Eu complex in the titration curve is 1:1.

[0057] In some embodiments, when performing application (2), the linear indication range of tetracycline hydrochloride in the sample to be tested is 6 μmol / L-22 μmol / L, and when σ=0.2, the detection limit of tetracycline hydrochloride is 9.5×10 -3 μmol / L.

[0058] Example 1

[0059] See also Figure 2 Embodiment 1 of the present invention provides a method for preparing an amphiphilic Eu complex, comprising the following steps:

[0060] S1.1. Add 1 g (5.16 mmol) of 1,4,7,10-tetraazacyclododecane-1,4,7-triacetate (CAS No.: 122555-91-3), 0.86 mL (6.19 mmol) of triethylamine (CAS No.: 121-44-8) and 20 mL of dichloromethane into a 50 mL round-bottom flask and stir in an ice bath for 0.5 h. Add 1.41 mL (6.19 mmol) of dodecanoyl chloride (CAS No.: 112-16-3) into 5 mL of dichloromethane. Add the mixed solution of dodecanoyl chloride dropwise into the dichloromethane solution within 5 min and stir at 0 °C for 10 min. h, add saturated sodium bicarbonate solution to quench, extract with dichloromethane and evaporate under reduced pressure, spin dry and pass through a column (SiO2, silica gel, eluent is ethyl acetate and petroleum ether in a volume ratio of 1:1) to obtain 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester-10-carbon dodecaneamide;

[0061] S1.2, dissolve 0.5 g (1.92 mmol) of 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester-10-carbon dodecaneamide and 0.64 mL (8.64 mmol) of trifluoroacetic acid in 5 mL of dichloromethane solution, stir at 25 °C for 10 h, and spin dry to obtain the ligand;

[0062] S2, 0.1 g (0.18 mmol) of ligand and 0.0524 g (0.2 mmol) of europium chloride (CAS No.: 13769-20-5) were dissolved in 15 mL of tetrahydrofuran, stirred at 50 °C for 10 h, cooled to room temperature, and anhydrous ether was added to precipitate a large amount of oily solids. The solids were washed with anhydrous ether three times, and dried to remove the organic solvent to obtain an amphiphilic Eu complex (DO3A-C12-Eu). The mass spectrum is shown in the following figure: Figure 4 shown.

[0063] The ligand prepared in step S1.2 was characterized by H NMR spectroscopy in dimethyl sulfoxide, such as Figure 3 As shown, its characterization data is:

[0064] 1 H NMR (400 MHz, Chloroform-d) δ 6.73 (t, J = 69.4 Hz, 13H), 3.81 (d,J = 55.2 Hz, 4H), 3.15 (s, 2H), 2.20 (s, 2H), 1.43–1.34 (m, 2H), 1.24 (d, J =7.1 Hz, 18H), 0.85 (t, J = 6.6 Hz, 3H).

[0065] Performance Testing

[0066] Response of the amphiphilic Eu complex DO3A-C12-Eu prepared in Example 1 to tetracycline hydrochloride:

[0067] Use pure water to prepare 2 mL of amphiphilic Eu complex solution with a concentration of 20 μmol / L. Add tetracycline hydrochloride to the amphiphilic Eu complex solution. After adding 1.5 μmol / L of tetracycline hydrochloride, perform fluorescence measurement until saturation is reached. During the increase of tetracycline hydrochloride concentration, the fluorescence intensity changes as shown in Figure 5 As shown, the results showed that the absorption peaks at 590 nm, 615 nm, 650 nm and 705 nm increased with the increase of tetracycline hydrochloride content and reached saturation when the tetracycline hydrochloride content was 22 μmol / L.

[0068] Titration curves were drawn based on different tetracycline hydrochloride concentrations and corresponding fluorescence intensities. The results are shown in Figure 2. Figure 6 As shown in the figure, it can be seen that in the range of 6 μmol / L-22 μmol / L, tetracycline hydrochloride has a good linear relationship with the fluorescence intensity, and the quantitative detection of tetracycline hydrochloride can be performed. It is calculated that when σ=0.2, the detection limit is 9.5×10 -3 μmol / L.

