CN-116803985-B - Rare earth fluorescent complex, time-resolved fluorescent microsphere, and preparation methods and applications thereof

CN116803985BCN 116803985 BCN116803985 BCN 116803985BCN-116803985-B

Abstract

The invention discloses a rare earth fluorescent complex, which relates to the field of luminescent materials and has a structural formula shown in a formula I. The rare earth fluorescent complex has the advantages that the luminous central atoms are bonded with two symmetrical tetradentate ligands, the overall structure of the complex is butterfly-shaped, and the complex is different from the existing beta-diketone ligand, one O coordination atom in the ligand is connected with N in pyridine, so that energy transfer is optimized, the triplet state energy level of the ligand can be matched with the excited state energy level of the central atoms, and the complex has a more efficient antenna effect under the irradiation of excitation light, and the fluorescence performance and fluorescence stability of the rare earth fluorescent complex are comprehensively improved. The preparation method of the rare earth fluorescent complex can efficiently and industrially synthesize the rare earth fluorescent complex. The time-resolved fluorescence microsphere has high fluorescence intensity and strong structural stability, and the fluorescence intensity does not obviously decline under the irradiation of 3-5 times of excitation light.

Inventors

LI DONGYAO
LI LIUHUI
Shao Binfen
MA CHUNMIAO
WANG HANMING

Assignees

广州万孚生物技术股份有限公司

Dates

Publication Date: 20260508
Application Date: 20230627

Claims (12)

1. The time-resolved fluorescence microsphere is characterized by comprising a carrier microsphere and a rare earth fluorescence complex embedded in the carrier microsphere, wherein the mass of the rare earth fluorescence complex is 10% -20% of the mass of the carrier microsphere; The structural formula of the rare earth fluorescent complex is shown as a formula II, Wherein R 1 、R 2 、R 3 is H, and R 5 is methylene or O; The preparation method of the time-resolved fluorescence microsphere comprises the following steps: 1) Uniformly mixing carrier microspheres and a surfactant in a solvent to form a dispersion system of the carrier microspheres; 2) Dissolving the rare earth fluorescent complex in a swelling agent, adding the swelling agent into a dispersion system of the carrier microsphere, carrying out swelling reaction under homogeneous stirring, distilling to remove the swelling agent, washing, and carrying out ultrasonic dispersion to obtain the time-resolved fluorescent microsphere; The carrier microsphere is selected from any one of polystyrene microsphere, hydrogel microsphere and polymethyl methacrylate microsphere, and the surface of the carrier microsphere is modified with any one of carboxyl, carboxyl of a middle arm, amino, sulfhydryl and streptavidin; The swelling agent is methylene dichloride, and the volume concentration of the swelling agent is 8% -12%; The surfactant comprises one or more than two of sodium dodecyl sulfate, sodium dodecyl sulfate and sodium dodecyl benzene sulfonate, and the mass concentration of the surfactant is 0.2% -1%.
2. The time-resolved fluorescence microsphere according to claim 1, wherein the rare earth fluorescent complex has a structural formula shown in formula III,
3. The time-resolved fluorescent microsphere as defined in claim 1 or 2, wherein the mass of the rare earth fluorescent complex is 10% -16% of the mass of the carrier microsphere.
4. The time-resolved fluorogenic microsphere according to claim 1 or 2, wherein the carrier microsphere is a surface modified carboxyl polystyrene microsphere.
5. The time-resolved fluorescence microsphere according to claim 1 or 2, wherein the volume concentration of the swelling agent is 8% -10%.
6. The time-resolved fluorescent microsphere according to claim 1 or 2, wherein the surfactant is sodium dodecyl sulfonate.
7. The time-resolved fluorescence microsphere according to claim 1 or 2, wherein the mass concentration of the surfactant is 0.2% -0.25%.
8. A method of producing time-resolved fluorogenic microspheres according to any one of claims 1-7, comprising the steps of: 1) Uniformly mixing carrier microspheres and a surfactant in a solvent to form a dispersion system of the carrier microspheres; 2) Dissolving the rare earth fluorescent complex in a swelling agent, adding the swelling agent into a dispersion system of the carrier microsphere, carrying out swelling reaction under homogeneous stirring, distilling to remove the swelling agent, washing, and carrying out ultrasonic dispersion to obtain the time-resolved fluorescent microsphere.
9. The method for preparing time-resolved fluorescence microsphere according to claim 8, wherein the swelling reaction time is 0.5-8 h.
10. The method for preparing time-resolved fluorescence microsphere according to claim 9, wherein the swelling reaction time is 0.5-2 h.
11. The method for preparing time-resolved fluorescence microsphere according to claim 10, wherein the swelling reaction time is 0.5-1 h.
12. The method for preparing time-resolved fluorescence microsphere according to claim 8, wherein in step 1), the carrier microsphere is a carboxyl polystyrene microsphere, and the preparation method comprises the following steps: Under the protection of inert gas, dissolving styrene and carboxylated monomers in an aqueous solution, stirring and heating to reflux, then adding potassium persulfate to initiate polymerization reaction, washing and performing ultrasonic dispersion after the reaction is complete to obtain the carboxyl polystyrene microsphere; The carboxylated monomer comprises any one or more than two of maleic anhydride, methacrylic acid esters, acrylic acid and acrylic esters, wherein the molar ratio of the styrene to the carboxylated monomer to the potassium persulfate is (0.2-0.4) (0.01-0.02) (0.5-1).

