Rare earth fluorescent complex and time-resolved fluorescent microsphere, and preparation method and application thereof

Abstract

The application discloses a rare earth fluorescent complex, relates to the field of luminescent materials, and discloses a rare earth fluorescent complex, a structural formula of which is shown as formula I. The rare earth fluorescent complex has a luminescent center atom bonded with two symmetrical tetradentate ligands, and the overall structure of the complex is butterfly-shaped. Unlike existing beta-diketone ligands, one of the O coordination atoms in the ligand of the application is connected with N in pyridine, thereby optimizing energy transfer, matching the triplet state energy level of the ligand with the excited state energy level of the center atom, and having a higher antenna effect under excitation light irradiation, comprehensively improving the fluorescent performance and fluorescent stability of the rare earth fluorescent complex. The preparation method of the rare earth fluorescent complex can efficiently and industrially synthesize the above rare earth fluorescent complex. The time-resolved fluorescent microspheres have high fluorescent intensity and strong structural stability, and the fluorescent intensity does not obviously decrease under 3-5 times of excitation light irradiation.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic materials, and in particular relates to rare earth fluorescent complexes and time-resolved fluorescent microspheres, their preparation methods and applications. Background Technology

[0002] Time-resolved fluorescence immunoassay (TRFI) is one of the three major ultrasensitive immunoassay methods. Its principle involves using rare-earth complexes with long fluorescence half-lives as fluorescent labels. Due to the different luminescence mechanisms of rare-earth complexes, their fluorescence lifetime is 2-3 orders of magnitude longer than that of background fluorescent substances (typically over 100 microseconds, more than 100 times). Therefore, in fluorescence signal detection, only long-lived fluorescence is measured, not short-lived fluorescence. That is, after the sample is excited by the light source, by extending the light information acquisition time and allowing the background fluorescence to decay before collecting the fluorescence signal, interference from non-specific fluorescence can be effectively eliminated, resulting in relatively higher detection sensitivity.

[0003] Time-resolved fluorescent microspheres are formed by filling polymer nanoparticles with rare-earth ion complexes. They are a special type of functional microsphere; each nanoscale polystyrene microsphere can encapsulate tens of thousands of rare-earth fluorescent molecules, effectively enhancing the fluorescence intensity of the labeling carrier and thus meeting the requirements for determining ultra-low concentrations of biological components.

[0004] Currently available time-resolved fluorescent microspheres have two main problems: First, the overall fluorescence intensity of the microspheres is not high, which cannot effectively improve the sensitivity of the detection products; second, the fluorescence stability of the rare earth complexes is insufficient: after being irradiated with excitation light, the overall fluorescence intensity of the microspheres will decay rapidly, which is very detrimental to the retesting and result verification of related detection products. Therefore, focusing on how to improve the luminescence efficiency and fluorescence stability of ff transition rare earth complexes in time-resolved microspheres is of great research significance.

[0005] The luminescent properties and stability of rare earth complexes are determined by their ligand structures. Taking europium complexes as an example, early studies on the fluorescence properties of europium complexes mainly focused on the formation of strongly fluorescent complexes with europium ions using bidentate ligands (β-diketones). Typical examples include: NTA (β-naphthoyltrifluoroacetone), TTA (2-thiophenecarboxyltrifluoroacetone), DNM (dinaphthoylmethane), and DBM (dibenzoylmethane).

[0006]

[0007] Patents CN108445219A, CN103011492A, CN111218270A, CN112745833A, and CN115466277A all fall into this category. NTA and TTA ligands are widely used due to their high light absorption efficiency, but these ligands still have shortcomings in fluorescence intensity and stability.

[0008] To address this issue, many novel ligand structures have been developed. However, due to the limitations of dentate ligands, finding a suitable ligand structure that can improve fluorescence performance, enhance structural stability, and efficiently encapsulate ligands remains a challenge. Currently, a relatively effective method is to repeat existing bidentate ligands to form multidentate ligands, as shown below:

[0009]

[0010] The rare-earth complexes of tetradentate ligands show some improvement in fluorescence stability, but no significant improvement in fluorescence intensity. Meanwhile, patent CN112521262A mentions a multidentate β-diketone ligand and its luminescent rare-earth complexes, the multidentate β-diketone ligand being as follows:

[0011]

[0012]

[0013] The aforementioned polydentate ligands can provide two or more atoms capable of donating lone pairs of electrons, forming low-coordination-ratio, cyclic chelates when coordinated with metals, thus reducing the adverse effects of the external environment on the luminescence of rare-earth complexes. Although these polydentate ligands exhibit extremely strong chelating ability and significantly improve fluorescence stability, the reduced matching degree between their triplet energy level and the excited state energy level of the central europium ion results in no significant improvement in fluorescence intensity. The time-resolved microspheres prepared using this strategy cannot simultaneously achieve both fluorescence intensity and stability, and therefore cannot meet the requirements for retesting in relevant detection products. Summary of the Invention

[0014] Based on this, the purpose of the present invention is to provide a rare earth fluorescent complex and its application in the preparation of time-resolved fluorescent microspheres. The time-resolved fluorescent microspheres prepared from the rare earth fluorescent complex have excellent fluorescence intensity and fluorescence stability.

[0015] A first aspect of the present invention is to provide a rare-earth fluorescent complex having the structural formula shown in Formula I.

[0016]

[0017] Wherein, R1 and R2 are independently selected from: H, halogen, nitro, cyano, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, amino, N-substituted amino, C1-C6 alkoxy, carboxyl, ester, amide, N-substituted amide, C6-C 10 Aryl or 5-10 membered heterocyclic aryl;

[0018] R3 is selected from: H or methoxy group;

[0019] R5 is selected from: O, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, amino, N-substituted amino, C1-C6 alkoxy, carboxyl, ester, amide, N-substituted amide, C6-C 10 Aryl or 5-10 membered heterocyclic aryl;

[0020] A is selected from: europium, samarium, dysprosium, terbium, lanthanum, lutetium, yttrium, or gadolinium.

[0021] In some embodiments, when A is selected from europium, its structural formula is shown in Formula II.

[0022]

[0023] R1 and R2 are independently selected from: H, halogen, nitro, cyano, C1-C3 alkyl, C1-C3 haloalkyl, C2-C4 alkenyl, C2-C4 alkynyl, amino, N-substituted amino, C1-C3 alkoxy, carboxyl, ester, amide, N-substituted amide, C6-C8 aryl or 5-8 membered heterocyclic aryl;

[0024] R3 is selected from: H or methoxy group;

[0025] R5 is selected from: O, C1-C3 alkyl, C1-C3 haloalkyl, C2-C4 alkenyl, C2-C4 alkynyl, amino, N-substituted amino, C1-C3 alkoxy, carboxyl, ester, amide, N-substituted amide, C6-C8 aryl or 5-8 membered heterocyclic aryl.

