A tridentate europium complex, preparation method and application thereof

Abstract

The present invention discloses a tridentate europium complex, a preparation method and application thereof. The general structural formula of the tridentate europium complex is Eu[PO]3, wherein PO is a long alkyl chain modified phosphorus-oxy-substituted 4-hydroxy-1,5-naphthyridine tridentate anionic ligand, and the synthesis method disclosed in the present invention has high yield and safe operation. After coordination of such ligands with europium, a tridentate europium complex with good thermal stability, good photostability and high solubility is obtained, and the complex is applied to fluorescent immunochromatography to obtain a highly sensitive fluorescent immunoassay paper test card for detecting SAA items, with a detection limit as low as 2μg / mL, lower than the clinical value of 10μg / mL, and a detection concentration of up to 220μg / mL.

Description

Technical Field

[0001] The invention relates to the technical field of rare earth complexes and luminescent materials, and in particular to a rare earth europium complex and a preparation method thereof, as well as application in the field of fluorescent immunochromatography. Background Art

[0002] Rare earth complex luminescent materials have high quantum efficiency, long luminescence lifetime, large Stokes shift, and narrow half-peak width, and have important application prospects in the fields of display, lighting, and immunoassay. As a photoluminescent material, due to the large molar absorption coefficient of the organic ligand, the light absorption capacity of the complex is increased by hundreds of times compared with pure inorganic rare earth materials. Moreover, rare earth complexes have higher quantum efficiency and lower toxicity than up-conversion rare earth materials. They are more suitable for fluorescent immunochromatographic detection.

[0003] In rare earth complexes, organic ligands play a decisive role in the luminescence properties of the complexes. Organic ligands chelate with rare earth ions, not only as antenna molecules to absorb and transfer energy, but also to protect the rare earth luminescence from being quenched by the external environment. At present, most rare earth complexes are bidentate complexes, but their photostability and thermal stability are very poor, and there are defects in their application in lighting, display and immunochromatography.

[0004] To solve this problem, researchers have designed tridentate anionic ligands. By increasing the chelating sites of the complex, when the complex is formed with the rare earth ion at a ratio of 3:1, the coordination saturation requirement can be met, avoiding the thermal stability problem caused by the introduction of neutral ligands, thereby obtaining thermally stable, light-stable and efficient rare earth complex luminescent materials. However, due to the increase of anionic ligands, the solubility of rare earth complexes is greatly reduced, affecting their application in immunochromatography, bioimaging and other fields. Therefore, this type of rare earth complex luminescent material still needs to make necessary improvements, and make it have better technical effects and broader application prospects. Summary of the invention

[0005] The present invention aims to solve the technical problem that the stability and solubility of existing rare earth complexes cannot be satisfied at the same time, and obtain a rare earth luminescent material with good stability and high solubility, so as to be better applied in the fields of fluorescent immunochromatography and the like.

[0006] As mentioned above, currently rare earth complexes are mainly bidentate, and even a small amount of tridentate has a significant decrease in solubility due to the rigidity of its ligand structure. In order to improve the solubility of rare earth complexes while ensuring their stability, the present invention designs and synthesizes chelating groups using a new synthesis method, successfully improves the solubility problem of tridentate rare earth complexes, and applies them in the field of fluorescent immunochromatography.

[0007] Specifically, the technical solution of the present invention is as follows:

[0008] A rare earth complex based on a tridentate 4-hydroxy-1,5-naphthyridine ligand, the general structural formula of which is Eu(PO)3, wherein PO represents a phosphorus-oxy-substituted 4-hydroxy-1,5-naphthyridine anionic ligand. The structure of the ligand PO is shown below:

[0009]

[0010] Among them, R1, R2, R3, and R4 are the same or different atoms or groups, and R1, R2, R3, and R4 are each independently selected from a hydrogen atom, a halogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aromatic group, and a heterocyclic group.

[0011] Preferably, the above-mentioned halogen atoms include F, Cl, Br, I; the alkyl group includes C1-C18 alkyl groups, such as methyl, ethyl, propyl, n-butyl and isobutyl; the alkenyl group includes C2-C18 alkenyl groups, such as ethylene, propylene and butene; the alkynyl group includes C2-C18 alkynyl groups, such as acetylene, propyne and butyne; the aromatic group includes C6-C36 aromatic groups, such as phenyl, benzoic acid and acetophenone; the heterocyclic group includes a heterocyclic group with a formula weight of less than 200, such as thiophene, thiazole, furan and pyridine.

