Polydentate ligand and complex thereof, and preparation method and application thereof

Abstract

The structural general formula of the rare earth complex is Ln (beta-diketon) 3 (Py-PO), Ln (beta-diketon) 3 (Bpy-PO), Ln (beta-diketon) 3 (Bpy-(PO) 2], Ln (beta-diketon) 3 (Phen-(PO) 2], beta-diketon is a beta-diketone auxiliary ligand, the structural general formula is Py-PO, Bpy-PO, Bpy-(PO) 2, Phen-PO and Phen-(PO) 2 as main ligands, and the structural formula of the europium complex is shown in the following formulas: the rare earth complex is subjected to multidentate chelating coordination, the complexing constant is large, the coordination ability is strong, and the rare earth complex can be used for preparing a rare earth complex According to the tridentate or tetradentate rare earth complex, the ligand is not easy to dissociate, and the solubility of the multidentate rare earth complex is well improved by simultaneously introducing phosphine oxide groups to two ends of a main ligand and modifying alkyl chains with different carbon atom numbers on an organic ligand in the tridentate or tetradentate rare earth complex.

Description

Technical Field

[0001] This invention relates to the field of luminescent materials technology, specifically to a rare earth complex and its preparation method, as well as its application in immunochromatography, fluorescent standard cards, and fluorescent additives for antifreeze. Background Technology

[0002] Rare earth complexes are complexes formed by coordination bonds between rare earth ions and organic compounds. They possess excellent optical, electrical, and magnetic properties, and have broad application prospects in many fields such as optics, biomedicine, sensors, and catalysis. As a photoluminescent material, due to the large molar absorptivity of organic ligands, the light absorption capacity of rare earth complexes can be increased by hundreds of times compared to pure inorganic rare earth materials, while significantly reducing the amount of rare earth required. Rare earth complex luminescent materials have unique advantages such as high luminous efficiency, long lifetime, good color purity, and large Stocks shift, and are widely used in information, display, military, medical, civilian lighting, nanoscience, agriculture, and other fields. Moreover, organic small-molecule rare earth complexes have better solubility than inorganic rare earth luminescent materials, making them easier to prepare into high-performance luminescent films or fluorescent microspheres and other functional luminescent materials. Therefore, rare earth organic complexes are a high-performance luminescent material that has attracted much attention and extensive research. Thus, the development of rare earth organic luminescent materials plays an important role in promoting the development of high technology and my country's national economy.

[0003] In recent years, thanks to the unremitting efforts of researchers, rare earth organic complexes have been put into practical application in some fields. At present, most rare earth complexes are still dominated by bidentate rare earth complexes. These complexes have disadvantages such as poor complexing ability, easy dissociation, easy affinity for oxygen, easy quenching of luminescence in water, low luminescence efficiency, and poor photostability and thermal stability, which greatly limit their application in complex environments.

[0004] Currently, Huang Chunhui's team at Peking University has designed a class of tridentate anionic organic ligands to form nine-coordinate chelates with rare earth europium ions (CN105017329A), avoiding the influence of neutral and auxiliary ligands on the photothermal stability of the complex. However, in practical applications, we have found that the increased anionic ligands in these rare earth complexes lead to enhanced ligand rigidity, resulting in poor solubility of the chelates after coordination with metallic europium, which affects their application in fields such as immunochromatography and bioimaging. Therefore, developing a rare earth complex with good photothermal stability and high solubility, transforming my country's rare earth resource advantages into a technological competitive advantage, is of significant practical importance for promoting my country's industrial transformation and upgrading, as well as enhancing its international competitiveness. Summary of the Invention

[0005] For tridentate or tetradentate rare earth complexes, the solubility is greatly reduced due to the introduction of rigid ligands. In order to solve the technical problem that the photothermal stability and solubility of existing rare earth complexes cannot be satisfied at the same time, this invention designs and synthesizes a multidentate rare earth complex. By introducing phosphine oxide groups on the main ligand and modifying the organic ligand with alkyl chains of different numbers of carbon atoms, the solubility of the rare earth complex is improved while ensuring its stability. It can be applied to immunochromatography, fluorescent standard cards, antifreeze fluorescent additives and other fields.

[0006] In a first aspect, the present invention provides a ligand with the following structure:

[0007]

[0008] in,

[0009] R1 is a C1-C18 alkyl, C6-C20 aryl, or C1-C15 heterocyclic group having N, O, or S.

[0010] R2, R3, R4, R5, R6, R7, and R8 are each independently hydrogen, halogen, hydroxyl, cyano, carboxyl, amino, C1-C18 alkyl, C1-C18 alkoxy, C6-C20 aryl, or C1-C15 heterocyclic groups having N, O, or S. Alternatively, any two adjacent groups of R2, R3, R4, R5, R6, R7, and R8 can participate in the formation of saturated or unsaturated aromatic rings and heterocycles containing heteroatoms.

[0011] Preferably, the heteroatom is N, O, or S.

[0012] Preferably, the aromatic ring or heterocyclic ring is a 5- to 10-membered aromatic monocyclic ring or an aromatic fused bicyclic ring.

[0013] Preferably, the H on the aromatic ring or heterocycle can be arbitrarily replaced by a halogen, carboxyl, C1-C18 alkyl, C6-C20 aryl, or C1-C15 heterocyclic group having N, O, or S.

[0014] Secondly, the present invention provides a rare earth complex with the general structural formula Ln(β-diketone)3(Py-PO), Ln(β-diketone)3(Bpy-PO), Ln(β-diketone)3[Bpy-(PO)2], Ln(β-diketone)3(Phen-PO) or Ln(β-diketone)3[Phen-(PO)2], wherein β-diketone is a β-diketone auxiliary ligand with the general structural formula […]. Py-PO is a monosubstituted phosphine oxide monopyridine main ligand, Bpy-PO is a monosubstituted phosphine oxide bipyridine main ligand, Bpy-(PO)2 is a disubstituted phosphine oxide bipyridine main ligand, Phen-PO is a monosubstituted phosphine oxide o-phenanthroline main ligand, and Phen-(PO)2 is a disubstituted phosphine oxide o-phenanthroline main ligand. The structural formulas of the rare earth complexes are shown below:

[0015]

[0016] in,

[0017] Ln is Eu, Tb, Sm, or Dy; preferably, Ln is Eu or Tb.

[0018] R1 is a C1-C18 alkyl, C6-C20 aryl, or C1-C15 heterocyclic group having N, O, or S.

[0019] R2, R3, R4, R5, R6, R7, and R8 are each independently hydrogen, halogen, hydroxyl, cyano, carboxyl, amino, C1-C18 alkyl, C1-C18 alkoxy, C6-C20 aryl, or C1-C15 heterocyclic groups having N, O, or S. Alternatively, R2, R3, R4, R5, R6, R7, and R8 can be paired to form saturated or unsaturated aromatic rings or heterocycles containing heteroatoms.

[0020] Preferably, the heteroatom is N, O, or S.

[0021] Preferably, the aromatic ring or heteroaromatic ring is a 5- to 10-membered aromatic monocyclic ring or an aromatic fused bicyclic ring.

[0022] Preferably, the H on the aromatic ring or aromatic heterocycle is substituted with a halogen, carboxyl, C1-C18 alkyl, C6-C20 aryl, or C1-C15 heterocyclic group having N, O, or S. R' and R” are each independently a halogen, C1-C18 alkyl, C1-C18 haloalkyl, C6-C20 aryl, or C1-C15 heterocyclic group having N, O, or S.