[0026] In some of these embodiments, R3 is selected from H.

[0027] In some embodiments, R1 and R2 are both selected from H, and R5 is methylene or O.

[0028] In some embodiments, the structural formula is shown in Formula III.

[0029]

[0030] A second aspect of the present invention is to provide a method for preparing rare earth fluorescent complexes, comprising the following steps:

[0031] 1) Using carboxylic acid pyridine compounds as reaction substrates, a condensation reaction is carried out with the addition of 2-mercaptothiazoline to obtain a mercaptothiazoline pyridine intermediate. The synthetic route is shown in Formula i.

[0032]

[0033] 2) A condensation reaction is carried out by adding an alkane or alkoxyl with a terminal amino group to the aforementioned mercaptothiazoline pyridine intermediate to obtain the HOPO intermediate. The synthetic route is shown in Formula ii.

[0034]

[0035] 3) The HOPO intermediate is subjected to an acidification reaction to remove the hydroxyl protecting group R4, yielding the HOPO ligand. The synthetic route is shown in Formula iii.

[0036]

[0037] 4) Under inert gas protection, the HOPO ligand and rare earth compound are dissolved in an organic solvent to carry out a coordination reaction. After filtration and washing, the rare earth fluorescent complex is obtained; its synthetic route is shown in Formula iv.

[0038]

[0039] Wherein, R1 and R2 are independently selected from: H, halogen, nitro, cyano, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, amino, N-substituted amino, C1-C6 alkoxy, carboxyl, ester, amide, N-substituted amide, C6-C 10 Aryl or 5-10 membered heterocyclic aryl;

[0040] R3 is selected from: H or methoxy group;

[0041] R4 is selected from: benzyloxy, p-methoxybenzyloxy, trimethylsiloxy, tert-butyldimethylsiloxy, triisopropylsiloxy;

[0042] R5 is selected from: O, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, amino, N-substituted amino, C1-C6 alkoxy, carboxyl, ester, amide, N-substituted amide, C6-C 10 Aryl or 5-10 membered heterocyclic aryl.

[0043] In some embodiments, R1 and R2 are selected from: H, halogen, nitro, cyano, C1-C3 alkyl, C1-C3 haloalkyl, C2-C4 alkenyl, C2-C4 alkynyl, amino, N-substituted amino, C1-C3 alkoxy, carboxyl, ester, amide, N-substituted amide, C6-C8 aryl or 5-8 membered heterocyclic aryl, wherein R1 is preferably H or C1-C3 alkoxy, and R2 is preferably H;

[0044] R3 is selected from H or methoxy, with H being the preferred choice;

[0045] R4 is selected from: benzyloxy, p-methoxybenzyloxy, trimethylsiloxy, tert-butyldimethylsiloxy, triisopropylsiloxy, preferably benzyloxy;

[0046] R5 is selected from: O, C1-C3 alkyl, C1-C3 haloalkyl, C2-C4 alkenyl, C2-C4 alkynyl, amino, N-substituted amino, C1-C3 alkoxy, carboxyl, ester, amide, N-substituted amide, C6-C8 aryl or 5-8 membered heterocyclic aryl, preferably O or methylene.

[0047] In some embodiments, the following steps are included:

[0048] 1) Under inert gas protection, 1-(benzyloxy)-6-oxo-1,6-dihydropyridine-2-carboxylic acid and 2-mercaptothiazoline in a molar ratio of (15-25):(17-27) were dissolved in a first solvent, and N,N'-dicyclohexylcarbodiimide and trace amounts of 4-dimethylaminopyridine were added. The molar ratio of 2-mercaptothiazoline to N,N'-dicyclohexylcarbodiimide was 1:(0.8-1.2). The reaction was stirred at room temperature, filtered, and the first solvent was evaporated to obtain the first precursor.

[0049] 2) Under inert gas protection, the first precursor and 2,2-oxodiethylamine in a molar ratio of (1.9-2.2):1 were dissolved in a second solvent, and the mixture was stirred at room temperature. The second solvent was evaporated and separated by chromatography to obtain the second precursor.

[0050] 3) Dissolve the second precursor in a hydrochloric acid-glacial acetic acid composite solution, stir and react at room temperature, evaporate to remove the solvent, wash and dry to obtain the HOPO ligand;

[0051] 4) Under inert gas protection, the HOPO ligand is dissolved in a third solvent, the pH is adjusted to 6.5-7.5, and europium chloride aqueous solution is added to carry out the reaction. The molar ratio of europium chloride to HOPO ligand is 1:(1.8-2.2). The mixture is filtered, washed, and dried to obtain rare earth fluorescent complex.

[0052] A third aspect of the present invention is to provide the application of rare earth fluorescent complexes in the preparation of time-resolved fluorescent microspheres.

[0053] A fourth aspect of the present invention is to provide a time-resolved fluorescent microsphere comprising a carrier microsphere and a rare earth fluorescent complex according to any one of claims 1-5 embedded within the carrier microsphere, wherein the mass of the rare earth fluorescent complex is 6% or more of the mass of the carrier microsphere.

[0054] In some embodiments, the mass of the rare earth fluorescent complex is more than 10% of the mass of the carrier microspheres, more preferably 10% to 20%.

[0055] In some embodiments, the mass of the rare earth fluorescent complex is 6% to 20% of the mass of the carrier microspheres, preferably 8% to 16%, and more preferably 10% to 16%.

[0056] In some embodiments, the carrier microspheres are selected from any one of polystyrene microspheres, hydrogel microspheres, and polymethyl methacrylate microspheres;

[0057] Furthermore, the surface of the carrier microspheres is modified with any one of carboxyl groups, interarm carboxyl groups, amino groups, thiol groups, and streptavidin.

[0058] Preferably, the carrier microspheres are polystyrene microspheres with carboxyl groups modified on their surface.

[0059] A fifth aspect of the present invention provides a method for preparing time-resolved fluorescent microspheres, comprising the following steps:

[0060] 1) Mix the carrier microspheres and surfactant evenly in a solvent to form a dispersion system of carrier microspheres;

[0061] 2) Dissolve the rare earth fluorescent complex in a swelling agent, then add it to the dispersion system of the carrier microspheres, carry out the swelling reaction under homogeneous stirring, remove the swelling agent by distillation, wash, and ultrasonically disperse to obtain time-resolved fluorescent microspheres;

[0062] The swelling agent includes any one or more of dichloromethane, trichloromethane, dichloroethane, tetrachloroethane, tetrahydrofuran, N,N-dimethylformamide, and acetone; the volume concentration of the swelling agent is 5% to 20%.