[0012] Wherein R5 is an aryl group. Preferably, the aryl group is a substituted phenyl group; further preferably, the substituent of the substituted phenyl group is selected from n-butyl, n-octyl, n-dodecyl, n-hexadecyl, and n-octadecyl; preferably n-octyl and n-dodecyl.

[0013] The synthetic route of the above ligand PO is as follows:

[0014]

[0015] Preferably, the ligand PO is selected from any one of the following:

[0016]

[0017] The structure of the tridentate europium complex is shown below:

[0018]

[0019] Preferably, the structures of the tridentate europium complexes are as follows, which have high solubility:

[0020]

[0021]

[0022] The synthetic route of the tridentate europium complex of the present invention is as follows:

[0023]

[0024] An embodiment of the present invention further provides a method for synthesizing a tridentate europium complex, the method comprising the following steps:

[0025] 1) Add the raw materials 2,2-dimethyl-1,3-dioxane-4,6-dione and triethyl orthoformate into a three-necked flask and react at 100°C for 1.5 hours. After the reaction, add 3-bromo-5-aminopyridine and the reaction is complete. Filter the reaction solution while hot and wash with ether to obtain compound A.

[0026] 2) Compound A and diphenyl ether were added into a three-necked flask, refluxed for 1.5 h, cooled to room temperature, filtered to obtain a solid product, and washed with dichloromethane to obtain a dark brown product B.

[0027] 3) The dark brown product B obtained in step 2) and potassium carbonate were added to a 100 mL three-necked flask, and DMF was added, and then benzyl chloride was added to react at 85° C. overnight. Extraction was performed, and DMF was drained, and compound C was obtained by column chromatography.

[0028] 4) Add Mg and I2 to a 100 mL three-necked flask, then add THF and heat the solution. Add 4-butyl bromobenzene in THF solution and continue stirring for 1 hour, then cool to 0°C and add diethylphosphine in THF solution for 30 minutes. Stir at room temperature for 2 hours. Quench the reaction with 2M HCl (20 mL), cool to 0°C and stir for 15 minutes. Filter with diatomaceous earth, extract the filtrate with dichloromethane three times, and obtain compound D by column chromatography.

[0029] 5) Compound C and compound D were added to a dioxane solution containing K2CO3, palladium acetate and ferrocene were added, and the mixture was refluxed at 100°C overnight. Compound E was obtained by extraction and column chromatography.

[0030] 6) Add compound E, dioxane and concentrated hydrochloric acid to a 25 mL single-mouth bottle and reflux for reaction. Adjust to neutral with NaOH solution. Extract with dichloromethane and obtain ligand PO by column chromatography.

[0031] 7) Add PO and NaOH to a single-mouth bottle, add DMSO and stir at room temperature until it becomes clear, reflux for 10 min, dropwise add EuCl3·6H2O ethanol solution, react at 70°C overnight. Filter to obtain the tridentate europium complex Eu[PO]3.

[0032] In the preparation of tridentate europium complexes, the main difficulty and key point lies in the synthesis of tridentate ligands. In the invention patent application CN105017329A, the Peking University team disclosed a rare earth europium complex luminescent material based on a tridentate anionic ligand and its preparation method and application, which involves the synthesis of tridentate ligands and discloses the synthesis route and yield of tridentate ligand L1 (tridentate ligand L1 is numbered 5f in the CN105017329A patent application). The present invention has made improvements on the relevant synthesis method and achieved significant beneficial effects:

[0033] (1) The synthesis process of the invention patent CN105017329A requires the use of active metallic sodium, which is flammable and explosive when in contact with water. However, the synthesis step of the present invention does not require the use of dangerous chemicals such as metallic sodium, and the preparation process is safer;

[0034] (2) The total yield of the tridentate ligand L1 in the invention patent CN105017329A is about 15%, while the present invention adopts a new synthesis method to greatly improve the synthesis yield of the tridentate ligand. For example, the total yield of the tridentate ligand L2 is about 50% (the tridentate ligand L2 is numbered PO4 in the present invention).

[0035] The present invention obtains a rare earth luminescent material with good stability and high solubility, which is suitable for application in the fields of fluorescent immunochromatography, bioimaging, luminescent films, etc. The present invention exemplarily applies the synthesized tridentate europium complex to the field of immunochromatography. Specifically, the europium complex is coated in polystyrene microspheres, and the surface of the microspheres is modified, and finally the antibody is coupled to the surface of the microspheres to synthesize fluorescent immunomicrospheres. It is used to detect SAA projects, and its detection sensitivity is high, the stability is good, and efficient and rapid in vitro monitoring is achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the H NMR spectrum of the ligand PO4.