[0023] The aforementioned aryl group also includes substituted aryl groups, wherein the substituted aryl group is an aryl group substituted by at least one of halogen, carboxyl, C1-C18 alkyl, C1-C18 alkoxy, and C1-C18 haloalkyl.

[0024] In this invention, the alkyl group is a straight-chain hydrocarbon group or a branched hydrocarbon group, and the alkyl group includes methyl, ethyl, propyl, n-butyl and isobutyl; the aryl group includes phenyl, naphthyl, benzoic acid, acetophenone and aryl groups substituted with different alkyl chains from C1 to C18; the heterocyclic group includes thiophene, thiazole, furan and pyridine, etc.

[0025] Preferably, R1 is butyl, octyl, phenyl, butyryl, or octylphenyl.

[0026] Preferably, R2, R3, R4, R5, R6, R7, and R8 are each independently hydrogen, methyl, or carboxyl groups.

[0027] Preferably, any two adjacent groups among R2, R3, R4, R5, R6, R7, and R8 participate in the formation of a pyrazine ring, a quinoxaline ring, a 1,2,5-thiadiazole ring, or a 2H-imidazolium ring.

[0028] Preferably, R' and R" are each independently methyl, trifluoromethyl, phenyl, naphthyl, or thiophene.

[0029] Preferably, formula I is one of the following structural formulas:

[0030]

[0031] Preferably, formula II is one of the following structural formulas:

[0032]

[0033]

[0034] Preferably, formula III is one of the following structural formulas:

[0035]

[0036] Preferably, formula IV is one of the following structural formulas:

[0037]

[0038]

[0039] Preferably, the above formula V is one of the following structural formulas:

[0040]

[0041]

[0042] Preferably, the auxiliary ligand β-diketone has one of the following structural formulas:

[0043] Preferably, the multidentate rare earth complex is one of the following structural formulas:

[0044]

[0045]

[0046]

[0047]

[0048]

[0049] Thirdly, the present invention provides a method for preparing multidentate rare earth complexes.

[0050] According to one embodiment of the present invention, the preparation method of Formula VI multidentate rare earth complex includes the following steps:

[0051] (1) Mix Mg, I2 and THF, heat, add a THF solution of bromine-substituted compound dropwise, cool to 0℃, add a THF solution of diethyl phosphite dropwise, quench the reaction solution with HCl solution, filter with diatomaceous earth, extract the filtrate with dichloromethane, and obtain phosphine oxide derivative by column chromatography.

[0052] (2) Monosubstituted bromopyridine derivatives and phosphine oxide derivatives were added to a dioxane solution containing K3PO4, and then NiCl2 (dppp) was added and refluxed overnight. The reaction solution was extracted with dichloromethane, dried, and column chromatography was used to obtain compound Py-PO (formula VI).

[0053] (3) Mix β-diketone and rare earth chloride hexahydrate, add methanol to react, add methanol solution of compound Py-PO dropwise, continue the reaction, after a large amount of solid precipitates, filter to obtain complex Ln(β-diketone)3(Py-PO).

[0054] The chemical reaction formula for the preparation method of Formula VII multidentate rare earth complex is shown below:

[0055]

[0056] Preferably, the molar ratio of the above-mentioned monosubstituted bromopyridine derivative and phosphine oxide derivative is 1:1.

[0057] Preferably, the molar ratio of Py-PO, LnCl3·6H2O, and β-diketone is 1:1:3.

[0058] The definitions of R1, R2, R3, R4, R5, R', and R" mentioned above are the same as those in the previous text.

[0059] According to one embodiment of the present invention, the preparation method of Formula VII multidentate rare earth complex includes the following steps:

[0060] (1) Mix Mg, I2 and THF, heat, add a THF solution of bromine-substituted compound dropwise, cool to 0℃, add a THF solution of diethyl phosphite dropwise, quench the reaction solution with HCl solution, filter with diatomaceous earth, extract the filtrate with dichloromethane, and obtain phosphine oxide derivative by column chromatography.

[0061] (2) The monosubstituted brominated bipyridine derivative and the phosphine oxide derivative were added to a dioxane solution containing K3PO4, and then NiCl2 (dppp) was added and refluxed overnight. The reaction solution was extracted with dichloromethane, dried, and column chromatography was used to obtain compound Bpy-PO (formula VII).

[0062] (3) Mix β-diketone and rare earth chloride hexahydrate, add methanol to react, add methanol solution of compound Bpy-PO dropwise, continue the reaction, after a large amount of solid precipitates, filter to obtain complex Ln(β-diketone)3(Bpy-PO).

[0063] The chemical reaction formula for the preparation method of Formula VII multidentate rare earth complex is shown below:

[0064]

[0065] Preferably, the molar ratio of the above-mentioned monosubstituted brominated bipyridine derivative and phosphine oxide derivative is 1:1.

[0066] Preferably, the molar ratio of Bpy-PO, LnCl3·6H2O, and β-diketone is 1:1:3.

[0067] The definitions of R1, R2, R3, R4, R5, R6, R7, R8, R', and R" mentioned above are the same as those in the previous text.

[0068] According to one embodiment of the present invention, the preparation method of the formula VIII multidentate rare earth complex includes the following steps:

[0069] (1) Mix Mg, I2 and THF, heat, add a THF solution of bromine-substituted compound dropwise, cool to 0℃, add a THF solution of diethyl phosphite dropwise, quench the reaction solution with HCl solution, filter with diatomaceous earth, extract the filtrate with dichloromethane, and obtain phosphine oxide derivative by column chromatography.

[0070] (2) The disubstituted brominated bipyridine derivative and the phosphine oxide derivative were added to a dioxane solution containing K3PO4, and then NiCl2 (dppp) was added and refluxed overnight. The reaction solution was extracted with dichloromethane, dried, and column chromatography was used to obtain compound Bpy-(PO)2 (formula VIII).

[0071] (3) Mix β-diketone and rare earth chloride hexahydrate, add methanol to react, add methanol solution of compound Bpy-(PO)2 dropwise, continue the reaction, after a large amount of solid precipitates, filter to obtain complex Ln(β-diketone)3[Bpy-(PO)2].

[0072] The chemical reaction formula for the preparation method of Formula VIII multidentate rare earth complex is shown below:

[0073]

[0074] Preferably, the molar ratio of the above-mentioned disubstituted brominated bipyridine derivative and phosphine oxide derivative is 1:2.

[0075] Preferably, the molar ratio of Bpy-(PO)2, LnCl3·6H2O, and β-diketone is 1:1:3.

[0076] The definitions of R1, R2, R3, R4, R5, R6, R7, R8, R', and R" mentioned above are the same as those in the previous text.

[0077] According to one embodiment of the present invention, the preparation method of formula IX multidentate rare earth complex includes the following steps:

[0078] (1) Mix Mg, I2 and THF, heat, add a THF solution of bromine-substituted compound dropwise, cool to 0℃, add a THF solution of diethyl phosphite dropwise, quench the reaction solution with HCl solution, filter with diatomaceous earth, extract the filtrate with dichloromethane, and obtain phosphine oxide derivative by column chromatography.

[0079] (2) The monosubstituted bromophenanthroline derivative and the phosphine oxide derivative were added to a dioxane solution containing K3PO4, and then NiCl2 (dppp) was added and refluxed overnight. The reaction solution was extracted with dichloromethane, dried, and column chromatography was used to obtain the compound Phen-PO (formula IX).

