CN112521262A

CN112521262A - Polydentate beta-diketone ligand, luminescent rare earth complex thereof and application

Info

Publication number: CN112521262A
Application number: CN202011316444.8A
Authority: CN
Inventors: 李富友; 徐�明; 郭琳娜; 李颖; 刘倩
Original assignee: Shanghai Taihui Biotechnology Co ltd; Fudan University
Current assignee: Shanghai Taihui Biotechnology Co ltd; Fudan University
Priority date: 2020-11-20
Filing date: 2020-11-20
Publication date: 2021-03-19
Anticipated expiration: 2040-11-20
Also published as: CN112521262B

Abstract

A multidentate beta-diketone ligand, the structure of which is shown as formula X, wherein m, n and k are independently selected from integers of 1-20, R₁Selected from hydrogen, hydroxyl, alkoxy, alkyl, alkenyl, alkynyl, alkenyloxy, alkynyloxy, phenyl, substituted group-containing benzene derivatives or aromatic hydrocarbon groups; r₂、R₃And R₄Independently selected from alkyl, alkenyl, alkynyl, phenyl, thiophene, substituted group-containing benzene derivatives, perfluoro-substituted alkyl or perfluoro-substituted phenyl. The polydentate beta-diketone ligand contains six carbonyl groups, and can coordinate with central rare earth ions through the six carbonyl groups in molecules to form a stable rare earth luminescent complex, so that luminescence can be enhanced, and luminescence quenching caused by the action of reagents such as a surfactant, a buffer solution and the like and the rare earth complex can be avoided. The invention also applies the prepared rare earth luminescent complex to the field of immunodetection, in particular to the time-resolved immunofluorescence assay, homogeneous chemiluminescence assay and fluorescence lateral immunochromatography assay, thereby improving the stability and sensitivity of the assay.

Description

Polydentate beta-diketone ligand, luminescent rare earth complex thereof and application

Technical Field

The invention belongs to the field of immunodetection and analysis, and particularly relates to a polydentate beta-diketone ligand, a luminescent rare earth complex containing the same and application of the polydentate beta-diketone ligand in immunodetection.

Background

The rare earth coordination compound is a rare earth complex for short, and is a compound formed by complexing or chelating rare earth central ions and organic ligands thereof. The rare earth complex has long luminescence life and narrow emission half-peak width, and therefore, the rare earth complex also becomes a luminescent material with great potential and plays an important role in the field of immunodetection. The immunoassay technology is an in vitro detection method established by utilizing the specific reaction of an antigen and an antibody, wherein the immunoassay method based on a luminescent rare earth complex is widely developed and applied, such as a homogeneous chemiluminescence method, a fluorescence lateral immunochromatography method, a time-resolved immunofluorescence method and the like. Therefore, the research of the rare earth luminescent complex has important significance and practical value.

In immunoassay application, a large amount of reagents such as surfactants and buffers are usually used, and these reagent components react with the rare earth complex to cause quenching of luminescence. For example, in immunoassay, PB or PBs buffer is often used, which contains phosphate ions, and the phosphate ions have strong complexation with central rare earth ions (e.g., europium Eu, terbium Tb, samarium Sm, etc.) of the complex, so that the luminescence signal is reduced or even completely quenched, thereby seriously affecting the immunoassay effect. Based on this, there is a strong need to develop an organic ligand with strong chelating ability, through which central rare earth ions are strongly chelated to form a rare earth complex, so as to reduce the adverse effect of the external environment on the luminescence of the rare earth complex.

Among the organic ligands disclosed in the prior art, those having strong chelating ability are claimed to be useful, for example, for binding separation of metal ions, but these ligands are not suitable for constructing luminescent rare earth complexes because they are associated with rare earth ions (e.g., Eu)³⁺) The complexes formed after chelation have no luminescent properties or are difficult to achieve luminescence. Therefore, it remains a challenge to develop organic ligands with strong chelating ability and efficiently form luminescent complexes with rare earth ions.

Disclosure of Invention

The invention provides a polydentate beta-diketone ligand with strong chelating capacity, a rare earth luminescent complex with high stability is prepared for the ligand based on the compound, and the prepared rare earth luminescent complex is applied to the field of immunodetection, such as a time-resolved immunofluorescence method, a homogeneous phase chemiluminescence method and a fluorescence lateral immunochromatography method, so that the luminescent performance of the rare earth complex in immunodetection is improved.

The following definitions apply within the scope of the invention.

If a ligand contains two or more atoms capable of providing a lone pair of electrons, the ligand is called a multidentate ligand. When the complex is coordinated with metal, a chelate with low coordination ratio and a cyclic structure is formed, the phenomenon of hierarchical complexation (coordination) is reduced or even eliminated, and the stability of the complex is improved. The invention discloses a polydentate beta-diketone ligand which contains six carbonyl groups, namely, the polydentate beta-diketone ligand can pass through six carbonyl groups in a molecule and a central rare earth ion (such as Eu)³⁺) Coordinating, thereby forming a multidentate coordination.

Organic ligands with strong chelating ability, such as the polydentate beta-diketone ligand disclosed by the invention, can be used for firmly chelating central rare earth ions to form stable rare earth complexes so as to reduce the adverse effect of external environment on the luminescence of the rare earth complexes. Advantageously, it is important to form stable rare earth coordination by the multidentate β -diketone ligand, both to enhance luminescence and to avoid interaction of the rare earth complexes with reagents, such as surfactants, buffers, which are typically used in large amounts, and to reduce quenching of luminescence.

In the field of in vitro diagnostics, immunodetection techniques are an important component. The immunoassay technology is a detection method established by utilizing the specific reaction of an antigen and an antibody. In clinical diagnosis, the immunodetection techniques mainly include immunoturbidimetry, electrochemiluminescence, homogeneous chemiluminescence, immunochromatography, time-resolved immunofluorescence, and the like. Among them, the photo-activated chemiluminescence method is considered to be a relatively high-sensitivity homogeneous immunoassay method. The method is based on the fact that the proximity between the donor (photosensitive particles) and the acceptor (luminescent particles) generates an effective energy transfer, thereby utilizing the effect to homogeneously detect the analyte. In this method, a donor (e.g. coated with phthalocyanine molecules) is usually excited by light of, for example, 680nm to generate singlet oxygen, and an acceptor approaches the donor by means of a specific immunoreaction, wherein a luminescent group rapidly absorbs singlet oxygen and emits light of a certain wavelength (e.g. 500-615nm), and then the presence or absence of an object to be detected in an actual detection sample is determined by detecting the intensity of a luminescent signal.