[0037] Figure 2 This is the NMR carbon spectrum of the ligand PO4.

[0038] Figure 3 This is the H NMR spectrum of the ligand PO7.

[0039] Figure 4 This is the NMR carbon spectrum of the ligand PO7.

[0040] Figure 5 This is the excitation and emission spectra of the tridentate europium complex Eu[PO]3-4.

[0041] Figure 6 These are the thermal stability test results of the tridentate europium complex Eu[PO]3-4.

[0042] Figure 7 These are the photostability test results of the tridentate europium complex Eu[PO]3-4.

[0043] Figure 8 These are the solubility test results of the tridentate europium complex before and after structural modification.

[0044] Fig. 9 This is the synthetic route map of immunochromatographic microspheres.

[0045] Fig.10 Schematic diagram of fluorescent immunochromatographic detection.

[0046] Fig.11 This is the standard curve for immunochromatographic detection. DETAILED DESCRIPTION

[0047] In order to explain the synthesis scheme of the present invention in more detail, the following will be described in detail in conjunction with the specific synthesis equation. Obviously, the described implementation case is only a part of the preferred implementation case of the present invention. All other similar implementation cases mentioned by researchers in this research field without making any innovative contribution should fall within the scope of protection of the present invention.

[0048] The invention obtains a tridentate europium complex with good stability and good solubility through seven-step chemical reaction synthesis.

[0049] Example 1

[0050] The synthetic route of this implementation is as follows:

[0051]

[0052] The specific synthesis steps are as follows:

[0053] Synthesis of compound A: 2,2-dimethyl-1,3-dioxane-4,6-dione (13.8 g, 96 mmol, 1.2 eq) and triethyl orthoformate (55 mL, 320 mmol, 4 eq) were reacted at 100°C for 2 h, and then 5-bromo-3-aminopyridine (13.76 g, 80 mmol, 1 eq) was added after the reaction to continue the reaction. The reaction solution was filtered while hot and washed with petroleum ether to obtain pure compound A. Yield: 95%.

[0054] Synthesis of compound B: Add compound A (24 g, 72 mmol) and diphenyl ether (350 mL) into a 1000 mL three-necked flask and reflux for 2 h. Cool to room temperature, filter to obtain a solid product, wash with dichloromethane to obtain a dark brown product. Then stir with 30 eq of NaOH aqueous solution for one hour, filter to obtain an aqueous solution, adjust to neutrality with dilute hydrochloric acid, and filter to obtain a solid product, compound B. Yield: 50%.

[0055] Synthesis of compound C: Add compound B (2g, 9.5mmol) and potassium carbonate (2g, 15mmol) into a 100mL three-necked flask, inject DMF (15mL), and then inject benzyl chloride (1.15mL, 10mmol) to react at 85℃ overnight. Extract, drain DMF, and obtain pure compound C by column chromatography. Yield: 70%.

[0056] Synthesis of compound D: Mg (0.8 g, 33.3 mmol, 3.3 eq) and I2 (5 mg, 0.02 mmol) were added to a 100 mL three-necked flask, and THF (20 mL) was added, and the solution was heated. A THF solution of 4-butyl bromobenzene (5.8 mL, 30 mmol, 3 eq) was added dropwise, and stirring continued for 1 h, then ice-bathed to 0 ° C, and a THF solution of diethylphosphine (1.3 mL, 10 mmol, 1.0 eq) was added dropwise for 30 min. Stir at room temperature for 2 h. The reaction was quenched with 2M HCl (20 mL), cooled to 0 ° C and stirred for 15 min. Filtered with diatomaceous earth, the filtrate was extracted three times with dichloromethane, and column chromatography was performed to obtain pure compound D. Yield: 70%.

[0057] Synthesis of compound E: Compound C (100 mg, 0.317 mmol, 1 eq) and compound D (150 mg, 0.476 mmol, 1.5 eq) were added to a dioxane solution (5 mL) containing K2CO3 (87 mg, 0.634 mmol, 2 eq), and refluxed at 100°C overnight with Pd(OAc)2 (8.3 mg, 0.0317 mmol 0.1 eq) and dppf (23 mg 0.0138 mmol 0.1 eq). Compound E was obtained by extraction and column chromatography. Yield: 80%.