[0080] (3) Mix β-diketone and rare earth chloride hexahydrate, add methanol to react, add methanol solution of compound Phen-PO dropwise, continue the reaction, after a large amount of solid precipitates, filter to obtain complex Ln(β-diketone)3(Phen-PO).

[0081] The chemical reaction formula for the preparation method of Formula IX multidentate rare earth complex is shown below:

[0082]

[0083] Preferably, the molar ratio of the above-mentioned monosubstituted bromophenanthroline derivative and phosphine oxide derivative is 1:1.

[0084] Preferably, the molar ratio of Phen-PO, LnCl3·6H2O, and β-diketone is 1:1:3.

[0085] The definitions of R1, R2, R3, R4, R5, R6, R7, R8, R', and R" mentioned above are the same as those in the previous text.

[0086] According to one embodiment of the present invention, the preparation method of the multidentate rare earth complex of formula X includes the following steps:

[0087] (1) Mix Mg, I2 and THF, heat, add a THF solution of bromine-substituted compound dropwise, cool to 0℃, add a THF solution of diethyl phosphite dropwise, quench the reaction solution with HCl solution, filter with diatomaceous earth, extract the filtrate with dichloromethane, and obtain phosphine oxide derivative by column chromatography.

[0088] (2) The disubstituted bromophenanthroline derivative and the phosphine oxide derivative were added to a dioxane solution containing K3PO4, and then NiCl2 (dppp) was added and refluxed overnight. The reaction solution was extracted with dichloromethane, dried, and column chromatography was used to obtain the compound Phen-(PO)2 (formula X).

[0089] (3) Mix β-diketone and rare earth chloride hexahydrate, add methanol to react, add methanol solution of compound Phen-(PO)2 dropwise, continue the reaction, after a large amount of solid precipitates, filter to obtain complex Ln(β-diketone)3[Phen-(PO)2].

[0090] The chemical reaction formula for the preparation method of Formula X multidentate rare earth complex is shown below:

[0091]

[0092] Preferably, the molar ratio of the above-mentioned monosubstituted bromophenanthroline derivative and phosphine oxide derivative is 1:2.

[0093] Preferably, the molar ratio of Phen-(PO)2, LnCl3·6H2O, and β-diketone is 1:1:3.

[0094] The definitions of R1, R2, R3, R4, R5, R6, R7, R8, R', and R" mentioned above are the same as those in the previous text.

[0095] Fourthly, the present invention provides applications of multidentate rare earth complexes.

[0096] The multidentate rare earth complexes provided by this invention are used in the fields of fluorescence immunochromatography, bioimaging, or luminescent thin films.

[0097] The multidentate rare earth complexes provided by this invention are used as fluorescent markers for fluorescence immunochromatography.

[0098] The aforementioned fluorescence immunochromatography includes the detection of serum amyloid A (SAA).

[0099] The multidentate rare earth complex provided by this invention is used to prepare fluorescent standard cards.

[0100] The multidentate rare earth complex provided by this invention is used as a fluorescent additive in antifreeze.

[0101] Compared with existing technologies, the technical solution of the present invention has the following advantages:

[0102] (1) The rare earth complex of the present invention has a multidentate chelate coordination, a large complexation constant, strong coordination ability, and the ligands themselves are not easy to dissociate. Furthermore, the tridentate or tetradentate rare earth complex of the present invention improves the solubility of the multidentate rare earth complex by introducing phosphine oxide groups at both ends of the main ligand and modifying the organic ligand with alkyl chains of different carbon atoms.

[0103] (2) The tetradentate rare earth ions of the present invention are coordinated and saturated, without the quenching effect of other solvent molecules, resulting in high luminescence quantum efficiency and excellent photothermal stability.

[0104] (3) The rare earth complex of the present invention does not use dangerous chemicals such as metallic sodium in the preparation process. The preparation process is safe, simple to operate, and has a high yield.

[0105] (4) The rare earth complex of the present invention has high luminescence quantum efficiency and can be used as a high-efficiency luminescent material for application in the field of fluorescence immunochromatography. The rare earth complex is coated in polystyrene microspheres and the surface of the microspheres is modified. Finally, the antibody is coupled to the surface of the microspheres to synthesize fluorescent immunochromatographic microspheres for the detection of SAA. The detection sensitivity is high and the stability is good, realizing efficient and rapid in vitro monitoring.

[0106] (5) The rare earth complex of the present invention has a large excitation wavelength (≥365nm), which expands its application range, extends the excitation wavelength and maintains the high luminescence quantum efficiency of the complex. Attached Figure Description

[0107] Figure 1 The image shows the 1H NMR spectrum of the ligand Phen-PO-4.

[0108] Figure 2 The image shows the 1H NMR spectrum of the ligand Phen-PO-5.

[0109] Figure 3 The 1H NMR spectrum of the ligand Phen-(PO)2-3.

[0110] Figure 4 The 1H NMR spectrum of the ligand Phen-(PO)2-5.

[0111] Figure 5 The UV-Vis absorption spectrum of the ligand Phen-PO-5 is shown.

[0112] Figure 6 The UV-Vis absorption spectrum of the ligand Phen-(PO)2-5 is shown.

[0113] Figure 7 Infrared spectra of the complexes Eu(TTA)3(Phen), Eu(TTA)3(Phen-PO)-5, and Eu(TTA)3[Phen-(PO)2]-5.

[0114] Figure 8 The absorption and emission spectrum of the complex Eu(TTA)3(Phen PO)-5 is shown.

[0115] Figure 9 The absorption and emission spectrum of the complex Eu(TTA)3[Phen(PO)2]-5 is shown.

[0116] Figure 10 To assess the solid-phase photostability of the complexes Eu(TTA)3(Phen), Eu(TTA)3(Phen-PO-5), and Eu(TTA)3[Phen(PO)2]-5.

[0117] Figure 11 The liquid phase photostability of the complexes Eu(TTA)3(Phen), Eu(TTA)3(Phen-PO-5), and Eu(TTA)3[Phen-(PO)2]-5 was determined.

[0118] Figure 12 This is a synthetic route diagram for immunochromatographic microspheres.

[0119] Figure 13 This is the standard curve for immunochromatographic detection.

[0120] Figure 14 This is a comparison of the photostability of PS-Eu(TTA)3[Phen-(PO)2]-5 fluorescent microspheres and commercial fluorescent microspheres. Detailed Implementation

[0121] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and the accompanying drawings. However, those skilled in the art will understand that this invention is not limited to the accompanying drawings and the following embodiments.

[0122] Example 1: Synthesis of Tb(PPA)3(Py-PO)-1

[0123] The synthesis route in this embodiment is as follows:

[0124]

[0125] The specific synthesis steps are as follows:

[0126] (1) Mg (11 mmol, 3.3 eq) and I2 (0.022 mmol) were added to a 100 mL three-necked flask, followed by THF (15 mL). The solution was heated to 60 °C, and 10 mL of a THF solution containing 10 mmol, 3 eq of bromobutane was added dropwise. The mixture was stirred for 1 h, then placed in an ice bath to 0 °C. Over 30 min, a THF solution containing 3.3 mmol, 1.0 eq of diethyl phosphite was added dropwise, and the mixture was stirred at room temperature for 2 h. The reaction solution was quenched with 2 M HCl (10 mL) at 0 °C, stirred for 15 min, filtered through diatomaceous earth, and the filtrate was extracted three times with dichloromethane. Compound A was obtained by column chromatography with a yield of 85%.