It is worth to be noted that the carrier microspheres are loaded with photosensitizers to prepare photosensitive particles, the photosensitizers react with oxygen under the action of exciting light to generate singlet oxygen, and the photosensitizers are known photosensitizers in the field and include, but are not limited to, porphyrins and phthalocyanins dyes, transition metal complexes, Quantum Dots (QDs), and derivatives or copolymers of these compounds; the carrier microsphere is loaded with a photochemical caching agent and a luminescent agent to prepare luminescent particles, wherein the photochemical caching agent is used for reacting with the singlet oxygen and releasing energy, the luminescent agent is used for receiving the energy and emitting light, the luminescent agent adopts an europium complex, and a ligand is a hexadentate ligand compound.

In addition, the invention also provides a long afterglow luminescent material which can be applied to the homogeneous immunoassay, the long afterglow luminescent material comprises the prepared photosensitive particles and luminescent particles, and energy input, energy buffer and energy output energy utilization paths can be realized through the screened photosensitizer, luminescent agent and photochemical buffer, namely, after an excitation light source is removed, excited state energy is slowly released in a light form and shows a phenomenon of persistent luminescence (the service life is more than 0.1 s).

The test paper strip used in the fluorescence lateral immunochromatography generally comprises a sample pad, a binding pad, a nitrocellulose membrane (NC membrane) and a water absorption pad, wherein the binding pad is sprayed with a fluorescent microsphere (or a long afterglow microsphere) for marking a first antibody (Ab1), the nitrocellulose membrane is marked with a T line of a second antibody (Ab2) and a C line of the second antibody, wherein the fluorescent microsphere can be prepared by coating a europium complex by a carrier microsphere, the long afterglow microsphere can be prepared by coating a selected photosensitizer, a luminescent agent and a photochemical buffering agent by the carrier microsphere, the luminescent agent in the invention is a europium complex, and a ligand is a hexadentate ligand compound, namely a polydentate beta-diketone ligand.

The basic principle of the fluorescence lateral immunochromatography is a double-antibody (or double-antigen) sandwich method and a competition method, which are briefly introduced by taking the double-antibody sandwich method as an example, when a sample of an antigen to be detected is added to a sample pad, the antigen to be detected is combined with a labeled antibody fluorescent microsphere (or a long afterglow microsphere of a labeled antibody) in the combined pad, a sandwich compound is formed by a second antibody which is chromatographed to a T line and the T line, the T line is stopped at the position of the T line, and the concentration of the antigen to be detected is quantitatively calculated by utilizing the positive correlation between the concentration and the fluorescence intensity (afterglow light intensity).

The time-resolved immunofluorescence method is one of the hypersensitive immunoassay methods which are compatible with chemiluminescence. The principle is that rare earth ions with longer fluorescence half-life are used as a marker, and the Stokes displacement of the marker is large, and the fluorescence life of the marker is 5-6 orders of magnitude higher than that of a background substance, so that the measurement time is delayed during measurement, and the interference of various non-specific fluorescence can be effectively eliminated by measuring the signal of the marker after the fluorescence of the background substance is fully attenuated, and the detection sensitivity is improved. In the invention, the fluorescent lifetime of the rare earth ion chelate adopting multidentate coordination is long, and the requirement of time-resolved immunofluorescence analysis can be met.

The time-resolved fluorescent microspheres (including the long-afterglow microspheres) can be wrapped with thousands of rare earth ion chelates, so that the fluorescence labeling efficiency is improved, carboxyl groups or other functional groups modified on the surfaces of the microspheres can be covalently coupled with proteins or antibodies, the microspheres can be applied to lateral immunochromatography, a quantitative detection technology with higher sensitivity than that of colloidal gold is developed, background signal interference is eliminated through time-delayed detection, the detection time is prolonged, and the detection sensitivity can be improved.

For the above-mentioned various in vitro diagnostic immunoassay techniques, immunoassay diluents are required in the production of the reagents/kits and in the immunoassay process. The diluent comprises reagent components such as buffer solution, surfactant, preservative and the like. The buffer solution includes Phosphate Buffered Saline (PBS), phosphate buffered saline (PB), Borate Buffered Saline (BBS), 2- (N-morpholino) ethanesulfonic acid buffer (MES), and the like. The surfactant includes Sodium Dodecylbenzenesulfonate (SDBS), Sodium Dodecyl Sulfate (SDS), cetyltrimethylammonium bromide (CTAB), and the like.

The present invention will be described in further detail below.

The invention discloses a polydentate beta-diketone ligand in a first aspect, wherein the polydentate beta-diketone ligand has a structure shown as the following formula:

wherein m, n and k are independently selected from integers of 1-20, preferably m, n and k are independently selected from integers of 1-10, more preferably m, n and k are independently selected from integers of 1-5;

R₁selected from hydrogen, hydroxyl, alkoxy, alkyl, alkenyl, alkynyl, alkenyloxy, alkynyloxy, phenyl, substituted group-containing benzene derivatives or aromatic hydrocarbon groups;

R₂、R₃and R₄Independently selected from alkyl, alkenyl, alkynyl, phenyl, thiophene, substituted group-containing benzene derivatives, perfluoro-substituted alkyl or perfluoro-substituted phenyl.

It is worth to be noted that the ligand of the hexadentate compound is prepared by combining three groups of beta-diketone fragments into the same compound by adopting a flexible alkyl chain, and the diketone compounds adopting the design principle of the invention can be prepared by the design and synthesis strategy of the invention.

In a preferred embodiment, in the formula X, m, n and k have the same value; r₂、R₃And R₄The same; preferably, R₂、R₃And R₄Selected from phenyl, CF₃、C₂F₅、C₃F₇Or C₆F₅。

According to one embodiment of the invention, for example, in said formula X, m, n and k are not all identical;

preferably, R₂、R₃And R₄Are not identical;

preferably, m, n and k are different from each other;

preferably, R₂、R₃And R₄Are different from each other;

preferably, R₂、R₃And R₄Selected from phenyl, CF₃、C₂F₅、C₃F₇Or C₆F₅。

According to one embodiment of the invention, for example, in said formula X, R₁Is hydrogen or hydroxy;

preferably, R₁Has a relative molecular mass of greater than or equal to 20; preferably, R₁Has a relative molecular mass of greater than or equal to 30; preferably, R₁Has a relative molecular mass of greater than or equal to 50; preferably, R₁Has a relative molecular mass of greater than or equal to 100;

preferably, R₁Is an unbranched hydrocarbon oxy group;

preferably, R₁Is a branched hydrocarbonoxy group;

preferably, R₁Is an unbranched alkoxy group;

preferably, R₁Is an alkoxy group with a branched chain.