[0063] In some embodiments, the swelling agent is dichloromethane; the volume concentration of the swelling agent is 5% to 15%, preferably 8% to 10%.

[0064] In some embodiments, the surfactant includes one or more of sodium dodecyl sulfonate, sodium dodecyl sulfate, and sodium dodecylbenzene sulfonate; the mass concentration of the surfactant is 0.1% to 1%.

[0065] Preferably, the surfactant is sodium dodecyl sulfonate, and the mass concentration of the surfactant is 0.1% to 0.5%, more preferably 0.2% to 0.25%.

[0066] In some embodiments, the swelling reaction takes 0.5 to 8 hours, preferably 0.5 to 2 hours, and more preferably 0.5 to 1 hour.

[0067] In some embodiments, in step 1), the carrier microspheres are carboxylated polystyrene microspheres, and the preparation method includes the following steps:

[0068] Styrene and carboxylated monomers were dissolved in an aqueous solution under inert gas protection, stirred and heated to reflux, and then potassium persulfate was added to initiate the polymerization reaction. After the reaction was complete, the mixture was washed and ultrasonically dispersed to obtain the carboxylated polystyrene microspheres.

[0069] The carboxylating monomer includes any one or more of maleic anhydride, methacrylic acid, methacrylates, acrylic acid and acrylates; the molar ratio of styrene, carboxylating monomer and potassium persulfate is (0.2-0.4):(0.01-0.02):(0.5-1).

[0070] To overcome the problem that existing time-resolved fluorescent microspheres with multidentate β-diketone ligands cannot simultaneously achieve a comprehensive improvement in both fluorescence intensity and ligand stability, thus failing to meet the requirements for multiple excitation detection or product retesting.

[0071] This invention proposes a rare-earth fluorescent complex in which the luminescent central atom is bonded to two symmetrical tetradentate ligands, resulting in a butterfly-shaped overall structure. Unlike existing β-diketone ligands, one of the O coordinating atoms in the ligands of this invention is connected to the N atom in pyridine, with a ketone as a coordinating atom at the adjacent position. This optimizes energy transfer, allowing the triplet energy level of the ligand to match the excited state energy level of the central atom. Furthermore, under excitation light irradiation, it exhibits a more efficient antenna effect, comprehensively improving the fluorescence quantum yield of the rare-earth fluorescent complex. Simultaneously, this complex exhibits good solubility in organic solvents and moderate aromaticity, avoiding aggregation-induced quenching that would lead to a decrease in fluorescence intensity.

[0072] The present invention provides a method for preparing rare earth fluorescent complexes, which can efficiently prepare the above-mentioned rare earth fluorescent complexes and realize the industrial production of rare earth fluorescent complexes.

[0073] The present invention relates to an application of a rare earth fluorescent complex, which has both high fluorescence performance and high fluorescence stability, and is easily soluble in organic solvents, enabling it to be efficiently embedded in a carrier to form time-resolved microspheres.

[0074] The present invention provides a time-resolved fluorescent microsphere, which optimizes the proportion of the rare earth fluorescent complex in the carrier microsphere, improves fluorescence intensity, enhances fluorescence stability, and shows no significant decline in fluorescence intensity under repeated excitation light irradiation.

[0075] The present invention discloses a method for preparing time-resolved fluorescent microspheres, wherein rare earth europium complexes are filled into the internal cavity of polystyrene microspheres through swelling and embedding, thereby giving the time-resolved microspheres high fluorescence intensity and stability. Attached Figure Description

[0076] Figure 1 This is a scanning electron microscope image of the time-resolved fluorescent microspheres in Example 1 of the present invention.

[0077] Figure 2 This is a scanning electron microscope image of the time-resolved fluorescent microspheres in Comparative Example 1 of this invention.

[0078] Figure 3 This is a comparison diagram of the fluorescence intensity of multiple resolving fluorescent microspheres of the present invention.

[0079] Figure 4 This is a comparison of the fluorescence intensity of the fluorescent microspheres of Example 1 of the present invention and domestic competitors under excitation light of 365nm and 340nm.

[0080] Figure 5 This is a comparison of the fluorescence intensity of the resolving fluorescent microspheres of Example 1 and Comparative Example 1 after 5 exposures to 365nm excitation light.

[0081] Figure 6 This is a fluorescence decay trend diagram of the resolving fluorescent microspheres of Example 1 and Comparative Example 1 of the present invention after 5 exposures to 365nm excitation light. Detailed Implementation

[0082] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments are merely illustrative of the present invention and should not be construed as limiting the invention.

[0083] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0084] As used herein, the term "alkyl" refers to both branched and straight-chain saturated aliphatic hydrocarbon groups having a specific number of carbon atoms. For example, the definition of "C1-C6" in "C1-C6 alkyl" includes groups having 1, 2, 3, 4, 5, or 6 carbon atoms arranged in a straight or branched chain. Specifically, "C1-C6 alkyl" includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl.

[0085] The term "cycloalkyl" refers to a monocyclic saturated aliphatic hydrocarbon group having a specific number of carbon atoms. For example, "cycloalkyl" includes cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0086] The term "alkoxy" refers to a group in which an alkyl group is directly attached to an oxygen group, that is, a group with an -O-alkyl structure, such as -OCH3, -OCH2CH3, -OCH2CH2CH3, -O-CH2CH(CH3)2, -OCH2CH2CH2CH3, -O-CH(CH3)2, etc.

[0087] The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic substituent in which one or more ring atoms are selected from heteroatoms of N, O, or S(O)m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon, such as morpholino, piperidinyl, tetrahydropyrrolithyl, pyrrolithyl, dihydroimidazolyl, etc., and their N-oxides. The connection of heterocyclic substituents can be achieved by carbon atoms or by heteroatoms.

[0088] The term "substituted" refers to the replacement of a hydrogen group in a specific structure with a specified substituent.

[0089] As will be understood by those skilled in the art, the term “halogen” as used herein refers to chlorine, fluorine, bromine, and iodine.

[0090] A rare-earth fluorescent complex, the structural formula of which is shown in Formula I.