[0127] (2) 6-Bromopyridinecarboxylic acid (1.158 mmol, 1 eq) and compound A (1.158 mmol, 1 eq) were added to 10 mL of dioxane solution containing K3PO4 (2.31 mmol, 2 eq), and then NiCl2 (dppp) (0.1158 mmol, 0.1 eq) was added and refluxed overnight. The reaction solution was extracted with dichloromethane, dried, and subjected to column chromatography to obtain compound Byp-PO-1 in 84% yield.

[0128] (3) PPA (0.267 mmol, 3 eq) and terbium trichloride hexahydrate (0.089 mmol, 1 eq) were added to a single-necked flask, followed by methanol (5 mL). The reaction was carried out at 70 °C. A methanol solution of compound Py-PO-1 (0.089 mmol, 1 eq) (5 mL) was added dropwise, and the reaction was continued. After half an hour, a large amount of solid precipitated out. The reaction solution was filtered to obtain the complex Tb(FFA)3(Py-PO)-1, with a yield of 92%.

[0129] Example 2: Synthesis of Eu(TTA)3(Byp-PO)-1

[0130] The synthesis route in this embodiment is as follows:

[0131]

[0132] The specific synthesis steps are as follows:

[0133] (1) Mg (11 mmol, 3.3 eq) and I2 (0.022 mmol) were added to a 100 mL three-necked flask, followed by THF (15 mL). The solution was heated to 60 °C, and 10 mL of a THF solution containing 10 mmol, 3 eq of bromobutane was added dropwise. The mixture was stirred for 1 h, then placed in an ice bath to 0 °C. Over 30 min, a THF solution containing 3.3 mmol, 1.0 eq of diethyl phosphite was added dropwise, and the mixture was stirred at room temperature for 2 h. The reaction solution was quenched with 2 M HCl (10 mL) at 0 °C, stirred for 15 min, filtered through diatomaceous earth, and the filtrate was extracted three times with dichloromethane. Compound A was obtained by column chromatography with a yield of 85%.

[0134] (2) 6-Bromo-2,2'-bipyridine (1.158 mmol, 1 eq) and compound A (1.158 mmol, 1 eq) were added to 10 mL of dioxane solution containing K3PO4 (2.31 mmol, 2 eq), and then NiCl2 (dppp) (0.1158 mmol, 0.1 eq) was added and refluxed overnight. The reaction solution was extracted with dichloromethane, dried, and subjected to column chromatography to obtain compound Byp-PO-1 in 80% yield.

[0135] (3) TTA (0.267 mmol, 3 eq) and europium trichloride hexahydrate (0.089 mmol, 1 eq) were added to a single-necked flask, followed by methanol (5 mL). The reaction was carried out at 70 °C. A methanol solution of compound Byp-PO-1 (0.089 mmol, 1 eq) (5 mL) was added dropwise, and the reaction was continued. After half an hour, a large amount of solid precipitated out. The reaction solution was filtered to obtain the complex Eu(TTA)3(Byp-PO)-1, with a yield of 95%.

[0136] Example 3: Synthesis of Eu(TTA)3(Byp-PO)-2

[0137] The synthesis route in this embodiment is as follows:

[0138]

[0139] The synthesis steps were the same as in Example 2, except that the bromobutane used in the first step was replaced with bromooctane, which yielded the target compound Eu(TTA)3(Byp-PO)-2. The yield of the third step was 85%.

[0140] Example 4: Synthesis of Eu(TTA)3(Byp-PO)-6

[0141] The synthesis route in this embodiment is as follows:

[0142]

[0143] The synthesis steps were the same as in Example 2, except that the bromobutane used in the first step was replaced with 1-bromo-4-octylbenzene, and the 6-bromo-2,2'-bipyridine used in the second step was replaced with 6-bromo-4,4'-dimethyl-2,2'-bipyridine, thus yielding the target compound Eu(TTA)3(Byp-PO)-6. The yield of the third step was 95%.

[0144] Example 5: Synthesis of Eu(TTA)3(Byp-PO)-7

[0145] The synthesis route in this embodiment is as follows:

[0146]

[0147] The synthesis steps were the same as in Example 2, except that the bromobutane used in the first step was replaced with 1-bromo-4-octylbenzene, and the 6-bromo-2,2'-bipyridine used in the second step was replaced with 6'-bromo-[2,2'-bipyridine]-3-carboxylic acid, thus yielding the target compound Eu(TTA)3(Byp-PO)-7. The yield of the third step was 95%.

[0148] Example 6: Synthesis of Eu(TTA)3[Byp-(PO)2]-3

[0149] The synthesis route in this embodiment is as follows:

[0150]

[0151] The specific synthesis steps are as follows:

[0152] (1) Mg (11 mmol, 3.3 eq) and I2 (0.022 mmol) were added to a 100 mL three-necked flask, followed by 15 mL of THF. The solution was heated to 60 °C, and 10 mL of a THF solution containing 10 mmol, 3 eq of bromobenzene was added dropwise. The mixture was stirred for 1 h, then cooled to 0 °C in an ice bath. Over 30 min, a THF solution containing 3.3 mmol, 1.0 eq of diethyl phosphite was added dropwise, and the mixture was stirred at room temperature for 2 h. The reaction solution was quenched with 10 mL of 2 M HCl at 0 °C, stirred for 15 min, filtered through diatomaceous earth, and the filtrate was extracted three times with dichloromethane. Compound D was obtained by column chromatography with a yield of 85%.

[0153] (2) 6,6'-dibromo-2,2'-bipyridine (1.158 mmol, 1 eq) and compound D (2.316 mmol, 2 eq) were added to 10 mL of dioxane solution containing K3PO4 (2.31 mmol, 2 eq), and then NiCl2 (dppp) (0.1158 mmol, 0.1 eq) was added and refluxed overnight. The reaction solution was extracted with dichloromethane, dried, and subjected to column chromatography to obtain compound Byp-(PO)2-3 in 78% yield.

[0154] (3) TTA (0.267 mmol, 3 eq) and europium trichloride hexahydrate (0.089 mmol, 1 eq) were added to a single-necked flask, followed by methanol (5 mL). The reaction was carried out at 70 °C. A methanol solution (5 mL) of compound Byp-(PO)2-3 (0.089 mmol, 1 eq) was added dropwise, and the reaction was continued. After half an hour, a large amount of solid precipitated out. The reaction solution was filtered to obtain the complex Eu(TTA)3[Byp-(PO)2]-3 with a yield of 95%.

[0155] Example 7: Synthesis of Eu(TTA)3[Byp-(PO)2]-4

[0156] The synthesis route in this embodiment is as follows:

[0157]

[0158] The synthesis steps were the same as in Example 6, except that the bromobenzene used in the first step was replaced with 1-bromo-4-butylbenzene, which yielded the target compound Eu(TTA)3[Byp-(PO)2]-4. The yield of the third step reaction was 75%.

[0159] Example 8: Synthesis of Eu(TTA)3[Byp-(PO)2]-5

[0160] The synthesis route in this embodiment is as follows:

[0161]

[0162] The synthesis steps were the same as in Example 6, except that the bromobenzene used in the first step was replaced with 1-bromo-4-octylbenzene, which yielded the target compound Eu(TTA)3[Byp-(PO)2]-5. The yield of the third step reaction was 75%.