According to one embodiment of the invention, for example, the multidentate β -diketone ligand of the invention is selected from any one of the following compounds:

the examples of the present invention also provide methods for preparing the multidentate β -diketone ligands described above, and it will be understood by those skilled in the art that other structures including compounds X-1 to X-20 and compound X can be synthesized by similar synthetic means by replacing the reaction raw materials.

The preparation of the polydentate beta-diketone ligands is illustrated here by the synthesis of the X-1 organic ligands, with the following scheme:

the method comprises the following specific steps:

1) preparation method of tribenzyl methanol

The raw materials are dibenzyl ketone and benzyl magnesium chloride, the solvent is diethyl ether, the reaction is carried out at room temperature, the reaction is carried out in an ice bath after the reaction is finished, a dilute sulfuric acid solution is added for quenching the reaction, the stirring is carried out for 15-30 min, the liquid is separated, an organic phase is taken, and the white solid which is tribenzyl methanol is obtained after the treatment.

Preferably, the feed ratio is:

dibenzyl ketone: the molar ratio of the benzyl magnesium chloride is 5: 1-2: 1;

the concentration of the dilute sulfuric acid solution was 5 wt%.

2) Process for preparing tri-substituted olefins

Raw materials are triethylsilane, boron trifluoride ethyl ether and the tribenzyl methanol prepared in the step 1) and a solvent is DCM, a saturated sodium chloride solution is used for quenching reaction, liquid separation is carried out, an organic phase is obtained, and colorless oily liquid is obtained after treatment.

Preferably, the feed ratio is:

tribenzyl methanol: triethyl silane: the molar ratio of boron trifluoride diethyl etherate is 1:6:4 to 1:10:6, preferably 1:8.6: 5.

3) Preparation method of tribenzylmethane

The raw materials are as follows: and 2) carrying out sealed reaction on the tri-substituted olefin prepared in the step 2), Pd/C and hydrogen in the presence of ethyl acetate and methanol at the temperature of 23-25 ℃, and carrying out column chromatography on the reaction product to obtain a white crystalline solid.

Preferably, the feeding molar ratio of the tri-substituted olefin to the palladium-carbon catalyst is 80:1 to 200:1, and preferably 100: 1.

4) Preparation of tri (p-acetyl) benzyl methane by Friedel-Crafts acylation reaction

The raw materials are as follows: aluminum trichloride, acetyl chloride and the tribenzyl methane prepared in the step 3) in the solvent of DCE, performing water quenching reaction, separating liquid, taking an organic phase, and processing to obtain light yellow viscous oily liquid, namely the tri (p-acetyl) benzyl methane.

Preferably, the feeding molar ratio of the tribenzyl methane to the acetyl chloride to the aluminum trichloride is as follows: 1:3:3 to 1:6:6, preferably 1:3.3: 3.3.

5) Knoevenagel condensation reaction

The raw materials are as follows: the preparation method comprises the following steps of carrying out quenching reaction on tri (p-acetyl) benzyl methane, ethyl pentafluoropropionate and sodium methoxide by using diethyl ether as a solvent, taking an organic phase from liquid separation, processing the organic phase, and adding ethanol for recrystallization to obtain the hexadentate ligand compound X-1.

Preferably, the molar ratio of the tri (p-acetyl) benzyl methane, the ethyl pentafluoropropionate and the sodium methoxide is: 1:3:3 to 1:6:6, preferably 1:3: 3.

Similarly, the synthetic procedures for X-13 to X-20 are substantially the same as for X-1 described above. For example, taking the synthesis of the X-13 ligand as an example: the following synthetic route for the X-13 ligand differs from the synthetic route for X-1 in that 1, 5-diphenyl-2-pentanone is used as the starting material and the other conditions are unchanged.

In a second aspect, the present invention provides a composition of luminescent complexes comprising a rare earth complex having as a ligand a multidentate β -diketone ligand according to the first aspect of the present invention. The beta-diketone hexadentate ligand (polydentate beta-diketone ligand) is coordinated with rare earth ions (such as europium Eu, terbium Tb, samarium Sm and the like) by strong chelation to form a rare earth luminescent complex.

Wherein the general formula of the rare earth luminescent complex is LnX_jJ is an integer selected from 1 to 10, and j is preferably 1. Ln is selected from Eu (europium), Tb (terbium) and Sm (samarium), and preferably, Ln is Eu (europium).

The embodiment of the invention also provides a rare earth complex with stable luminescenceThe general formula of the rare earth complex is LnX_jWherein X is a multidentate β -diketone ligand as defined in any one of claims 1 to 6; j is an integer selected from 1 to 10; preferably, j is 1; ln is selected from Eu (europium), Tb (terbium) and Sm (samarium); preferably, Ln is Eu (europium); after the rare earth complex with stable luminescence is mixed with the immunodetection diluent for a period of time, the luminescence intensity of the rare earth complex is greater than or equal to 50% of the initial luminescence intensity;

preferably, the rare earth complex has a luminous intensity of greater than or equal to 70% of the initial luminous intensity;

preferably, the rare earth complex has a luminous intensity of greater than or equal to 80% of the initial luminous intensity;

preferably, the rare earth complex has a luminous intensity greater than or equal to 90% of the initial luminous intensity;

preferably, the rare earth complex has a luminous intensity greater than or equal to 95% of the initial luminous intensity;

preferably, the rare earth complex has a luminous intensity of greater than or equal to 98% of the initial luminous intensity;

preferably, the rare earth complex has a luminous intensity of greater than or equal to 99% of the initial luminous intensity;

wherein the immunoassay diluent comprises at least one of a buffer reagent, a surfactant, a hemolytic agent, and a preservative;

the period of time is more than 1 hour, preferably more than 2 hours, preferably more than 5 hours;

preferably, the buffer reagent comprises at least one of PBS, PB, BBS, MES;

preferably, the surfactant comprises at least one of SDBS, SDS, CTAB. The embodiment of the invention also provides a preparation method of the rare earth luminescent complex, which takes europium complex Eu (X-1) as an example:

1) dissolving ligand X-1 in reaction medium to form reaction system, and dissolving europium ion-containing salt (such as EuCl)₃·6H₂O) is dissolved in a solvent, slowly added into a reaction system, and the pH value of the reaction system is adjusted to be lower by using an alkaline substance at a certain temperature4-5, wherein the alkaline substance comprises but is not limited to sodium hydroxide;

2) after continuing to react for a certain time, adding water into the reaction system until precipitation particles are generated in the reaction system;

3) stopping adding water, continuously stirring the reaction system, and performing post-treatment to obtain white precipitate after the precipitate particles are aggregated into large particles.