Description

Rare earth fluorescent complex, time-resolved fluorescent microsphere, and preparation methods and applications thereof Technical Field The invention belongs to the field of in-vitro diagnostic materials, and particularly relates to a rare earth fluorescent complex and a time-resolved fluorescent microsphere as well as a preparation method and application thereof. Background The time-resolved fluoroimmunoassay is one of the three current hypersensitive immunoassay methods, and the principle is that a rare earth complex with longer fluorescence half-life is adopted as a fluorescent marker, wherein the fluorescence lifetime of the rare earth complex is 2-3 orders of magnitude (usually more than 100 microseconds and more than one hundred times) higher than that of a background fluorescent substance due to the difference of the rare earth complex in the luminescence principle. Therefore, only long-life fluorescence is measured in the fluorescent signal detection, but short-life fluorescence is not measured, namely, after the sample is excited by a light source, the background fluorescence is decayed by prolonging the light information acquisition time, and then the fluorescent signal is acquired, so that the interference of nonspecific fluorescence can be effectively eliminated, and the fluorescent signal has relatively higher detection sensitivity. The time-resolved fluorescence microsphere (Time Resolved Fluorescent Microsphere) is formed by filling rare earth ion complex into polymer nano particles. The fluorescent microsphere is a special functional microsphere, and thousands of rare earth fluorescent molecules can be wrapped in each nanoscale polystyrene microsphere, so that the fluorescent intensity of a labeling carrier is effectively improved, and the measurement of ultralow-content biological components is satisfied. The time-resolved fluorescence microsphere sold at present has the problems that firstly, the fluorescence intensity of the whole microsphere is not high, the sensitivity of a detection product cannot be effectively improved, secondly, the fluorescence stability of the rare earth complex is insufficient, namely, the fluorescence intensity of the whole microsphere is rapidly attenuated after being irradiated by excitation light, and the time-resolved fluorescence microsphere is very unfavorable for retest and result verification of related detection products, so that the time-resolved fluorescence microsphere has great research significance on how to improve the luminous efficiency and fluorescence stability of the f-f transition rare earth complex in the time-resolved microsphere. The luminescent properties of the rare earth complex and the stability of the complex are determined by the ligand structure. Taking the rare earth europium complex as an example, aiming at the research on the fluorescence property of the europium complex, the early stage takes the strong fluorescence complex formed by a bidentate ligand (beta-diketone) and europium ions as the main part, and more typical examples include NTA (beta-naphthoyl trifluoroacetone), TTA (2-thiophenyl trifluoroacetone), DNM (dinaphthoxymethane) and DBM (dibenzoylmethane). This is the case for example with patent CN108445219a, patent CN103011492a, patent CN111218270a, patent CN112745833A, CN115466277 a. Among them, NTA and TTA ligands are widely used because of their efficient light absorption properties, but the ligands still have disadvantages in fluorescence intensity and stability. To solve this problem, many novel ligand structures have been developed, but it is still a challenge to find a suitable ligand structure that can improve both its fluorescence properties and its structural stability, and also can be efficiently entrapped, due to the constraints of the ligand. At present, one method which is more effective is to repeat the existing bidentate ligand to form a polydentate ligand, which is specifically shown as follows: The fluorescence stability of the rare earth complex of the tetradentate ligand is improved to a certain extent, but the fluorescence intensity is not obviously improved. Meanwhile, patent CN112521262A mentions a multidentate beta-diketone ligand and a luminescent rare earth complex thereof, wherein the multidentate beta-diketone ligand is as follows: The polydentate ligand can provide two or more atoms capable of providing lone electron pairs, and forms chelate with a low coordination ratio and a cyclic structure when being coordinated with metal, so that adverse influence of external environment on light emission of the rare earth complex is reduced. Although the polydentate ligand has extremely strong chelating ability and greatly improves fluorescence stability, the matching degree of the triplet state energy level and the excited state energy level of the central europium ion is reduced, so that the fluorescence intensity is not obviously improved. The time-resolved microspheres prepared by the strategy ar