[0091]

[0092] Wherein, R1 and R2 are independently selected from: H, halogen, nitro, cyano, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, amino, N-substituted amino, C1-C6 alkoxy, carboxyl, ester, amide, N-substituted amide, C6-C 10 Aryl or 5-10 membered heterocyclic aryl;

[0093] R3 is selected from: H or methoxy group;

[0094] R5 is selected from: O, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, amino, N-substituted amino, C1-C6 alkoxy, carboxyl, ester, amide, N-substituted amide, C6-C 10 Aryl or 5-10 membered heterocyclic aryl;

[0095] A is selected from: europium, samarium, dysprosium, terbium, lanthanum, lutetium, yttrium, or gadolinium.

[0096] In some embodiments, when A is selected from europium, its structural formula is as shown in Formula II.

[0097]

[0098] R1 and R2 are independently selected from: H, halogen, nitro, cyano, C1-C3 alkyl, C1-C3 haloalkyl, C2-C4 alkenyl, C2-C4 alkynyl, amino, N-substituted amino, C1-C3 alkoxy, carboxyl, ester, amide, N-substituted amide, C6-C8 aryl or 5-8 membered heterocyclic aryl;

[0099] R3 is selected from: H or methoxy group;

[0100] R5 is selected from: O, C1-C3 alkyl, C1-C3 haloalkyl, C2-C4 alkenyl, C2-C4 alkynyl, amino, N-substituted amino, C1-C3 alkoxy, carboxyl, ester, amide, N-substituted amide, C6-C8 aryl or 5-8 membered heterocyclic aryl.

[0101] In some of these embodiments, R3 is selected from H.

[0102] In some embodiments, R1 and R2 are both selected from H, and R5 is methylene or O.

[0103] In some embodiments, the structural formula is shown in Formula III.

[0104]

[0105] A method for preparing rare earth fluorescent complexes includes the following steps:

[0106] 1) Using carboxylic acid pyridine compounds as reaction substrates, a condensation reaction is carried out with the addition of 2-mercaptothiazoline to obtain a mercaptothiazoline pyridine intermediate. The synthetic route is shown in Formula i.

[0107]

[0108] 2) A condensation reaction is carried out by adding an alkane or alkoxyl with a terminal amino group to the aforementioned mercaptothiazoline pyridine intermediate to obtain the HOPO intermediate. The synthetic route is shown in Formula ii.

[0109]

[0110] 3) The HOPO intermediate is subjected to an acidification reaction to remove the hydroxyl protecting group R4, yielding the HOPO ligand. The synthetic route is shown in Formula iii.

[0111]

[0112] 4) Under inert gas protection, the HOPO ligand and rare earth compound are dissolved in an organic solvent to carry out a coordination reaction. After filtration and washing, the rare earth fluorescent complex is obtained; its synthetic route is shown in Formula iv.

[0113]

[0114] Wherein, R1 and R2 are independently selected from: H, halogen, nitro, cyano, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, amino, N-substituted amino, C1-C6 alkoxy, carboxyl, ester, amide, N-substituted amide, C6-C 10 Aryl or 5-10 membered heterocyclic aryl;

[0115] R3 is selected from: H or methoxy group;

[0116] R4 is selected from: benzyloxy, p-methoxybenzyloxy, trimethylsiloxy, tert-butyldimethylsiloxy, triisopropylsiloxy;

[0117] R5 is selected from: O, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, amino, N-substituted amino, C1-C6 alkoxy, carboxyl, ester, amide, N-substituted amide, C6-C 10 Aryl or 5-10 membered heterocyclic aryl.

[0118] In some embodiments, R1 and R2 are selected from: H, halogen, nitro, cyano, C1-C3 alkyl, C1-C3 haloalkyl, C2-C4 alkenyl, C2-C4 alkynyl, amino, N-substituted amino, C1-C3 alkoxy, carboxyl, ester, amide, N-substituted amide, C6-C8 aryl or 5-8 membered heterocyclic aryl, wherein R1 is preferably H or C1-C3 alkoxy, and R2 is preferably H;

[0119] R3 is selected from H or methoxy, with H being the preferred choice;

[0120] R4 is selected from: benzyloxy, p-methoxybenzyloxy, trimethylsiloxy, tert-butyldimethylsiloxy, triisopropylsiloxy, preferably benzyloxy;

[0121] R5 is selected from: O, C1-C3 alkyl, C1-C3 haloalkyl, C2-C4 alkenyl, C2-C4 alkynyl, amino, N-substituted amino, C1-C3 alkoxy, carboxyl, ester, amide, N-substituted amide, C6-C8 aryl or 5-8 membered heterocyclic aryl, preferably O or methylene.

[0122] In some embodiments, the following steps are included:

[0123] 1) Under inert gas protection, 1-(benzyloxy)-6-oxo-1,6-dihydropyridine-2-carboxylic acid and 2-mercaptothiazoline in a molar ratio of (15-25):(17-27) were dissolved in a first solvent, and N,N'-dicyclohexylcarbodiimide and trace amounts of 4-dimethylaminopyridine were added. The molar ratio of 2-mercaptothiazoline to N,N'-dicyclohexylcarbodiimide was 1:(0.8-1.2). The reaction was stirred at room temperature, filtered, and the first solvent was evaporated to obtain the first precursor.

[0124] 2) Under inert gas protection, the first precursor and 2,2-oxodiethylamine in a molar ratio of (1.9-2.2):1 were dissolved in a second solvent, and the mixture was stirred at room temperature. The second solvent was evaporated and separated by chromatography to obtain the second precursor.

[0125] 3) Dissolve the second precursor in a hydrochloric acid-glacial acetic acid composite solution, stir and react at room temperature, evaporate to remove the solvent, wash and dry to obtain the HOPO ligand;

[0126] 4) Under inert gas protection, the HOPO ligand is dissolved in a third solvent, the pH is adjusted to 6.5-7.5, and europium chloride aqueous solution is added to carry out the reaction. The molar ratio of europium chloride to HOPO ligand is 1:(1.8-2.2). The mixture is filtered, washed, and dried to obtain rare earth fluorescent complex.

[0127] Application of a rare earth fluorescent complex in the preparation of time-resolved fluorescent microspheres.

[0128] A time-resolved fluorescent microsphere comprises a carrier microsphere and a rare earth fluorescent complex according to any one of claims 1-5 embedded within the carrier microsphere, wherein the mass of the rare earth fluorescent complex is more than 6% of the mass of the carrier microsphere.

[0129] In some embodiments, the mass of the rare earth fluorescent complex is more than 10% of the mass of the carrier microspheres, more preferably 10% to 20%.

[0130] In some embodiments, the mass of the rare earth fluorescent complex is 6% to 20% of the mass of the carrier microspheres, preferably 8% to 16%, and more preferably 10% to 16%.