Wherein the europium ion-containing salt is preferably Eu³⁺Chloride salt of (4) or Eu³⁺Nitrate of (2); the europium ion-containing salt may contain crystal water, such as EuCl₃·6H₂O；

Preferably, the ligands X-1 and EuCl₃·6H₂The feeding molar ratio of O is 10: 1-1: 10; more preferably, ligands X-1 and EuCl₃·6H₂The feeding molar ratio of O is 2: 1-1: 5;

preferably, the reaction medium can be chosen from acetone, N-Dimethylformamide (DMF), Tetrahydrofuran (THF), EuCl₃·6H₂Dissolving O in ethanol and acetone;

preferably, the temperature of the reaction system is determined according to the boiling point of the reaction medium, in order to reflux the reaction medium.

In a third aspect of the invention, a reagent composition for immunoassay is disclosed, which comprises three types. The three types are respectively: time-resolved immunofluorescence assay reagents, homogeneous chemiluminescence assay reagents, and fluorescence lateral immunochromatography assay reagents. In all of the above three types of immunoassay reagents, the unit that ultimately emits light is a composition of the rare earth luminescent complex according to the second aspect of the present invention. The nature of the rare earth luminescent complex is very important as the last step in energy transfer or conversion and has a critical influence on the final luminescent signal intensity.

Type I: time-resolved immunofluorescence detection reagent

The time-resolved immunofluorescence detection reagent mainly comprises luminescent microspheres, and the luminescent microspheres can be time-resolved luminescent microspheres, for example. The time-resolved luminescent microsphere comprises a carrier microsphere and the rare earth luminescent complex of the second aspect of the invention.

The core component of the time-resolved luminescent microsphere is a rare earth luminescent complex, and the service life of the common rare earth luminescent complex can reach the level of microsecond to millisecond, so that luminescent signals can be acquired by using a time-resolved technology. Therefore, when the time resolution technology is not used, the time resolution luminescent microsphere can also be used for direct luminescent intensity test and analysis, and has the functions of common fluorescent or phosphorescent luminescent microspheres.

Wherein the carrier microsphere is at least one selected from hydrogel microspheres, styrene polymer microspheres, microspheres formed by protein, silicon nano-microspheres and polymethyl methacrylate microspheres; preferably, the carrier microspheres are selected from styrene polymer microspheres, especially from styrene polymer microspheres with amino, carboxyl, amido and/or aldehyde groups on the surface.

According to an embodiment of the present invention, for example, in the luminescent microsphere, the mass of the rare earth complex is 0.01% to 50% of the mass of the support microsphere;

preferably, the mass of the rare earth complex is 0.1-20% of that of the carrier microsphere;

preferably, the mass of the rare earth complex is 1-10% of that of the carrier microsphere;

preferably, the mass of the rare earth complex is 5-10% of the mass of the carrier microsphere.

According to one embodiment of the present invention, for example, the luminescent intensity of the luminescent microsphere is greater than or equal to 50% of the initial luminescent intensity for a period of time after the luminescent microsphere is mixed with the immunoassay diluent;

preferably, the luminescent intensity of the luminescent microsphere is greater than or equal to 70% of the initial luminescent intensity;

preferably, the luminescent intensity of the luminescent microsphere is greater than or equal to 80% of the initial luminescent intensity;

preferably, the luminescent intensity of the luminescent microsphere is greater than or equal to 90% of the initial luminescent intensity;

preferably, the luminescent intensity of the luminescent microsphere is greater than or equal to 95% of the initial luminescent intensity;

preferably, the luminescent intensity of the luminescent microsphere is greater than or equal to 98% of the initial luminescent intensity;

preferably, the luminescent intensity of the luminescent microsphere is greater than or equal to 99% of the initial luminescent intensity;

wherein the immunoassay diluent comprises at least one of a buffer reagent, a surfactant, a preservative, and a hemolytic agent;

preferably, the buffer reagent comprises at least one of PBS, PB, BBS, MES;

preferably, the surfactant comprises at least one of SDBS, SDS, CTAB.

The preparation method of the time-resolved luminescent microsphere comprises the following steps:

1) providing carrier microsphere and rare earth luminescent complex LnX_j；

2) In dispersion or solution, rare earth luminescent complex LnX_jDispersing or adsorbing the microspheres into carrier microspheres to prepare time-resolved luminescent microspheres;

wherein, the rare earth luminescent complex LnX can be prepared_jRespectively dissolved or dispersed in a suitable solvent to form a solution, and the above suitable solvent is not limited, and may be, for example, liquid paraffin, a mixture of benzyl alcohol-ethylene glycol and water, a mixture of mesitylene and ethanol, tetrahydrofuran, dichloromethane, or the like. Forming the above materials into stable solution or dispersion, adding carrier microsphere or dispersion of carrier microsphere to obtain rare earth luminescent complex LnX_jAnd adsorbing or coating to prepare the time-resolved luminescent microsphere. Preferably, water or other suitable solvent may be used to disperse the carrier microspheres, such as deionized water, Phosphate Buffered Saline (PBS), Borate Buffered Saline (BBS), and the like.

In the preparation of the solution or dispersion, if necessary, an auxiliary device such as ultrasonic waves, a high-pressure homogenizer, or the like may be used, or appropriate heating may be performed with stirring.

Type II: homogeneous phase chemiluminescence method detection reagent

The homogeneous phase chemiluminescence detection reagent mainly comprises: light absorbing donor microspheres and light emitting acceptor microspheres;

the light absorption donor microspheres comprise carrier microspheres and a light absorbent;

the luminescence acceptor microsphere comprises a carrier microsphere, a photochemical buffer agent and the composition of the rare earth luminescence complex of the second aspect of the invention.

Wherein the carrier microsphere is at least one selected from hydrogel microspheres, styrene polymer microspheres, microspheres formed by protein, silicon nano-microspheres and polymethyl methacrylate microspheres; preferably, the carrier microspheres are styrene polymer microspheres; further preferably, the carrier microsphere is a styrene polymer microsphere with amino, carboxyl, amido and/or aldehyde groups on the surface.