[0069] Selective testing

[0070] Selectivity test of the amphiphilic Eu complex DO3A-C12-Eu prepared in Example 1 for tetracycline hydrochloride:

[0071] A solution containing 20 μmol / L of the amphiphilic Eu complex DO3A-C12-Eu was prepared using pure water, and 100 μmol / L of PPi, F - , Cl - Br - ,I - 、NO3 - 、HCO3 - 、SO3 2- 、SO4 2- 、CO3 2- , TC analytes, such as Figure 7As shown, the fluorescence spectra after adding the above analytes to the amphiphilic Eu complex were measured. The results showed that after adding tetracycline hydrochloride to the Eu complex, the fluorescence intensity at 590 nm, 615 nm, 650 nm and 705 nm increased, while the emission peaks at 590 nm, 615 nm, 650 nm and 705 nm did not change significantly after adding other analytes, indicating that the amphiphilic Eu complex DO3A-C12-Eu has extremely excellent selectivity for tetracycline hydrochloride.

[0072] Stability test

[0073] Stability test of the amphiphilic Eu complex DO3A-C12-Eu prepared in Example 1:

[0074] A solution containing 20 μmol / L of the amphiphilic Eu complex DO3A-C12-Eu was prepared using pure water, and its particle size from 0h to 10h was measured, such as Figure 8 As shown, the results showed that the particle size of DO3A-C12-Eu did not fluctuate significantly from 0 h to 10 h, indicating that the amphiphilic Eu complex has excellent stability in aqueous media.

[0075] Although the embodiments of the present invention are described in detail above, it is obvious to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations are within the scope and spirit of the present invention as described in the claims. Moreover, the present invention described herein may have other embodiments and may be implemented or realized in a variety of ways.

Claims

1. An amphiphilic Eu complex, characterized in that: The structure is shown in Formula I: 。 2. A method for preparing the amphiphilic Eu complex as claimed in claim 1, characterized in that: The method comprises the following steps: in a solvent, 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester is subjected to alkane amidation and then reduced, and then coordinated with europium salt to obtain an amphiphilic Eu complex.

3. The preparation method according to claim 2, characterized in that: In a dichloromethane solvent, 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester is subjected to an alkanoyl amidation reaction with dodecanoyl chloride to generate 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester-10-carbon dodecanoic acid amide.

4. The preparation method according to claim 3, characterized in that: After 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester and dodecanoyl chloride are reacted at 0°C for 10 h-12 h, 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester-10-carbon dodecaneamide is separated.

5. The preparation method according to claim 3 or 4, characterized in that: The molar ratio of the 1,4,7,10-tetraazacyclododecane-1,4,7-triacetate tri-tert-butyl ester to the dodecanoyl chloride is 1: (0.8-2).

6. The preparation method according to claim 3, characterized in that: Trifluoroacetic acid was added to 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester-10-carbon dodecaneamide, and the mixture was reduced at 25°C for 10 h-12 h to obtain the ligand.

7. The preparation method according to claim 6, characterized in that: The molar ratio of the 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester-10-carbon dodecaneamide to the trifluoroacetic acid is 1: (3.2-4.5).

8. The preparation method according to claim 6, characterized in that: In tetrahydrofuran solvent, the ligand is reacted with europium salt at 50°C for 10 h-12 h to obtain an amphiphilic Eu complex.

9. The preparation method according to claim 8, characterized in that: The europium salt includes any one of europium nitrate and europium chloride; the molar ratio of the ligand to the europium salt is 1: (2.5-4.5).

10. Use of the amphiphilic Eu complex as claimed in claim 1 in the detection of tetracycline hydrochloride, characterized in that: Includes qualitative testing and quantitative testing.

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