[0131] In some embodiments, the carrier microspheres are selected from any one of polystyrene microspheres, hydrogel microspheres, and polymethyl methacrylate microspheres;

[0132] Furthermore, the surface of the carrier microspheres is modified with any one of carboxyl groups, interarm carboxyl groups, amino groups, thiol groups, and streptavidin.

[0133] Preferably, the carrier microspheres are polystyrene microspheres with carboxyl groups modified on their surface.

[0134] A method for preparing time-resolved fluorescent microspheres includes the following steps:

[0135] 1) Mix the carrier microspheres and surfactant evenly in a solvent to form a dispersion system of carrier microspheres;

[0136] 2) Dissolve the rare earth fluorescent complex in a swelling agent, then add it to the dispersion system of the carrier microspheres, carry out the swelling reaction under homogeneous stirring, remove the swelling agent by distillation, wash, and ultrasonically disperse to obtain time-resolved fluorescent microspheres;

[0137] The swelling agent includes any one or more of dichloromethane, trichloromethane, dichloroethane, tetrachloroethane, tetrahydrofuran, N,N-dimethylformamide, and acetone; the volume concentration of the swelling agent is 5% to 20%.

[0138] In some embodiments, the swelling agent is dichloromethane; the volume concentration of the swelling agent is 5% to 15%, preferably 8% to 10%.

[0139] In some embodiments, the surfactant includes one or more of sodium dodecyl sulfonate, sodium dodecyl sulfate, and sodium dodecylbenzene sulfonate; the mass concentration of the surfactant is 0.1% to 1%.

[0140] Preferably, the surfactant is sodium dodecyl sulfonate, and the mass concentration of the surfactant is 0.1% to 0.5%, more preferably 0.2% to 0.25%.

[0141] In some embodiments, the swelling reaction takes 0.5 to 8 hours, preferably 0.5 to 2 hours, and more preferably 0.5 to 1 hour.

[0142] In some embodiments, in step 1), the carrier microspheres are carboxylated polystyrene microspheres, and the preparation method includes the following steps:

[0143] Styrene and carboxylated monomers were dissolved in an aqueous solution under inert gas protection, stirred and heated to reflux, and then potassium persulfate was added to initiate the polymerization reaction. After the reaction was complete, the mixture was washed and ultrasonically dispersed to obtain the carboxylated polystyrene microspheres.

[0144] The carboxylating monomer includes any one or more of maleic anhydride, methacrylic acid, methacrylates, acrylic acid and acrylates; the molar ratio of styrene, carboxylating monomer and potassium persulfate is (0.2-0.4):(0.01-0.02):(0.5-1).

[0145] Example 1

[0146] A method for preparing time-resolved microspheres with high fluorescence stability and high fluorescence intensity includes the following steps:

[0147] 1. Preparation of rare earth europium complexes

[0148] Specifically, the following steps are included:

[0149] 1) Under an inert gas atmosphere, 1-(benzyloxy)-6-oxo-1,6-dihydropyridine-2-carboxylic acid (4.90 g, 20.0 mmol) and 2-mercaptothiazoline (2.62 g, 22.0 mmol) were dissolved in 50 mL of tetrahydrofuran. N,N'-dicyclohexylcarbodiimide (4.60 g, 22.0 mmol) and trace amounts of 4-dimethylaminopyridine were added, and the mixture was stirred overnight at room temperature. After the reaction was complete, solid impurities such as dicyclohexylurea were removed by filtration, and the solvent was removed by rotary evaporation to obtain a yellow first solid product.

[0150] 2) Under an inert gas atmosphere, the first solid product (1.93 g, 6.3 mmol) and 2,2-oxodiethylamine (0.31 g, 3.0 mmol) were dissolved in dry dichloromethane (20 mL) and stirred overnight at room temperature. After removing the solvent by rotary evaporation, impurities were removed by silica gel column chromatography with dichloromethane:methanol = 100:1 as the eluent, yielding a pale yellow second solid product.

[0151] 3) The second solid product (1.67 g, 3.0 mmol) was dissolved in concentrated HCl (12 M) / glacial acetic acid (1:1, 20 mL) and stirred overnight at room temperature. After removing the solvent by rotary evaporation, a beige solid residue was obtained. The residue was washed three times with diethyl ether, and the product was collected and dried under vacuum to obtain a white powder, which was the ligand 1,2-HOPO.

[0152] The synthetic route for the 1,2-HOPO ligand in this embodiment is shown below:

[0153]

[0154] 4) Under inert gas protection, the self-made 1,2-HOPO ligand (1.14 g, 3.0 mmol) was first dissolved in 5 mL of methanol. The pH was then adjusted to 7.0 by adding sodium hydroxide aqueous solution dropwise, followed by heating to reflux with magnetic stirring. Half an equivalent of europium chloride hexahydrate (0.55 g, 1.5 mmol) was dissolved in 1 mL of deionized water and then added dropwise to the above reaction solution of 1,2-HOPO ligand. A precipitate was formed immediately, and the reaction was continued with stirring for 1-2 hours. Finally, the precipitate was separated by filtration and washed several times with deionized water and methanol. The product was then dried in an oven overnight to obtain a white solid, namely the rare earth europium complex Eu(1,2-HOPO)2.

[0155] The synthetic route for the europium complex Eu(1,2-HOPO)2 in this embodiment is shown below:

[0156]

[0157] 2. Preparation of carboxylated polystyrene microspheres

[0158] Specifically, the following steps are included:

[0159] In a 500 mL three-necked flask, under nitrogen / argon bubbling or direct nitrogen protection, add 22.9 mL (0.20 mol) styrene, 0.82 mL (0.01 mol) methacrylic acid, and 200 mL deionized water. Heat to reflux while stirring at 400 rpm, then add 0.135 g (0.50 mmol) potassium persulfate to initiate polymerization. After reacting for 3 hours, stop heating and cool the reaction solution to room temperature. Centrifuge the synthesized microspheres at 18000 rpm for 15 min, remove the supernatant, add 250 mL of ultrapure water, and sonicate to disperse. Repeat the above operation 3-5 times to obtain monodisperse carboxylated polystyrene microspheres with a diameter of 200 nm ± 10 nm, uniform particle size, and regular surface.

[0160] 3. Preparation of time-resolved microspheres

[0161] Specifically, the following steps are included:

[0162] Take 250 ml of carboxylated polystyrene microspheres with a solid content of 2% and mix them with 200 ml of sodium dodecyl sulfate solution with a mass concentration of 0.625% to form a carboxylated polystyrene microsphere dispersion system. Take 0.5 g of the above europium complex Eu(1,2-HOPO)2 and dissolve it in 50 ml of dichloromethane. Then add it to the carboxylated polystyrene microsphere dispersion system to obtain a time-resolved microsphere premix. After homogenizing and stirring for 30 min, remove the dichloromethane by vacuum distillation at 30 °C. Centrifuge the synthesized microspheres at 18000 r / min for 15 min. After removing the supernatant, add 250 ml of ultrapure water and ultrasonically disperse. Repeat the above operation 3-5 times. Wash with deionized water by centrifugation 3-5 times to obtain time-resolved microspheres with a diameter of 210 nm ± 10 nm, uniform particle size, and regular surface.