The light absorber is selected from porphyrin and phthalocyanine dyes, transition metal complexes, Quantum Dots (QDs), and derivatives or copolymers of these compounds.

The photochemical buffering agent is selected from the following structural formula:

wherein G and T are heteroatoms selected from O, S, Se and N;

R₁' and R₂' and R₄' to R₈' are each independently selected from H, hydroxyl, carboxyl, amino, mercapto, ester, nitro, sulfonic, halogen, amide, or alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, aryl, aralkyl, heteroaryl or heteroaralkyl having N, O or S, or combinations thereof, having 1 to 50, preferably 1 to 24, carbon atoms, such as 2 to 14, wherein the aryl, aralkyl, heteroaryl or heteroaralkyl optionally has one or more substituents L; and

l is selected from hydroxyl, carboxyl, amino, thiol, ester, nitro, sulfonic, halogen, amide, or alkyl, alkenyl, alkynyl, alkoxy, and alkylamino groups having 1 to 50, preferably 1 to 24, such as 2 to 14, or 6 to 12 carbon atoms, or combinations thereof; and

R₃' is an electron withdrawing group or an aryl group comprising an electron withdrawing group.

According to one embodiment of the present invention, for example, in the luminescence acceptor microsphere of the homogeneous chemiluminescence detection reagent, the mass of the rare earth complex is 0.01-50% of the mass of the support microsphere;

preferably, the mass of the rare earth complex is 5-10% of that of the carrier microsphere;

the molar ratio of the photochemical buffering agent to the rare earth complex is 1: 100-10: 1, preferably 1: 50-1: 1, and more preferably 1: 10-1: 1.

The preparation method of the light absorption donor microsphere and the light emitting acceptor microsphere comprises the following steps:

1) providing carrier microsphere, light absorbing agent, photochemical buffering agent and rare earth luminescent complex LnX_j；

2) Dispersing or adsorbing a light absorbing agent into carrier microspheres in dispersion liquid or solution to prepare light absorbing donor microspheres;

3) in dispersion or solution, a photochemical buffer agent and a rare earth luminescent complex LnX_jDispersing or adsorbing the carrier microspheres to prepare luminescent receptor microspheres;

the light absorbing agent, the photochemical buffering agent and the rare earth luminescent complex may be dissolved or dispersed in a suitable solvent, such as liquid paraffin, a mixture of benzyl alcohol-ethylene glycol and water, a mixture of mesitylene and ethanol, tetrahydrofuran, dichloromethane, etc. to form a solution. Forming the above materials into stable solution or dispersion, adding carrier microsphere or dispersion of carrier microsphere to obtain light absorbent, photochemical buffer agent and rare earth luminescent complex LnX_jAdsorbing or coating to obtain light-absorbing donor microsphere or light-emitting acceptorA bulk microsphere. Preferably, water or other suitable solvent may be used to disperse the carrier microspheres, such as deionized water, Phosphate Buffered Saline (PBS), Borate Buffered Saline (BBS), and the like.

Type III: fluorescence lateral immunochromatography detection reagent

The fluorescence lateral immunochromatography detection reagent mainly comprises long-afterglow luminescent microspheres. The long-afterglow luminescent microsphere comprises a carrier microsphere, a light absorbing agent, a photochemical buffering agent and the composition of the rare earth luminescent complex of the second aspect of the invention.

The long afterglow lateral immunochromatography is an improved technology of fluorescence lateral immunochromatography, long afterglow luminescent microspheres are used for replacing traditional luminescent (fluorescent and phosphorescent) microspheres, luminescent signals can be collected after exciting light is turned off, interference caused by background fluorescence in the immunoassay process can be effectively avoided, the detection sensitivity is improved, the detection limit is reduced, and accurate and high-sensitivity immunoassay is favorably realized.

Wherein the carrier microsphere is at least one selected from hydrogel microspheres, styrene polymer microspheres, microspheres formed by protein, silicon nano-microspheres and polymethyl methacrylate microspheres; preferably, the carrier microsphere is a styrene polymer microsphere, and further preferably, the carrier microsphere is a styrene polymer microsphere with an amino group, a carboxyl group, an amido group and/or an aldehyde group on the surface.

The photochemical buffering agent may be, for example, a compound represented by the following structural formula:

wherein G and T are heteroatoms selected from O, S, Se and N;

According to one embodiment of the present invention, for example, in the fluorescence lateral immunochromatography detection reagent, the mass of the rare earth complex is 0.01% to 50% of the mass of the carrier microsphere;

the molar ratio of the light absorber, the photochemical buffering agent and the rare earth complex is 0.001:1: 100-0.01: 1:1, preferably 0.001:1: 50-0.001: 1:1, and more preferably 0.001:1: 10-0.001: 1: 1.

The preparation method of the long afterglow luminescent microsphere comprises the following steps:

2) In dispersion or solution, light-absorbing agent, photochemical buffer agent and rare earth luminescent complex LnX_jDispersing or adsorbing to carriersPreparing long afterglow luminescent microsphere;

wherein the light absorbing agent, photochemical buffering agent and rare earth luminescent complex LnX can be added_jRespectively, in a suitable solvent such as liquid paraffin, a mixture of benzyl alcohol-ethylene glycol and water, a mixture of mesitylene and ethanol, tetrahydrofuran, dichloromethane, etc. to form a solution. Forming the above materials into stable solution or dispersion, adding carrier microsphere or dispersion of carrier microsphere to obtain light absorbent, photochemical buffer agent and rare earth luminescent complex LnX_jAnd adsorbing or coating to obtain the long-afterglow luminescent microsphere. Preferably, water or other suitable solvent may be used to disperse the carrier microspheres, such as deionized water, Phosphate Buffered Saline (PBS), Borate Buffered Saline (BBS), and the like.

In a fourth aspect, the invention discloses a kit for immunoassay, which comprises the reagent composition disclosed in the third aspect of the invention, and a standard substance and a diluent which are suitable for an immunoassay method. The immunoassay methods include, for example, time-resolved immunofluorescence, homogeneous chemiluminescence, and fluorescence lateral immunochromatography. The diluent comprises reagent components such as buffer solution, surfactant, preservative and the like. The buffer solution includes Phosphate Buffered Saline (PBS), phosphate buffered saline (PB), Borate Buffered Saline (BBS), 2- (N-morpholino) ethanesulfonic acid buffer (MES), and the like. The surfactant includes Sodium Dodecylbenzenesulfonate (SDBS), Sodium Dodecyl Sulfate (SDS), cetyltrimethylammonium bromide (CTAB), and the like.