[0163] Example 9: Synthesis of Eu(TTA)3(Phen-PO)-3

[0164] The synthesis route in this embodiment is as follows:

[0165]

[0166] The specific synthesis steps are as follows:

[0167] (1) Mg (11 mmol, 3.3 eq) and I2 (0.022 mmol) were added to a 100 mL three-necked flask, followed by 15 mL of THF. The solution was heated to 60 °C, and 10 mL of a THF solution containing 10 mmol, 3 eq of bromobenzene was added dropwise. The mixture was stirred for 1 h, then cooled to 0 °C in an ice bath. Over 30 min, a THF solution containing 3.3 mmol, 1.0 eq of diethyl phosphite was added dropwise, and the mixture was stirred at room temperature for 2 h. The reaction solution was quenched with 10 mL of 2 M HCl at 0 °C, stirred for 15 min, filtered through diatomaceous earth, and the filtrate was extracted three times with dichloromethane. Compound D was obtained by column chromatography with a yield of 85%.

[0168] (2) 2-Bromo-1,10-phenanthroline (1.158 mmol, 1 eq) and compound D (1.158 mmol, 1 eq) were added to 10 mL of dioxane solution containing K3PO4 (2.31 mmol, 2 eq), and then NiCl2 (dppp) (0.1158 mmol, 0.1 eq) was added and refluxed overnight. The reaction solution was extracted with dichloromethane, dried, and subjected to column chromatography to obtain compound Phen-PO-3 in 80% yield.

[0169] (3) TTA (0.267 mmol, 3 eq) and europium trichloride hexahydrate (0.089 mmol, 1 eq) were added to a single-necked flask, followed by methanol (5 mL). The reaction was carried out at 70 °C. A methanol solution of compound Phen-PO-3 (0.089 mmol, 1 eq) (5 mL) was added dropwise, and the reaction was continued. After half an hour, a large amount of solid precipitated out. The reaction solution was filtered to obtain the complex Eu(TTA)3(Phen-PO)-3, with a yield of 85%.

[0170] Example 10: Synthesis of Eu(TTA)3(Phen-PO)-4

[0171] The synthesis route in this embodiment is as follows:

[0172]

[0173] The synthesis steps were the same as in Example 9, except that the bromobenzene used in the first step was replaced with 1-bromo-4-butylbenzene, thus obtaining the target compound Eu(TTA)3(Phen-PO)-4. The yield of the third step reaction was 95%.

[0174] Example 11: Synthesis of Eu(TTA)3(Phen-PO)-5

[0175] The synthesis route in this embodiment is as follows:

[0176]

[0177] The synthesis steps were the same as in Example 9, except that the bromobenzene used in the first step was replaced with 1-bromo-4-octylbenzene, thus yielding the target compound Eu(TTA)3(Phen-PO)-5. The yield of the third step reaction was 95%.

[0178] Example 12: Synthesis of Eu(NTA)3(Phen-PO)

[0179] The synthesis route in this embodiment is as follows:

[0180]

[0181] The synthesis steps are the same as in Example 9, except that the TTA used in the third step of the reaction is replaced with NTA, thus obtaining the target compound.

[0182] The product Eu(NTA)3(Phen-PO) was reacted in the third step with a yield of 95%.

[0183] Example 13: Synthesis of Eu(DBM)3(Phen-PO)

[0184] The synthesis route in this embodiment is as follows:

[0185]

[0186] The synthesis steps are the same as in Example 9, except that the TTA used in the third step of the reaction is replaced with DBM, thus obtaining the target compound.

[0187] The product Eu(DBM)3(Phen-PO) was reacted in the third step with a yield of 95%.

[0188] Example 14: Synthesis of Eu(DBM)3(Phen-PO)

[0189] The synthesis route in this embodiment is as follows:

[0190]

[0191] The synthesis steps were the same as in Example 9, except that the TTA used in the third step was replaced with ACAC, thus obtaining the target compound Eu(ACAC)3(Phen-PO). The yield of the third step reaction was 95%.

[0192] Example 15: Synthesis of Eu(TTA)3[Phen-(PO)2]-3

[0193] The synthesis route in this embodiment is as follows:

[0194]

[0195] The specific synthesis steps are as follows:

[0196] (1) Mg (11 mmol, 3.3 eq) and I2 (0.022 mmol) were added to a 100 mL three-necked flask, followed by 15 mL of THF. The solution was heated to 60 °C, and 10 mL of a THF solution containing 10 mmol, 3 eq of bromobenzene was added dropwise. The mixture was stirred for 1 h, then cooled to 0 °C in an ice bath. A THF solution containing 3.3 mmol, 1.0 eq of diethyl phosphite was added dropwise over 30 min, and the mixture was stirred at room temperature for 2 h. The reaction solution was quenched with 10 mL of 2 M HCl at 0 °C, stirred for 15 min, filtered through diatomaceous earth, and the filtrate was extracted three times with dichloromethane. Compound D was obtained by column chromatography with a yield of 85%.

[0197] (2) 2,9-Dibromo-1,10-phenanthroline (1.158 mmol, 1 eq) and compound D (2.316 mmol, 2 eq) were added to 10 mL of dioxane solution containing K3PO4 (2.31 mmol, 2 eq), and then NiCl2 (dppp) (0.1158 mmol, 0.1 eq) was added and refluxed overnight. The reaction solution was extracted with dichloromethane, dried, and subjected to column chromatography to obtain compound Phen-(PO)2-3 in 80% yield.

[0198] (3) TTA (0.267 mmol, 3 eq) and europium trichloride hexahydrate (0.089 mmol, 1 eq) were added to a single-necked flask, followed by methanol (5 mL). The reaction was carried out at 70 °C. A methanol solution (5 mL) of compound Phen-(PO)2-3 (0.089 mmol, 1 eq) was added dropwise, and the reaction was continued. After half an hour, a large amount of solid precipitated out. The reaction solution was filtered to obtain the complex Eu(TTA)3[Phen-(PO)2]-3 with a yield of 95%.

[0199] Example 16: Synthesis of Eu(TTA)3[Phen-(PO)2]-4

[0200] The synthesis route in this embodiment is as follows:

[0201]

[0202] The synthesis steps were the same as in Example 15, except that the bromobenzene used in the first step was replaced with 1-bromo-4-butylbenzene, which yielded the target compound Eu(TTA)3(Phen-PO)-4. The yield of the third step was 95%.

[0203] Example 17: Synthesis of Eu(TTA)3[Phen-(PO)2]-5

[0204] The synthesis route in this embodiment is as follows:

[0205]

[0206] The synthesis steps are the same as in Example 15, except that the bromobenzene used in the first step reaction is replaced with 1-bromo-4-octylbenzene, thus obtaining...

[0207] The target compound Eu(TTA)3(Phen-PO)-5 was obtained with a 95% yield in the third step reaction.

[0208] Example 18: NMR spectrum of polydentate ligands

[0209] The NMR spectra of the tridentate ligands Phen-PO-4 and Phen-PO-5 are as follows: Figure 1 and Figure 2 As shown, the NMR spectra of tetradentate Phen-(PO)₂⁻⁃ and Phen-(PO)₂⁻⁵ are characterized as follows: Figure 3 and Figure 4 As shown in the attached figures, the structures of each ligand are confirmed.

[0210] Example 19: Absorption spectrum of multidentate ligands

[0211] The absorption spectrum characterization of the tridentate ligand Phen-PO-5 is as follows: Figure 5 As shown, the absorption spectrum characterization of the tetradentate ligand Phen-(PO)2-5 is as follows: Figure 6 As shown, by appendix Figure 5 The results confirmed the structure of the tridentate ligand Phen-PO-5, which consists of attached... Figure 6 The results confirmed the structure of the tetradentate ligand Phen-(PO)2-5.