[0163] Comparative Example 1

[0164] The method for preparing time-resolved microspheres based on β-diketone ligands (2-thiophenecarboxyltrifluoroacetone) includes the following steps:

[0165] (1) Preparation of rare earth europium complexes

[0166] Under an inert gas atmosphere, 2-thiophenecarboxyltrifluoroacetone (2.22 g, 10 mmol) and 1,10-o-phenanthroline (1.80 g, 10 mmol) in a 1:1 ratio were first dissolved in 50 mL of ethanol. The pH was then adjusted to 7.0 by adding NaOH aqueous solution dropwise, followed by heating to the reaction temperature with magnetic stirring. An equal amount of europium trichloride (3.66 g, 10 mmol) was dissolved in 20 mL of deionized water and added dropwise to the reaction solution, immediately forming a precipitate. The mixture was stirred continuously at the reaction temperature for 1-2 hours. Finally, the europium complex was separated by filtration and washed several times with deionized water and ethanol. The product was then dried overnight in an oven to obtain the rare earth complex Eu(TTA)3phen.

[0167] The synthetic route for the europium complex Eu(TTA)3phen is shown below:

[0168]

[0169] (2) Preparation of carboxylated polystyrene microspheres

[0170] The same as step 2 in Example 1.

[0171] (3) Preparation of time-resolved microspheres by encapsulation method

[0172] Take 250 ml of carboxylated polystyrene microspheres with a solid content of 2% and mix them with 200 ml of sodium dodecyl sulfate solution with a mass concentration of 0.625%. Dissolve 0.5 g of rare earth europium complex Eu(TTA)3phen in 50 ml of dichloromethane and then add it to the dispersion system of carboxylated polystyrene microspheres. After homogenizing and stirring for 30 min, remove the dichloromethane by vacuum distillation at 30 °C. Centrifuge the synthesized microspheres at 18000 r / min for 15 min. After removing the supernatant, add 250 ml of ultrapure water and sonicate to disperse. Repeat the above operation 3-5 times. Wash with deionized water by centrifugation 3-5 times to obtain time-resolved microspheres with a diameter of 210 nm ± 10 nm, uniform particle size, and regular surface.

[0173] Comparative Example 2

[0174] The method for preparing time-resolved microspheres based on β-diketone ligands (2-naphthoyltrifluoroacetone) includes the following steps:

[0175] (1) Preparation of rare earth europium complexes

[0176] Under an inert gas atmosphere, 2-naphthoyltrifluoroacetone (2.66 g, 10 mmol) and 1,10-phenanthroline (1.80 g, 10 mmol) in a 1:1 ratio were first dissolved in 50 mL of ethanol. The pH was then adjusted to 7.0 by adding NaOH aqueous solution dropwise, followed by heating to the reaction temperature with magnetic stirring. An equal amount of europium trichloride (3.66 g, 10 mmol) was dissolved in 20 mL of deionized water and added dropwise to the reaction solution, immediately forming a precipitate. The mixture was stirred at the reaction temperature for 1-2 hours. Finally, the europium complex was separated by filtration and washed several times with deionized water and ethanol. The product was then dried overnight in an oven to obtain the rare earth complex Eu(NTA)3phen.

[0177] The synthetic route for the europium complex Eu(NTA)3phen is shown below:

[0178]

[0179] (2) Preparation of carboxylated polystyrene microspheres

[0180] The same as step 2 in Example 1.

[0181] (3) Preparation of time-resolved microspheres by encapsulation method

[0182] Take 250 ml of carboxylated polystyrene microspheres with a solid content of 2% and mix them with 200 ml of sodium dodecyl sulfate solution with a mass concentration of 0.625%. Dissolve 0.5 g of rare earth europium complex Eu(NTA)3phen in 50 ml of dichloromethane and then add it to the dispersion system of carboxylated polystyrene microspheres. After homogenizing and stirring for 30 min, remove the dichloromethane by vacuum distillation at 30 °C. Centrifuge the synthesized microspheres at 18000 r / min for 15 min, remove the supernatant, add 250 ml of ultrapure water, and sonicate to disperse. Repeat the above operation 3-5 times. Wash with deionized water by centrifugation 3-5 times to obtain time-resolved microspheres with a diameter of 210 nm ± 10 nm, uniform particle size, and regular surface.

[0183] Performance testing

[0184] 1. Microscopic morphology testing

[0185] The time-resolved microspheres of Example 1 and Comparative Example 1 were subjected to scanning electron microscopy (SEM) tests, and the SEM test results are as follows: Figure 1-2 As shown, the time-resolved microspheres obtained in Example 1 and Comparative Example 1 have uniform particle size and regular surface.

[0186] 2. Microsphere particle size and monodispersity test

[0187] 2.1 Microsphere size testing

[0188] Samples of the same concentration were tested using a Malvern Zeta sizer Nano-S90 laser particle size analyzer. The steps included:

[0189] 1) Prepare time-resolved microsphere samples from Example 1, Comparative Examples 1-2, and a domestic competitor. Dilute 2 μL of fluorescent microspheres with a solid content of 1% in 2 mL of aqueous solution.

[0190] 2) The dispersion medium for the laser particle size analyzer was set to polystyrene, and the dispersant was set to water.

[0191] 3) The values ​​of particle size and dispersibility (PDI) are the average values ​​of three test results.

[0192] 2.2 Test methods for fluorescence intensity and stability of microspheres

[0193] Samples of the same concentration were tested using a Shimadzu RF-5301PC fluorescence spectrophotometer:

[0194] 1) Prepare time-resolved microsphere samples from Example 1, Comparative Examples 1-2, and a domestic competitor. Dilute 5 μL of fluorescent microspheres with a solid content of 1% in 2 mL of aqueous solution.

[0195] 2) Set the excitation wavelength (Excitation / Ex) to 365nm or 340nm, and the emission wavelength (Emission / Em) range to 500nm-700nm;

[0196] 3) In the stability test, the interval between each test is 1 minute.

[0197] The test results are shown in Table 1 and Figure 3-4 As shown.