In a fifth aspect, the invention provides a method of immunoassay. The immunoassay methods include, for example, time-resolved immunofluorescence, homogeneous chemiluminescence, and fluorescence lateral immunochromatography. The invention provides a polydentate beta-diketone ligand with strong chelating capacity, a rare earth luminescent complex with high stability is prepared for the ligand based on the compound, and the prepared rare earth luminescent complex is applied to the field of immunoassay, so that the luminescent performance of the rare earth complex in immunoassay is improved, and a better immunoassay effect is realized.

Drawings

FIG. 1 is a nuclear magnetic spectrum of an organic ligand X-1 prepared in an example of the present invention.

FIG. 2 is an infrared spectrum of complex Eu (X-1) prepared according to an example of the present invention.

FIG. 3 shows the emission luminance test of Eu (X-1) complex prepared according to an example of the present invention, wherein the Eu (X-1) solution is shown on the left, and Eu (TTA) is shown on the right as a control₃Phen solution.

FIG. 4 is a photostability test of the complex Eu (X-1), Eu (TTA), prepared according to an example of the present invention₃Phen was used as a control.

FIG. 5 shows the emission intensity test of Eu (X-13) complex prepared according to an example of the present invention, wherein the solid line represents Eu (X-13), and the dotted line represents Eu (TTA)₃Phen。

FIG. 6 shows the photostability test of complex Eu (X-13) prepared according to an example of the present invention.

FIG. 7 shows the luminescence of europium complexes prepared in the examples of the present invention after 1 hour of incubation in the usual dilutions for immunoassays, using Eu (TTA)₃Phen complex (left) and Eu (X-13) complex (right), the results show that Eu (X-13) complex (right) exhibits an overwhelming advantage, and Eu (TTA)₃The luminescence of the Phen complex is essentially quenched and precipitates are produced.

FIG. 8 is an electron micrograph of PS-Eu-X1 microspheres prepared according to example 17 of the present invention.

FIG. 9 shows the stability test of the components of the anti-immunoassay diluent in the examples of the present invention, in which both the luminescent microspheres made of Eu (X-13) and the luminescent microspheres made of Eu (X-1) were used, the stability was good.

FIG. 10 is a graph of luminescence signal versus time for a comparative example of the present invention, consisting of a conventional rare earth complex Eu (TTA)₃The luminescent microsphere prepared by Phen has poor performance of anti-dilution liquid.

FIG. 11 is an electron micrograph of a photoreceptor microsphere prepared in example 27 of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Preparation of multidentate beta-diketone ligand X-1

1-1) preparation method of tribenzyl methanol

Adding dibenzyl ketone (10.5g, 50mmol) and diethyl ether into a 250mL three-necked bottle, and stirring to dissolve; dropwise adding benzyl magnesium bromide (100mmol, 100mL) into the bottle at room temperature, continuously stirring at room temperature, and monitoring the reaction process by a TLC silica gel plate; after the reaction, placing the reaction bottle in an ice bath, dropwise adding a 5% sulfuric acid solution (150mL), continuously stirring for 30min, and separating liquid; extracting the water phase with ethyl acetate for three times, and then combining the organic phases; the organic phase was dried, spun dried and column chromatographed to give 5.6g of a white powdery solid in 92.7% yield and the structure was confirmed as follows:

¹H NMR(400MHz,CDCl₃)δ7.31(m,1H),7.30–7.27(m,5H),7.26–7.23(m,3H),7.23–7.19(m,7H),2.76(s,6H),1.49(s,1H)。

1-2) preparation method of tri-substituted olefin

Triethylsilane (27.4mL,172mmol) and DCM (ultra-dry, 100mL) were added to a 250mL eggplant-shaped flask and dissolved with stirring at room temperature; boron trifluoride etherate (12.4mL, 100mmol) was added dropwise to the flask and the product of step 1) (6.04g in 20mL DCM) was added dropwise at 0 deg.C, stirring was continued and the progress of the reaction was monitored by TLC. After the reaction is finished, dropwise adding a saturated sodium chloride solution into the reaction system, quenching and separating liquid; extracting the water phase with dichloromethane for three times, and combining the organic phases; the organic phase is dried, dried by spinning and subjected to column chromatography to obtain 5.13g of colorless clear transparent oily liquid, the yield is 90 percent, and the structure is confirmed as follows:

¹H NMR(400MHz,CDCl₃)δ7.31–7.28(m,2H),7.27–7.23(m,5H),7.23–7.19(m,2H),7.19–7.14(m,6H),6.56(s,1H),3.54(s,2H),3.37(s,2H)。

1-3) preparation method of tribenzylmethane

In a 25mL eggplant type bottle, 1-2) of the obtained product (2.83g, 10mmol), ethyl acetate (4.4mL) and methanol (0.6mL) were added and dissolved with stirring at room temperature; adding Pd/C (0.212g, 1% mmol) into the reaction flask, sealing the reaction system with an insulating tape, and inserting a hydrogen balloon (hydrogen balloon does not have to be inserted below the liquid level); placing the reaction bottle in an oil bath at 25 ℃ and stirring at constant temperature overnight; after TLC monitoring the disappearance of the starting material, the reaction solution was filtered on silica gel, the filtrate was spin-dried, and column chromatography gave 2.3g of a white crystalline solid in 81% yield, with the following structure:

¹H NMR(400MHz,CDCl₃)δ7.28(m,1H),7.26(m,2H),7.25–7.23(m,3H),7.19–7.14(m,3H),7.11(m,6H),2.55(d,J＝6.9Hz,6H),2.29(dt,J＝14.0,7.0Hz,1H)。

1-4) Friedel-Crafts acylation reaction to prepare tri (p-acetyl) benzyl methane

To a 100mL eggplant-shaped flask were added aluminum trichloride (3.9g, 16.5mmol) and DCE (15mL), and to the flask was added acetyl chloride (1.3g, 16.5mmol) with stirring, and stirring was continued for 10 min. After 10min, the product obtained in step 3) (1.42g, 5mmol in 15mL DCE) was added to the reaction flask, stirred at 0 ℃ and the progress of the reaction was monitored by TLC; after the reaction is finished, adding water for quenching, and separating liquid; the aqueous phase was washed three times with dichloromethane and the organic phases combined; washing the organic phase with dilute hydrochloric acid, and separating liquid; washing the organic phase with saturated sodium carbonate solution, and separating liquid; the organic phase is dried, spin-dried and column chromatographed to give 1.6g of pale yellow oily liquid, yield: 97.8%, the structure was confirmed as follows:

¹H NMR(400MHz,CDCl₃)δ7.85(d,J＝8.3Hz,6H),7.17(d,J＝8.2Hz,6H),2.59(d,J＝7.0Hz,6H),2.55(s,9H)。

1-5) Knoevenagel condensation reaction

Adding the product (0.325g, 1mmol) obtained in the step 4) and THF (5mL) into a 15mL sealed tube, and stirring to dissolve; ethyl pentafluoropropionate (0.55g, 3mmol) and sodium methoxide (0.16g, 3mmol) were added to the reaction flask at room temperature, stirred at room temperature, and the progress of the reaction was monitored by TLC; after the reaction is finished, dropwise adding a dilute sulfuric acid solution into the reaction system to neutralize the reaction solution, extracting the water phase with ethyl acetate for three times, and combining organic phases; the organic phase was dried, spun dry, and recrystallized by the addition of ethanol to give 0.52g of a pale yellow powdery solid, 67.9% yield, structure confirmed as follows:

¹H NMR(400MHz,CDCl₃) δ 7.88(d, J ═ 8.4Hz,6H),7.23(d, J ═ 8.4Hz,6H),6.60(s,3H),2.65(d, J ═ 7.0Hz,6H),2.40(dd, J ═ 13.7,6.9Hz, 1H). The NMR spectrum is shown in FIG. 1.

Example 2

Preparation of multidentate beta-diketone ligand X-2

Based on the preparation of ligand X-1 in example 1, steps 1-2) and 1-3) were omitted and the ethyl pentafluoropropionate of step 1-5) was replaced with C₂H₅COOC₂H₅And other reaction conditions are similar, so that the hexadentate ligand X-2 is obtained.

Example 3

Preparation of multidentate beta-diketone ligand X-3

On the basis of the preparation of the ligand X-2 in the embodiment 2, the tribenzyl methanol obtained in the first step is converted into tribenzyl sodium methoxide, and then the tribenzyl sodium methoxide is reacted with methyl iodide to generate tribenzyl methyl ether, and other reaction conditions are similar to obtain the hexadentate ligand X-3.

Example 4

Preparation of multidentate beta-diketone ligand X-8

Based on the preparation of the ligand X-1 in example 1, the raw material dibenzyl ketone used in 1-1) was replaced by dibenzyl acetone, and the raw material ethyl pentafluoropropionate used in 1-5) was replaced by PhCOOC₂H₅Wherein Ph is phenyl and the other reaction conditions are similar, to give the hexadentate ligand X-8.

Example 5

Preparation of multidentate beta-diketone ligand X-13

Similarly, the synthetic procedures for X-13 to X-20 and other analogous X ligands are essentially the same as for X-1 above. Taking X13 as an example: the following synthetic route for the X-13 ligand differs from the synthetic route for X-1 in that the initial starting material dibenzyl ketone is replaced with 1, 5-diphenyl-2-pentanone.

Example 6

Preparation of multidentate beta-diketone ligand X-14

The following synthetic route for the X-14 ligand differs from the synthetic route for X-13 in that the methyl iodide in the synthetic route is replaced by ethyl iodide.

Example 7

Preparation of multidentate beta-diketone ligand X-18

The following synthetic route for the X-18 ligand differs from the synthetic route for X-13 in that the starting material 1, 5-diphenyl-2-pentanone is replaced by 1, 16-diphenyl-6-hexadecanone and the other starting materials are replaced as shown in the synthetic route.

Thus, using a method analogous to that of examples 1 to 7, X-1 to X-20 and other analogous X ligands can be synthetically obtained.

Example 8

Preparation of rare earth complexes

Firstly, taking X-1 as a ligand and europium (Eu) as a coordination center to prepare a rare earth complex Eu (X-1).

The preparation method comprises the following steps:

8-1) adding 0.038g X-1 and acetone as a solvent into a 250mL eggplant-shaped bottle, and stirring at room temperature to dissolve;

8-2) 0.018g of EuCl₃·6H₂Dissolving O in ethanol, and adding into a reaction system;

8-3) placing the reaction system in an oil bath at 80 ℃, heating and stirring, and adding a dilute sodium hydroxide solution into the reaction system to adjust the pH of the reaction solution to 4-5;

8-4) placing the reaction system in an oil bath at 80 ℃, heating, stirring and refluxing overnight, and then adding deionized water into a reaction bottle until an orange precipitate just appears;

8-5) heating and stirring the reaction system at 80 ℃, and after precipitating particles are gradually condensed into large particles, treating to obtain 0.039g of orange-white precipitate which is a composition containing the complex Eu (X-1);

8-6) recrystallizing and purifying to obtain the complex Eu (X-1).

During the synthesis, the Eu (X-1) complex was found to have a very bright red emission under UV lamp illumination, and its structure was further confirmed by IR spectrum data, as shown in FIG. 2.

Eu (X-2) to Eu (X-20) and other europium complexes with X as the ligand are prepared in the same manner as Eu (X-1).

In addition, the rare earth ions are made of europium (Eu) by the same method³⁺) By replacing with other rare earth ions (Ln)³⁺) Can prepare other rare earth complexes, such as terbium complex, samarium complex and the like, and the rare earth complexes have better luminescent property, and the luminescent wavelength can be adjusted according to the requirementAnd (e.g., terbium complex can emit green light, samarium complex can emit deep red light). Therefore, the Tb (X-1) complex and the Sm (X-1) complex are also prepared by the present invention in the same manner.

Example 9

This example tests the luminescence of a Eu (X-1) rare earth complex as Eu (TTA)₃Phen (using conventional bidentate ligands) for comparison, gives the complexes Eu (X-1) and Eu (TTA)₃The light emission luminance of Phen was compared.

Wherein, Eu (TTA)₃Phen is a reported complex, collectively referred to as: (1, 10-phenanthroline) tris [4,4, 4-trifluoro-1- (2-thienyl) -1, 3-butanedione]Europium (III), the structure is as follows:

as shown in FIG. 3, in DMF solution, Eu (X-1) in the left bottle and Eu (TTA) in the right bottle₃The concentrations of Phen were all 10. mu.M. However, the Eu (X-1) in the bottle on the left side has a significantly higher luminous intensity under the irradiation of the ultraviolet lamp than the Eu (TTA) on the right side₃Phen. The patent shows that the high-performance rare earth luminescent complex has obvious advantages in constructing the high-performance rare earth luminescent complex, and particularly has the advantage of improving the luminescent brightness. The improvement of the light-emitting brightness has important significance for many practical applications, such as increasing the signal intensity in immunoassay, and improving the accuracy and sensitivity of the assay.