[0212] Example 20: Infrared spectrum of multidentate complex

[0213] In this embodiment, the infrared spectra of Eu(TTA)3(Phen-PO)-5 and Eu(TTA)3[Phen-(PO)2]-3 were tested, and Eu(TTA)3Phen (a traditional bidentate complex) was used as a comparison. Eu(TTA)3Phen is a reported complex, with the full name: (1,10-phenanthroline)tris[4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione]eup(III), and its structure is as follows:

[0214]

[0215] like Figure 7 As shown in the attached figures, the structures of each complex are confirmed.

[0216] Example 21: Absorption-emission peaks of multidentate complexes

[0217] The absorption and emission spectrum of the tridentate complex Eu(TTA)3(PhenPO)-5 is as follows: Figure 8As shown, the absorption and emission peaks of the tetradentate complex Eu(TTA)3[Phen(PO)2]-5 are as follows Figure 9 As shown, the excitation wavelength of this europium complex can extend to about 380 nm, while the emission peak is 612 nm. The longer excitation wavelength and larger Stocks shift are beneficial for applications in fields such as immunoassay and bioimaging.

[0218] Example 22: Solid-phase light stability of multidentate complexes

[0219] This embodiment tested the solid-phase photostability of Eu(TTA)3(Phen-PO)-5 and Eu(TTA)3[Phen-(PO)2]-5, with Eu(TTA)3Phen as a comparison.

[0220] Eu(TTA)3(Phen-PO)-5, Eu(TTA)3[Phen-(PO)2]-5, and Eu(TTA)3Phen were fixed at different positions on the same NC membrane to prepare detection cards. These cards were scanned 10 times consecutively using a dry time-resolved fluorescence analyzer to record the fluorescence decay of the solid phases of the three Eu(III) complexes. Figure 10 As shown, under the same illumination, the modified tridentate and tetradentate europium complexes exhibit significantly improved photostability compared to commercially available bidentate europium complexes. Specifically, after 10 consecutive irradiations with the same ultraviolet light, the fluorescence intensity of Eu(TTA)3[Phen-(PO)2]-5 decreased by only 5%, Eu(TTA)3(Phen-PO)-5 decreased by 10%, while Eu(TTA)3Phen decreased by nearly 40%. This indicates that the modified tridentate and tetradentate europium complexes have better photostability, and the higher the coordination number, the better the stability. This suggests that increasing the coordination number between the ligand and the central ion is beneficial for improving the stability of the complex.

[0221] Example 23: Liquid phase photostability of multidentate complexes

[0222] This embodiment tested the liquid phase photostability of Eu(TTA)3(Phen-PO)-5 and Eu(TTA)3[Phen-(PO)2]-5, with Eu(TTA)3Phen as a comparison.

[0223] Three dichloromethane solutions of Eu(III) complexes were prepared at a concentration of 0.025 mM. Two solutions were irradiated at a distance of 20 cm for 5 min using an 18 W 365 nm UV lamp, and the fluorescence decay of the three Eu(III) complex dichloromethane solutions was recorded. Figure 11As shown, after irradiation with 18W UV light for 5 minutes, the fluorescence intensity of Eu(TTA)3[Phen-(PO)2]-5 decreased by 25%, the fluorescence intensity of Eu(TTA)3(Phen-PO)-5 decreased by 55%, while the fluorescence intensity of Eu(TTA)3Phen decreased by nearly 100%. This indicates that the modified tridentate and tetradentate europium complexes have better photostability in solution, and the higher the coordination number, the better the stability.

[0224] Example 24: Solubility of multidentate europium complexes

[0225] The solubility of Eu(TTA)3(Phen-PO)-5 and Eu(TTA)3[Phen-(PO)2]-5 complexes was tested using toluene, dichloromethane, acetone, and tetrahydrofuran solvents, and compared with the europium complex Eu5 disclosed in invention patent CN105017329A (obtained by the preparation method in Example 5 of CN105017329A).

[0226] Table 1 Solubility of multidentate europium complexes

[0227]

[0228]

[0229] As shown in Table 1, the solubility of the multidentate europium complex proposed in this invention is greatly improved after structural modification, while the solubility of Eu5 without modification of the long carbon chain is relatively low.

[0230] Example 25: Preparation of fluorescent microspheres by coating microspheres with multidentate europium complexes

[0231] Polystyrene microspheres (1.185 mL), deionized water (2.82 mL), and acetone (5 mL) were added to a reaction flask and stirred at room temperature for 20 min. Then, an acetone solution of Eu(TTA)3[Phen-(PO)2]-5 tetradentate europium complex (1 mL, 20 mg / mL) was added and the mixture was stirred at room temperature for another 5 h. After the reaction, the mixture was centrifuged (12000 r / min, 10 min) and washed three times with deionized water / ethanol (v:v = 9:1). Finally, 10 mL of deionized water was added for storage, with a solid content of 1%.

[0232] Example 26: Preparation of amino fluorescent microspheres

[0233] Take 20 mL of the fluorescent microspheres obtained in Example 25 (10 mg / mL), centrifuge (12000 rpm, 10 min), remove the supernatant, add 20 mL of NaOH aqueous solution (pH = 11), and sonicate (10 min). Weigh 30 mg of mercaptoethylamine and add it to the above microsphere solution at once. After shaking, react at room temperature for 24 h. After the reaction, centrifuge (8000 rpm, 10 min), add water / ethanol (v:v = 9:1), centrifuge and wash three times repeatedly, add 20 mL of borate buffer (BBS, pH = 7.5) for storage, with a solid content of 1%.

[0234] Example 27: Preparation of aldehyde-based fluorescent microspheres

[0235] Take 15 mL of 2-(N-morpholino)ethanesulfonic acid buffer (MES, pH=6.0) (10 mg / mL) containing amino fluorescent microspheres obtained in Example 25, add aldehyde dextran (500,000, 1.25 mmol / g, 100 mg), react for 20 min, then add sodium cyanoborohydride (100 μL, 50 mg / mL), continue the reaction overnight, centrifuge (7000 r / min, 5 min), wash three times with deionized water, add 15 mL of BBS buffer solution (pH=7.5) for storage, with a solid content of 1%. The obtained microspheres are named Eu(TTA)3[Phen-(PO)2-5]-4NP, which are fluorescent microspheres with aldehyde groups on the surface.

[0236] Example 28: Preparation of Immunospheres

[0237] Eu(TTA)3[Phen-(PO)2]-5-4NP aldehyde fluorescent microspheres obtained in Example 27 were added to BBS solution (3 mL, pH = 7.5) in a solution of 625 μL (10 mg / mL). Chicken IgY (5 mg / mL, 200 μL) was added to the microspheres and reacted for 20 min. Sodium cyanoborohydride (20 μL, 50 mg / mL) was then added and reacted at room temperature for 6 h. The mixture was then centrifuged (7000 r / min, 5 min), the supernatant was removed, 4 mL of BBS buffer solution (pH = 7.5) was added, and the mixture was sonicated for 5 min. The mixture was then blocked with BSA (300 μL, 10%) for 1 h, centrifuged, and 5 mL of BBS was added, resulting in a solid content of 0.25%.