[0198] Table 1

[0199]

[0200] As can be seen from Table 1, the average particle size of the time-resolved microspheres of Example 1, Comparative Examples 1-2, and a domestic competitor is similar. Among them, Example 1 and the domestic competitor (NW competitor) have lower PDI and better monodispersity.

[0201] Combining Table 1 and Figure 3 , 4 It can be seen that, under 365nm excitation light irradiation, the fluorescence intensity (RFU value: 914.437) of the time-resolved microspheres prepared in Example 1 (Eu(1,2-HOPO)2) is much higher than that of the time-resolved microspheres (RFU values: 773.216 and 615.324) prepared by the same method using common β-diketone europium complexes. Furthermore, the fluorescence intensity of the time-resolved microspheres prepared in Example 1 is comparable to that of a competing domestic product at its maximum emission wavelength. However, as shown in Table 1 and... Figure 4 It can be seen that under 340nm excitation light irradiation, the fluorescence intensity (RFU value: 1015.848) of the time-resolved microspheres prepared in Example 1 is higher than that of a domestic competitor (RFU value: 1005.776), indicating that the time-resolved microspheres of the present invention have superior fluorescence intensity at shorter ultraviolet wavelengths.

[0202] 3. Fluorescence intensity and stability test

[0203] Time-resolved microsphere samples from Example 1 and Comparative Examples 1-2 were excited five times under 365 nm excitation light, and the fluorescence intensity of the samples after each excitation was recorded. The results are shown in Table 2 and... Figure 5 , 6 As shown.

[0204] Table 2

[0205]

[0206] From Table 1 and Figure 5 , 6It can be seen that the time-resolved microspheres prepared in Example 1 of this invention exhibit extremely high fluorescence stability, with only a 0.41% fluorescence decay after 5 excitations. In contrast, the time-resolved microspheres in Comparative Examples 1 and 2 show a fluorescence decay of approximately 8%, with stability far lower than that of the time-resolved microspheres of this invention. This demonstrates that the time-resolved microspheres prepared using Eu(1,2-HOPO)2 as the fluorescent molecule in this invention, compared to β-diketone europium complex time-resolved microspheres, possess both higher fluorescence intensity and more significant fluorescence stability. Under repeated excitation light irradiation, the fluorescence intensity of the microspheres did not change significantly. This characteristic can help the labeling carrier to be used for retesting during the detection process.

[0207] Examples 2-4

[0208] The only difference between Examples 2-4 and Example 1 is that the substituents of the ligands in the europium complexes of Examples 2-4 are replaced, and the structural formula of the europium complexes is shown in Formula II;

[0209]

[0210] Referring to Table 3, the structures of the ligands in Examples 2 to 4 were obtained by replacing the R3 and R5 substituents of the ligand in Example 1. The final fluorescent microspheres were then compared. The fluorescence intensity of samples of the same concentration was tested using an RF-5301PC fluorescence spectrophotometer manufactured by Shimadzu Corporation at an excitation wavelength of 365 nm.

[0211] Table 3

[0212] ligand structure microsphere fluorescence intensity Example 1 <![CDATA[R3 = hydrogen atom, R5 = oxygen atom]]> 910 Example 2 <![CDATA[R3 = hydrogen atom, R5 = methylene]]> 900 Example 3 <![CDATA[R3 = methoxy, R5 = methylene]]> 770 Example 4 <![CDATA[R3 = methoxy, R5 = oxygen atom]]> 780

[0213] As can be seen from the table above, the fluorescence intensity of the microspheres in the europium complex of the present invention is better when R3 is a hydrogen atom than when R3 is a methoxy group; meanwhile, there is no significant change in the fluorescence intensity of the microspheres when R5 is a methylene or oxygen atom. Therefore, Examples 1 and 2, where R3 is a hydrogen atom, exhibit excellent fluorescence intensity and are relatively preferred technical solutions.

[0214] Examples 5-8

[0215] The only difference between Examples 5-8 and Example 1 is that in step 3 of the time-resolved microsphere preparation method, the content of europium complex Eu(1,2-HOPO)2 is added according to the content in Table 4. Comparative Examples 3 and 4 were obtained by adjusting the content of europium complex Eu(1,2-HOPO)2 while keeping other conditions and parameters unchanged. The fluorescence intensity of samples with the same concentration was tested using a Shimadzu RF-5301PC fluorescence spectrophotometer for Comparative Examples 3 and 4 and Examples 5-8.

[0216] Table 4

[0217]

[0218]

[0219] As shown in Table 4, the amount of Eu(1,2-HOPO)2 fluorescent molecules has a significant impact on the fluorescence intensity of the microspheres. When the mass ratio of added PS mother spheres to fluorescent molecules reaches 5g:0.3g, the microspheres exhibit high fluorescence intensity. When the mass ratio of added PS mother spheres to fluorescent molecules reaches 5g:0.5g (Example 1), the fluorescence intensity of the microspheres is basically at its maximum. Further increasing the amount of fluorescent molecules has no significant effect on the fluorescence intensity of the microspheres.

[0220] Examples 9-13

[0221] The only difference between Examples 9-13 and Example 1 is that, in step 3 of the time-resolved microsphere preparation method, the mass concentration of sodium dodecyl sulfate (SDS) in the time-resolved microsphere premix is ​​added according to the contents in Table 5. Fluorescence intensity and particle size and dispersibility tests were performed on samples of the same concentration using a Shimadzu RF-5301PC fluorescence spectrophotometer and a Malvern Zeta sizer Nano-S90 laser particle size analyzer.

[0222] Table 5

[0223]

[0224] As shown in Table 5, the amount of surfactant has a certain influence on the fluorescence intensity of the microspheres. When the mass concentration of SDS is 0.1%, the microspheres can have good dispersibility (monodispersibility of microspheres < 0.05). When the amount of SDS exceeds 0.5%, it has a certain impact on the monodispersity and fluorescence intensity of the microspheres. The microspheres with the best dispersibility and fluorescence intensity are obtained when the surfactant ratio is controlled between 0.2% and 0.25%.

[0225] Examples 14-18

[0226] The only difference between Examples 14-18 and Example 1 is that in step 3 of the time-resolved microsphere preparation method, the dichloromethane in Example 1 is replaced with the swelling agent types listed in Table 6. Fluorescence intensity and particle size and dispersibility tests were performed on samples of the same concentration using a Shimadzu RF-5301PC fluorescence spectrophotometer and a Malvern Zeta sizer Nano-S90 laser particle size analyzer.