[0058] Synthesis of compound PO, taking tridentate ligand PO4 as an example: Add compound E (50 mg, 0.09 mmol), CH2Cl2 (2 mL), and concentrated hydrochloric acid (1.13 mL) into a 25 mL single-mouth bottle and stir at room temperature, reflux reaction, and change to dioxane for reflux. Adjust to neutral with NaOH solution. Extract with dichloromethane, and column chromatography to obtain pure compound PO4. Yield: 95%.

[0059] Synthesis of compound Eu[PO]3-4: Add compound PO4 (150 mg, 0.263 mmol, 3 eq) to a single-mouth bottle, add methanol (3 mL) and stir at room temperature, add dropwise a methanol solution (1 mL) of EuCl3·6H2O (32 mg, 0.0876 mmol, 1 eq), and react at 70°C overnight. Filter to obtain pure compound Eu[PO]3-4. Yield: 90%.

[0060] Example 2

[0061] The synthetic route in this embodiment is as follows:

[0062]

[0063] The synthesis steps are the same as those in Example 1, except that R5 is different.

[0064] Example 3

[0065] The synthetic route in this embodiment is as follows:

[0066]

[0067] The synthesis steps are the same as those in Example 1, except that R1 and R2 are different.

[0068] Example 4

[0069] The synthetic route in this embodiment is as follows:

[0070]

[0071] The synthesis steps are the same as those in Example 1, except that R2, R4 and R5 are different.

[0072] Example 5

[0073] The synthetic route in this embodiment is as follows:

[0074]

[0075] The synthesis steps are the same as those in Example 1, except that R1, R2, R4 and R5 are different.

[0076] Example 6 NMR Spectrum Characterization of Tridentate Ligand PO4

[0077] 1 H NMR (400MHz, CDCl3) δ8.64(s,1H),8.35(s,1H),7.95(s,1H),7.65(s, 4H), 7.20 (s, 4H), 6.83 (s, 1H), 2.57 (t, J = 7.3Hz, 4H), 1.55 (s, 4H), 1.26 (m, J = 8.2 Hz, 20H), 0.87 (t, J = 6.7Hz, 6H). 13C NMR (101MHz, CDCl3) δ155.28,153.97, 148.15,142.97,139.28,137.45,132.10,129.12,128.85,128.12,127.0 6,111.72,35.93,31.79,30.97,29.33,29.26,29.14,22.57,13.99.HRMS for[M-PF6] + :calcd 1855.2734, found 1855.2965.

[0078] The NMR spectrum of the tridentate ligand PO4 is shown in Figure 1 and Figure 2 The NMR spectrum of the tridentate ligand PO7 is shown in Figure 3 and Figure 4 As shown. Figure 1 and attached Figure 2 The results confirmed the structure of the tridentate ligand PO4; Figure 4 The results confirmed the structure of the tridentate ligand PO7.

[0079] Example 7 Characterization of thermal and photostability of tridentate europium complexes

[0080] The technical effects of the present invention are mainly reflected by the emission spectrum, thermal stability and ultraviolet tolerance of the rare earth complexes in the embodiments. Among them, the ultraviolet tolerance is measured by testing the luminous intensity attenuation of the polymer film (PMMA) doped with the rare earth complex under ultraviolet light irradiation.

[0081] The excitation and emission spectra of the tridentate europium complex Eu[PO]3-4 are shown in Figure 5 shown.

[0082] The thermal stability of the tridentate europium complex Eu[PO]3-4 was tested as follows Figure 6 shown.

[0083] The photostability of the tridentate europium complex Eu[PO]3-4 was tested as follows Figure 7 shown.

[0084] In the thermal stability and photostability tests, two common europium complexes were used as controls: Eu(TTA)3Phen and Eu(DBM)3Phen.

[0085] like Figure 6 As shown in Figure 2, compared with the two common europium complexes, the thermal stability of the tridentate europium complex Eu[PO]3-4 is significantly enhanced. Figure 7As shown, compared with the two common europium complexes, the photostability of the tridentate europium complex Eu[PO]3-4 is significantly enhanced.

[0086]

[0087] Example 8 Solubility of tridentate europium complex

[0088] The solubility of Eu[PO]3-4 and Eu[PO]3-7 complexes was tested using acetone, toluene and other reagents. The solubility of the tridentate europium complex proposed in the present invention is greatly improved after structural modification. Figure 8 As shown, when the number of C atoms in the carbon chain of the tridentate ligand R5 benzene ring is n=8, it is Eu[PO]3-4; when the number of C atoms in the carbon chain of the tridentate ligand R5 benzene ring is n=4, it is Eu[PO]3-7; when the number of C atoms in the carbon chain of the tridentate ligand R5 benzene ring is n=0, it is the europium complex Eu5 disclosed in invention patent CN105017329A, without modifying the long carbon chain, and its solubility is relatively small as a comparison.