[0238] Example 29: Preparation of Immunochromatographic Test Strips

[0239] The immunochromatographic test strip consists of four parts: a PVC plastic base plate, an NC membrane, a sample pad, and absorbent paper. The preparation method is as follows:

[0240] (1) Attach the NC film to the PVC plastic base plate.

[0241] (2) Prepare SAA antibody 5F2 protein with a 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 goat anti-chicken IgY protein with a concentration of 1 mg / mL and draw it on the NC membrane at a speed of 1 μL / cm as the C line.

[0242] (3) Eu(TTA)3[Phen-(PO)2]-5-4NP-3H8 immunoglobulins and Eu(TTA)3[Phen-(PO)2]-5-4NP-Chicken IgY were prepared with gold spray diluent at concentrations of 25 μg / mL and 5 μg / mL, respectively. The above diluents were sprayed onto the sample plate using an XYZ three-dimensional gold spray spectrometer, with 5F2 and 3H8 immunoglobulins being paired.

[0243] (4) Place the NC membrane with T / C lines and the sample plate sprayed with immunoglobulins in a constant temperature incubator at 37°C overnight.

[0244] (5) Attach the sample pad and absorbent paper to the fixed position of the NC membrane respectively.

[0245] (6) Use a microcomputer automatic cutter to cut the test strip to a width of 0.38cm, and install it in a plastic slot. Store it in a dry, room-temperature environment for later use.

[0246] Example 30: SAA Project Testing

[0247] Using normal human serum as the sample diluent, SAA standards were 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 and 98.75 μL of each concentration of standard were taken, mixed thoroughly, and added to the sample dispensing port. After reacting for 11 min, the fluorescence intensity of the T and C lines was detected and recorded, and Ig was calculated. T / I C The value was measured and repeated three times. The concentration of the SAA standard was plotted against the corresponding I value. T / I C The curve of the value (results as follows) Figure 13 (As shown).

[0248] The multidentate europium complex prepared in this invention exhibits good stability and solubility. Its application in fluorescence immunochromatography yielded a highly sensitive fluorescence immunoassay strip for detecting SAA (Self-Acting Antibiotic Acid) markers. Figure 13 It is known that its detection limit is as low as 5 μg / mL, which is lower than the clinical value of 10 μg / mL, and the detection concentration is as high as 220 μg / mL.

[0249] Table 2. Analytical evaluation results of the immunochromatographic platform for detecting SAA.

[0250]

[0251] Example 31: Photostability of Fluorescent Microspheres

[0252] use Figure 12 The method for preparing fluorescent microspheres involves using a tetradentate europium complex of Eu(TTA)3[Phen-(PO)2]-5 to prepare PS-Eu(TTA)3[Phen-(PO)2]-5 microspheres, and using Eu(TTA)3Phen as a comparative example to prepare another control fluorescent microsphere, named PS-Eu(TTA)3Phen microspheres.

[0253] Eu(TTA)3[Phen-(PO)2]-5 was coated onto blank epoxy microspheres, and then aldehyde-dextran fluorescent microspheres were prepared by surface amylation and chemical coupling with aldehyde dextran. Finally, SAA antibody was coupled to the aldehyde groups on the surface of the microspheres to prepare immunofluorescent microspheres for immunochromatographic detection. PS-Eu(TTA)3[Phen-(PO)2]-5 microspheres and commercial fluorescent microspheres were prepared to a concentration of 0.25 mg / mL and placed in a transparent glass bottle. Both solutions were irradiated with an 18W 365nm UV lamp at 20cm for 5 min, and the fluorescence decay of the two microspheres was recorded. Figure 14 As shown, the fluorescence intensity of PS-Eu(TTA)3[Phen-(PO)2]-5 microspheres hardly decreased after 1 min of irradiation, and decreased by 2% after 5 min of irradiation, which was not significant. However, the fluorescence intensity of PS-Eu(TTA)3Phen microspheres decreased by nearly 70% after 1 min, which was more significant. This indicates that the present invention has significant advantages in constructing high-performance rare earth luminescent complexes, especially in improving photostability. The improvement of photostability is of great significance for many practical applications. For example, it can increase the stability of signal acquisition in immunoassay, thereby reducing errors caused by signal acquisition and improving the accuracy of detection.

[0254] Example 32: Preparation of Fluorescent Standard Cards

[0255] Preparation of single-line standard cards (NC membrane): Aqueous solutions of fluorescent microspheres with concentrations of 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, and 2 mg / mL were prepared. Using a membrane drawing instrument (XYZ-300), the microsphere aqueous solution was drawn onto the NC membrane on a plastic substrate at a speed of 1 μL / cm. The substrate was then incubated overnight at 37°C. Sample pads and absorbent pads were attached to the top and bottom ends of the plastic substrate. The NC membrane was cut into 0.38 mm wide test strips using a microcomputer-controlled automatic cutter (ZQ2002), and the strips were installed in their corresponding card cases and stored in a dry, room-temperature environment.

[0256] Preparation of the dual-line standard card (NC membrane): Prepare aqueous solutions with fluorescent microsphere concentrations of 0.1 mg / mL and 0.5 mg / mL. Using a membrane scribing instrument (XYZ-300), the above-mentioned 0.1 mg / mL and 0.5 mg / mL microsphere aqueous solutions were scribed onto the NC membrane on a plastic substrate at a speed of 1 μL / cm, respectively, to serve as the C-line and T-line. The substrate was then incubated overnight at 37°C. The sample pad and absorbent pad were attached to the top and bottom ends of the plastic substrate. The NC membrane was cut into 0.38 mm wide test strips using a microcomputer-controlled automatic cutter (ZQ2002), and the strips were installed in the matching card holders and stored in a dry environment at room temperature.

[0257] Preparation of fluorescent standard cards (PDMS film): Weigh 0.2g of rare earth complex and place it in a 3mL glass bottle. Add 0.1g of polydimethylsiloxane and 0.5mL of chloroform to the glass bottle. Stir the mixture in the glass bottle at 50℃ for at least two hours to ensure uniform mixing, thus obtaining the first suspension. Add 0.1g of dimethylsiloxane curing agent to the first suspension and stir the mixture at room temperature to ensure uniform mixing, thus obtaining the second suspension. Pour the prepared second suspension into a container. After static curing, a fluorescent film is formed on the bottom inner wall of the container. Fluorescent standard cards with different linewidths or cross-sectional areas are prepared according to different needs.

[0258] Preparation of fluorescent standard cards (PE film): Weigh 15g of rare earth complex, first impregnate it with a mixed solvent consisting of 20g of anhydrous ethanol and 25g of white oil, then add it together with 20g of PEG 600 to 920g of low-density polyethylene granules and mix evenly. Heat and melt the mixture, blow it into a film at a blow ratio of 2.0, and prepare fluorescent standard cards with different line widths or cross-sectional areas according to different needs.

[0259] Fluorescent standard cards were prepared using Eu(TTA)3[Phen-(PO)2]-5 tetradentate europium complex. The resulting fluorescent standard cards have a uniform distribution of fluorescent material, can be stably stored for a long time, and have a coefficient of variation (CV) ≤ 0.5%, which can ensure the consistency and accuracy of the test signal of the standard cards.

[0260] Example 33: Preparation of Antifreeze

[0261] The antifreeze is formulated as follows: 20-30 parts of 1,3-propanediol, 5-10 parts of isopropyl acetate, 5-5 parts of emulsifier OP-102, 1-2 parts of rare earth complex, and 70-80 parts of water.