[0227] Table 6

[0228] Types of swelling agents Microsphere particle size Monodispersity of microspheres microsphere fluorescence intensity Example 1 dichloromethane 210±10nm ＜0.05 910 Example 14 chloroform 220±10nm ＜0.05 700 Example 15 Tetrahydrofuran 280±20nm ＞0.1 150 Example 16 N,N-Dimethylformamide 350±20nm ＞0.2 120 Example 17 Cyclohexane 250±20nm ＜0.1 320 Example 18 1,2-Dichloroethane 270±10nm ＜0.05 400

[0229] As can be seen from Table 6, different types of swelling agents have a significant impact on the encapsulation of Eu(1,2-HOPO)2 molecules in this invention. In comparison, dichloromethane and trichloromethane have good solubility for Eu(1,2-HOPO)2, and the microspheres can effectively shrink after swelling, maintaining good monodispersity. The microspheres prepared by using dichloromethane as a swelling agent in Example 1 have higher fluorescence intensity.

[0230] Examples 19-23

[0231] The only difference between Examples 9-23 and Example 1 is that in step 3 of the time-resolved microsphere preparation method, the content of dichloromethane in Example 1 is replaced with the content shown in Table 7. Fluorescence intensity and particle size and dispersibility tests were performed on samples of the same concentration using a Shimadzu RF-5301PC fluorescence spectrophotometer and a Malvern Zeta sizer Nano-S90 laser particle size analyzer.

[0232] Table 7

[0233]

[0234] As shown in Table 7, different concentrations of swelling agents have a significant impact on the encapsulation of europium complex Eu(1,2-HOPO)2 molecules. Dichloromethane with a concentration below 5% cannot effectively dissolve europium complex Eu(1,2-HOPO)2, while dichloromethane with a concentration above 15% will cause some damage to the microspheres during the swelling process. Choosing a dichloromethane concentration of 8% to 10% as a swelling agent is more conducive to the preparation of time-resolved microspheres with high fluorescence intensity.

[0235] Examples 24-27

[0236] The only difference between Examples 24-27 and Example 1 is that in step 3 of the time-resolved microsphere preparation method, the swelling time is set according to Table 8. Then, using a Shimadzu RF-5301PC fluorescence spectrophotometer and a Malvern Zeta sizer Nano-S90 laser particle size analyzer, fluorescence intensity and particle size and dispersibility tests were performed on samples of the same concentration.

[0237] Table 8

[0238]

[0239]

[0240] The experimental results in Table 8 show that different swelling times have little effect on the encapsulation of europium complex Eu(1,2-HOPO)2. The fluorescence intensity of the microspheres can reach the maximum value when the swelling time is 0.5 to 1 h. Extending the swelling time does not significantly change the fluorescence loading of the microspheres, but excessive swelling may reduce the monodispersity of the microspheres.

[0241] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0242] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A time-resolved fluorescent microsphere, characterized in that, The invention comprises a carrier microsphere and a rare earth fluorescent complex embedded within the carrier microsphere, wherein the mass of the rare earth fluorescent complex is 10% to 20% of the mass of the carrier microsphere; The structural formula of the rare earth fluorescent complex is shown in Formula II. Among them, R1, R2, and R3 are all H, and R5 is methylene or O; The preparation method of the time-resolved fluorescent microspheres includes the following steps: 1) Mix the carrier microspheres and surfactant evenly in a solvent to form a dispersion system of carrier microspheres; 2) Dissolve the rare earth fluorescent complex in a swelling agent, then add it to the dispersion system of the carrier microspheres, carry out the swelling reaction under homogeneous stirring, remove the swelling agent by distillation, wash, and ultrasonically disperse to obtain time-resolved fluorescent microspheres; The carrier microspheres are selected from any one of polystyrene microspheres, hydrogel microspheres, and polymethyl methacrylate microspheres; and the surface of the carrier microspheres is modified with any one of carboxyl groups, mesocarboxyl groups, amino groups, thiol groups, and streptavidin. The swelling agent is dichloromethane; the volume concentration of the swelling agent is 8% to 12%. The surfactant includes any one or more of sodium dodecyl sulfonate, sodium dodecyl sulfate, and sodium dodecylbenzene sulfonate; the mass concentration of the surfactant is 0.2% to 1%.

2. The time-resolved fluorescent microspheres according to claim 1, characterized in that, The structural formula of the rare earth fluorescent complex is shown in Formula III.

3. The time-resolved fluorescent microspheres according to claim 1 or 2, characterized in that, The mass of the rare earth fluorescent complex is 10%-16% of the mass of the carrier microspheres.

4. The time-resolved fluorescent microspheres according to claim 1 or 2, characterized in that, The carrier microspheres are polystyrene microspheres with carboxyl groups modified on their surface.

5. The time-resolved fluorescent microspheres according to claim 1 or 2, characterized in that, The volume concentration of the swelling agent is 8% to 10%.

6. The time-resolved fluorescent microspheres according to claim 1 or 2, characterized in that, The surfactant is sodium dodecyl sulfonate.

7. The time-resolved fluorescent microspheres according to claim 1 or 2, characterized in that, The surfactant has a mass concentration of 0.2% to 0.25%.

8. A method for preparing time-resolved fluorescent microspheres according to any one of claims 1-7, characterized in that, Includes the following steps: 1) Mix the carrier microspheres and surfactant evenly in a solvent to form a dispersion system of carrier microspheres; 2) Dissolve the rare earth fluorescent complex in a swelling agent, then add it to the dispersion system of the carrier microspheres, carry out the swelling reaction under homogeneous stirring, remove the swelling agent by distillation, wash, and ultrasonically disperse to obtain time-resolved fluorescent microspheres.

9. The method for preparing time-resolved fluorescent microspheres according to claim 8, characterized in that, The swelling reaction takes 0.5 to 8 hours.

10. The method for preparing time-resolved fluorescent microspheres according to claim 9, characterized in that, The swelling reaction takes 0.5 to 2 hours.

11. The method for preparing time-resolved fluorescent microspheres according to claim 10, characterized in that, The swelling reaction takes 0.5 to 1 hour.

12. The method for preparing time-resolved fluorescent microspheres according to claim 8, characterized in that, In step 1), the carrier microspheres are carboxylated polystyrene microspheres, and the preparation method includes the following steps: Styrene and carboxylated monomers were dissolved in an aqueous solution under inert gas protection, stirred and heated to reflux, and then potassium persulfate was added to initiate the polymerization reaction. After the reaction was complete, the mixture was washed and ultrasonically dispersed to obtain the carboxylated polystyrene microspheres. The carboxylating monomer includes any one or more of maleic anhydride, methacrylic acid, methacrylates, acrylic acid and acrylates; the molar ratio of styrene, carboxylating monomer and potassium persulfate is (0.2-0.4):(0.01-0.02):(0.5-1).

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