[0089] Example 9: Preparation of fluorescent microspheres by encapsulating tridentate europium complexes in microspheres

[0090] Take polystyrene microspheres (1.185mL), deionized water (2.82mL), and acetone (5mL) and add them to the reaction bottle. Stir at room temperature for 20min. Add acetone solution of Eu[PO]3-4 tridentate europium complex (1mL, 20 mg / mL) and continue stirring at room temperature for 5h. Centrifuge after reaction (12000r / min, 10min), add deionized water / ethanol (v:v=9:1) and wash repeatedly for 3 times. Add 10mL deionized water for storage. Solid content 1%.

[0091] Example 10 Preparation of amino fluorescent microspheres

[0092] Take the fluorescent microspheres (20mL, 10mg / mL) obtained in Example 9 and centrifuge (12000r / min, 10min) to remove the supernatant, add NaOH aqueous solution (20mL, pH=11), and sonicate (10min). Weigh mercaptoethylamine (30mg) and add it to the above microsphere solution at one time, shake and react at room temperature for 24h. Centrifuge after reaction (8000r / min, 10min), add water / ethanol (9:1) and centrifuge repeatedly and wash 3 times. Add 20mL BBS (pH=7.5) for storage, solid content 1%.

[0093] Example 11 Preparation of aldehyde fluorescent microspheres

[0094] Take the MES solution (pH=6.0) (15mL, 10 mg / mL) containing amino fluorescent microspheres obtained in Example 10, add aldehyde dextran (500,000, 1.25mmol / g, 100mg) and react for 20min, then add sodium cyanoborohydride (100μL, 50mg / mL) and continue to react overnight. Centrifuge (7000r / min, 5min), add deionized water and wash repeatedly three times. Add 15mL BBS (pH=7.5) for storage. Solid content 1%. The obtained microspheres are named Eu[PO]3-4NP, which are fluorescent microspheres containing aldehyde groups on the surface.

[0095] Example 12 Preparation of immune microspheres

[0096] Take the Eu[PO]3-4NP aldehyde fluorescent microspheres obtained in Example 11, add the aldehyde fluorescent microspheres (625μL, 10mg / mL) to BBS solution (3mL, pH=7.5). Add chicken IgY (5mg / mL, 200μL) to the above microspheres and react for 20min, then add sodium cyanoborohydride (20μL, 50mg / mL) and react at room temperature for 6h. Then centrifuge (7000r / min, 5min) to remove the supernatant, add 4mL BBS buffer solution (pH=7.5), ultrasonicate for 5min, block with BSA (300μL, 10%) for 1h, centrifuge, add 5mL BBS, and the solid content is 0.25%.

[0097] Example 13 Preparation of Immunochromatographic Test Strips

[0098] The immunochromatographic test strip consists of four parts: a PVC plastic base, a NC membrane, a sample pad, and absorbent paper. The preparation method is as follows: ① Paste the NC membrane on the PVC plastic base. ② Prepare the SAA antibody 5F2 protein concentration of 1.5 mg / mL and draw it on the NC membrane at a speed of 1 μL / cm as the T line; prepare the sheep anti-chicken IgY protein concentration of 1 mg / mL and draw it on the NC membrane at a speed of 1 μL / cm as the C line. ③ Use the gold spray diluent to prepare Eu[PO]3-4NP-3H8 immune microspheres and Eu[PO]3-4NP-Chicken IgY with concentrations of 25 μg / mL and 5 μg / mL, and use the XYZ three-dimensional film spray gold instrument to spray the above diluent on the sample plate, where 5F2 and 3H8 are immunopaired. ④ Place the NC membrane with the T / C line and the sample plate sprayed with the immune microspheres in a constant temperature box at 37°C overnight. ⑤ Paste the sample pad and absorbent paper on the fixed position of the NC membrane respectively. ⑥ Use a microcomputer automatic cutter to cut the test strip into pieces with a width of 0.38 cm, install it in a plastic card slot, and store it in a dry and room temperature environment for future use.