[0262] The rare-earth complex of this invention, when added to antifreeze as a fluorescent additive, exhibits excellent oil solubility and can be added to antifreeze in any proportion. Furthermore, this complex has high fluorescence quantum efficiency, requiring only a small addition ratio as a fluorescent agent, significantly reducing costs. The rare-earth complex of this invention emits 615nm red light, providing a clearer signal and effectively indicating the antifreeze's usage status to the user.

Claims

1. A ligand, characterized in that, The ligand structure is shown in the following formula: in, R1 is a C1-C18 alkyl, C6-C20 aryl, or C1-C15 heterocyclic group having N, O, or S. R2, R3, R4, R5, R6, R7, and R8 are each independently hydrogen, halogen, hydroxyl, cyano, carboxyl, amino, C1-C18 alkyl, C1-C18 alkoxy, C6-C20 aryl, or C1-C15 heterocyclic groups having N, O, or S, or R2, R3, R4, R5, R6, R7, and R8 in pairs of adjacent groups participate in the formation of saturated or unsaturated aromatic rings and heterocycles containing heteroatoms; Preferably, the heteroatom is N, O, or S; Preferably, the aromatic ring or heteroaromatic ring is a 5- to 10-membered aromatic monocyclic ring or an aromatic fused bicyclic ring; Preferably, the H on the aromatic ring or aromatic heterocycle is arbitrarily substituted with a halogen, carboxyl, C1-C18 alkyl, C6-C20 aryl, or C1-C15 heterocyclic group having N, O, or S.

2. A rare earth complex, characterized in that, The rare earth complexes have the general structural formulas Ln(β-diketone)3(Py-PO), Ln(β-diketone)3(Bpy-PO), Ln(β-diketone)3[Bpy-(PO)2], Ln(β-diketone)3(Phen-PO), or Ln(β-diketone)3[Phen-(PO)2], wherein β-diketone is a β-diketone auxiliary ligand with the general structural formula […]. With Py-PO, Bpy-PO, Bpy-(PO)2, Phen-PO, and Phen-(PO)2 as the main ligands, the structural formulas of the rare earth complexes are shown below: The structural formulas of the main ligands Py-PO, Bpy-PO, Bpy-(PO)2, Phen-PO, and Phen-(PO)2 are shown in the following equations: in, Ln is Eu, Tb, Sm or Dy; preferably, Ln is Eu or Tb; R1 is a C1-C18 alkyl, C6-C20 aryl, or C1-C15 heterocyclic group having N, O, or S. R2, R3, R4, R5, R6, R7, and R8 are each independently hydrogen, halogen, hydroxyl, cyano, carboxyl, amino, C1-C18 alkyl, C1-C18 alkoxy, C6-C20 aryl, or C1-C15 heterocyclic groups having N, O, or S, or R2, R3, R4, R5, R6, R7, and R8 in pairs of adjacent groups participate in the formation of saturated or unsaturated aromatic rings and heterocycles containing heteroatoms; Preferably, the heteroatom is N, O, or S; Preferably, the aromatic ring or heteroaromatic ring is a 5- to 10-membered aromatic monocyclic ring or an aromatic fused bicyclic ring; Preferably, the H on the aromatic ring or aromatic heterocycle is arbitrarily replaced by a halogen, carboxyl, C1-C18 alkyl, C6-C20 aryl, or C1-C15 heterocyclic group having N, O, or S. R' and R" are each independently a halogen, a C1-C18 alkyl, a C1-C18 haloalkyl, a C6-C20 aryl, or a C1-C15 heterocyclic group having N, O, or S.

3. The rare earth complex according to claim 2, characterized in that, The aryl group further includes substituted aryl groups, wherein the substituted aryl group is an aryl group substituted by at least one selected from halogen, carboxyl, C1-C18 alkyl, C1-C18 alkoxy, and C1-C18 haloalkyl. Preferably, the alkyl group is a straight-chain hydrocarbon group or a branched hydrocarbon group; more preferably, the alkyl group includes methyl, ethyl, propyl, n-butyl and isobutyl; the aryl group includes phenyl, naphthyl, benzoic acid, acetophenone and aryl groups substituted with different alkyl chains from C1 to C18; the heterocyclic group includes thiophene, thiazole, furan and pyridine. Preferably, R1 is butyl, octyl, phenyl, butyryl, or octylphenyl; Preferably, R2, R3, R4, R5, R6, R7, and R8 are each independently hydrogen, methyl, or carboxyl groups; Preferably, two adjacent groups of R2, R3, R4, R5, R6, R7, and R8 participate in the formation of a pyrazine ring, a quinoxaline ring, a 1,2,5-thiadiazole ring, or a 2H-imidazolium ring; Preferably, R' and R'' are each independently methyl, trifluoromethyl, phenyl, naphthyl, or thiophene; Preferably, Formula I is one of the following structural formulas: Preferably, Formula II is one of the following structural formulas: Preferably, Formula III is one of the following structural formulas: Preferably, formula IV is one of the following structural formulas: Preferably, formula V is one of the following structural formulas: Preferably, the auxiliary ligand β-diketone has one of the following structural formulas:

4. The rare earth complex according to claim 2, characterized in that, Rare earth complexes are one of the following structural formulas:

5. The method for preparing rare earth complexes according to claim 2, characterized in that, The preparation method includes the following steps: (1) Mix Mg, I2 and THF, heat, add THF solution of bromine-substituted compound dropwise, cool to 0℃, add THF solution of diethyl phosphite dropwise, quench the reaction solution with HCl solution, filter with diatomaceous earth, extract the filtrate with dichloromethane, and obtain phosphine oxide derivative by column chromatography. (2) The brominated derivative and the phosphine oxide derivative were added to a dioxane solution containing K3PO4, and then NiCl2 (dppp) was added and refluxed overnight. The reaction solution was extracted with dichloromethane, dried, and the phosphine oxide derivative main ligand was obtained by column chromatography. (3) Mix β-diketone auxiliary ligand and rare earth chloride hexahydrate, add methanol to react, add methanol solution of the main ligand of the phosphine oxide derivative of the compound dropwise, continue the reaction, after a large amount of solid precipitates, filter to obtain the target complex. Preferably, the molar ratio of the above-mentioned phosphine oxide derivative main ligand, LnCl3·6H2O, and β-diketone is 1:1:

3.

6. The method for preparing rare earth complexes according to claim 5, characterized in that, The brominated derivative is a monobrominated derivative or a dibrominated derivative; The monobrominated derivative is a monobrominated pyridine derivative, a monobrominated bipyridine derivative, or a monobrominated o-phenanthroline derivative. The dibromo derivative is a dibromobipyridine derivative or a dibromo-o-phenanthroline derivative; Preferably, the molar ratio of the monobrominated derivative to the phosphine oxide derivative is 1:1; Preferably, the molar ratio of the dibromo derivative to the phosphine oxide derivative is 1:

2.

7. The application of the rare earth complex according to any one of claims 2-4, characterized in that, The rare earth complexes are used in the fields of fluorescence immunochromatography, bioimaging, or luminescent thin films.

8. The application of the rare earth complex according to any one of claims 2-4, characterized in that, The rare earth complex was used as a fluorescent marker in fluorescence immunochromatography. Preferably, the fluorescence immunochromatography is used for the detection of serum amyloid A (SAA).

9. A fluorescent standard card, characterized in that, The fluorescent standard card comprises the rare earth complex according to any one of claims 2-4.

10. An antifreeze, characterized in that, The rare earth complex according to any one of claims 2-4 is used as a fluorescent additive in antifreeze.

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