[0099] Example 14 SAA Project Detection

[0100] Using normal human serum as sample diluent, the SAA standard was diluted to 0 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 55 μg / mL, 110 μg / mL, 220 μg / mL, and 440 μg / mL. 1.25 μL of standard with different concentrations and 98.75 μL of SAA sample diluent were mixed and added to the sample injection port. After reacting for 11 minutes, the fluorescence intensity of the T line and C line was detected and recorded, and the I T / I C The test was repeated three times, and the SAA standard concentration and the corresponding I T / I C The curve of the value (the result is as follows Fig.11 shown).

[0101] The tridentate europium complex prepared by the present invention has good stability and good solubility, and is applied to fluorescent immunochromatography to obtain a highly sensitive fluorescent immunoassay test card for detecting SAA items. Fig.11 It can be seen that its detection limit is as low as 2μg / mL, which is lower than the clinical value of 10μg / mL, and the detection concentration is as high as 220μg / mL.

[0102] Table 1 Analysis and evaluation results of immunochromatographic platform for detecting SAA

[0103]

[0104] Example 14 Tridentate Ligand Synthesis Method

[0105] In the preparation of tridentate europium complexes, the main difficulty and key point lies in the synthesis of tridentate ligands.

[0106] In the invention patent application CN105017329A, the Peking University team disclosed a rare earth europium complex luminescent material based on a tridentate anionic ligand and its preparation method and application, which involves the synthesis of tridentate ligands and discloses the synthesis route and yield of the tridentate ligand L1 (the tridentate ligand L1 is numbered 5f in the CN105017329A patent disclosure).

[0107]

[0108] The present invention improves the relevant synthesis method and obtains significant beneficial effects:

[0109] (1) The synthesis process of the invention patent CN105017329A requires the use of active metallic sodium, which is flammable and explosive when in contact with water. However, the synthesis step of the present invention does not require the use of dangerous chemicals such as metallic sodium, and the preparation process is safer;

[0110] (2) The total yield of the tridentate ligand L1 in the invention patent CN105017329A is about 15%, while the present invention adopts a new synthesis method to greatly improve the synthesis yield of the tridentate ligand. For example, the total yield of the tridentate ligand L2 is about 50% (the tridentate ligand L2 is numbered PO4 in the present invention).

[0111]

Claims

1. A tridentate europium complex, It is characterized in that The tridentate europium complex is selected from any one of the following:

2. The method for preparing the tridentate europium complex according to claim 1, It is characterized in that The route of the preparation method is as follows:

3. The preparation method according to claim 2, It is characterized in that The preparation method comprises the following steps: 1) Add the raw materials 2,2-dimethyl-1,3-dioxane-4,6-dione and triethyl orthoformate into a three-necked flask and react at 100°C for 1.5 hours. After the reaction, add 3-bromo-5-aminopyridine and the reaction is complete. Filter the reaction solution while hot and wash with ether to obtain compound A. 2) Compound A and diphenyl ether were added to a three-necked flask, refluxed for 1.5 h, cooled to room temperature, filtered to obtain a solid product, and washed with dichloromethane to obtain a dark brown product B; 3) The dark brown product B obtained in step 2) and potassium carbonate were added to a 100 mL three-necked flask, DMF was added, and then benzyl chloride was added to react at 85° C. overnight; extraction was performed, the DMF was drained, and compound C was obtained by column chromatography; 4) Mg, I 2 Add to a 100 mL three-necked flask, then add THF, and heat the solution; dropwise add 4-butyl bromobenzene in THF solution and continue stirring for 1 h, then ice bath to 0°C, dropwise add diethylphosphine in THF solution for 30 min; stir at room temperature for 2 h, quench the reaction with 2M HCl (20 mL), cool to 0°C and stir for 15 min, filter with diatomaceous earth, extract the filtrate with dichloromethane three times, and obtain compound D by column chromatography; 5) Add compound C and compound D to the K-containing 2 CO 3 Add palladium acetate and ferrocene to a dioxane solution, reflux at 100°C overnight, extract, and perform column chromatography to obtain compound E; 6) Compound E, dioxane and concentrated hydrochloric acid were added to a 25 mL single-necked bottle for reflux reaction, adjusted to neutral with NaOH solution, extracted with dichloromethane, and subjected to column chromatography to obtain ligand PO; 7) Add PO4 and NaOH to a single-mouth bottle, add DMSO and stir at room temperature until it becomes clear, reflux for 10 minutes, and drop EuCl 3 6H 2 O ethanol solution, react at 70℃ overnight, filter to obtain tridentate europium complex Eu[PO] 3 .

4. Use of the tridentate europium complex according to any one of claims 1 to 3, It is characterized in that The application is to manufacture fluorescent immunochromatographic test strips.

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