Example 10

This example tests the photostability of Eu (X-1) rare earth complexes, and uses Eu (TTA)₃Phen for comparison, the complexes Eu (X-1) and Eu (TTA)₃Comparison of the photostability of Phen.

In DMF solution, Eu (X-1) and Eu (TTA)₃Phen concentrations were 10. mu.M, and both samples were irradiated with UV LED lamps (365 nm wavelength) of the same power density, respectively. As shown in FIG. 4, Eu (X-1) showed a decrease in luminescence intensity of 2% of initial intensity after half an hour of irradiation (i.e., 98% of initial intensity, where luminescence initial intensity is 445540), and a decrease in luminescence intensity after one hour of irradiationA reduction of 6% (i.e. 94% of the initial strength) is not significant, whereas Eu (TTA)₃The half hour drop in the luminescence intensity of Phen (i.e., 50% of the initial intensity) was more pronounced. The patent shows that the high-performance rare earth luminescent complex has obvious advantages in constructing the high-performance rare earth luminescent complex, and particularly has the aspect of improving the light stability. The improvement of the light stability has important significance for many practical applications, for example, the stability of signal acquisition in immunoassay can be increased, and then the error caused by the signal acquisition is reduced and the accuracy of detection is improved.

Example 11

This example tests the luminescence of a Eu (X-13) rare earth complex as Eu (TTA)₃Phen for comparison, the complexes Eu (X-13) and Eu (TTA)₃Comparison of the luminescence intensity of Phen.

In DMF solution, Eu (X-13) and Eu (TTA)₃The concentrations of Phen were all 10. mu.M. However, as shown in FIG. 5, Eu (X-13) emitted light is significantly higher than Eu (TTA) under the same test conditions using the Edinburgh FS-5 instrument₃Phen. The patent shows that the method has remarkable advantages in constructing high-performance rare earth luminescent complexes, particularly in improving the luminescent intensity, and the method has better universality and universality in the aspect.

Example 12

This example tested the photostability of Eu (X-13) rare earth complex under the same test conditions as in example 10.

Eu (X-13) concentration in DMF solution was 10 μ M, and the sample was irradiated with an ultraviolet LED lamp (wavelength: 365nm) having the same power density as in example 10. As shown in FIG. 6, Eu (X-13) showed little decrease in emission intensity after one hour of irradiation, and the emission intensity was stable during one hour of irradiation. This result confirmed that Eu (X-13) has very strong photostability in combination with example 10. The method has obvious advantages in constructing high-performance rare earth luminescent complexes, particularly in improving light stability, and has good universality and universality.

Example 13

This example tests the luminescent properties of the rare earth luminescent complexes in common diluents for immunodetection. As shown in FIG. 7, Eu (X-13) (Right) and Eu (TTA) were added to two bottles containing the diluent for immunoassay₃Phen (left) in acetone, both complexes at a concentration of 10. mu.M, in a dilution containing 10mmol/L PBS buffer and 1mmol/L surfactant SDBS. Then, the plate was left for one hour in the dark, after which the luminescence was measured. Eu (X-13) (right) was tested to have an initial emission intensity of 623760, and to have an emission intensity of 620000 or more after one hour. Also, as shown in FIG. 7, the Eu (X-13) bottle on the right exhibits bright luminescence under irradiation of the ultraviolet lamp, while the Eu (TTA) bottle on the left₃The Phen bottle had essentially no luminescence. Eu (TTA) on the left₃The Phen luminescence was severely quenched and precipitates were found in the bottom of the bottle, mainly due to the buffer and surfactant pairs Eu (TTA) in the dilution₃Complexation of the Phen rare earth complex leads to quenching, and makes Eu (TTA)₃Phen bound to it and agglomerated to settle. Based on the strategy of the invention, the strong chelating coordination effect of the ligand and the rare earth ensures that the complex is very stable and is not easily interfered by the external environment, so that the complex still has bright luminescence in a diluent containing a buffer reagent and a surfactant and keeps clear and transparent. The patent shows that the rare earth luminescent complex has obvious advantages in constructing high-performance rare earth luminescent complexes, and particularly improves the stability of the rare earth luminescent complexes in buffer solution.

Example 14

This example tests the luminescence properties of the rare earth luminescent complex Eu (X-14) in a dilution commonly used in immunoassays. The experimental conditions and test methods were the same as in example 13. Eu (X-14) (right) tested had an initial emission intensity of 667420, which was 660000 or higher after one hour.

Example 15

This example tests the luminescence properties of the rare earth luminescent complex Eu (X-18) in a dilution commonly used in immunoassays. The experimental conditions and test methods were the same as in example 13. Eu (X-14) (right) tested had an initial emission intensity of 605047, and was 600000 or more after one hour.

Example 16

A plurality of bottles filled with immunoassay diluent are respectively added with acetone solutions of Eu (X-1), Eu (X-2), Eu (X-3), Eu (X-7), Eu (X-8), Eu (X-11), Eu (X-13), Eu (X-14) and Eu (X-18), wherein the concentration of each complex is 10 mu M, and the diluent contains 10mmol/L PBS buffer reagent and 1mmol/L surfactant SDBS. Then, the mixture is placed for one hour in a dark place, each bottle is bright and luminous under the irradiation of an ultraviolet lamp, no precipitate is generated, and the complex is very stable and is not easily interfered by the external environment due to the strong chelating coordination of the ligand and the rare earth, so that the mixture still can brightly emit light in a diluent containing a buffer reagent and a surfactant and is kept clear and transparent.

Comparative example 1

Although some ligands of the prior art may have strong chelating ability, such as may be used for binding separation of metal ions, for example, aminocarboxylic polydentate ligands or amide polydentate ligands are typical representatives of these ligands, these ligands are not suitable for constructing luminescent rare earth complexes because they are associated with rare earth ions (e.g., Eu)³⁺) The complexes formed after chelation have no luminescent properties or are difficult to achieve luminescence. For example, the multidentate ligand MNO is reacted with the rare earth Eu³⁺The ionic complex emits very weak light, and tests show that the luminescent intensity of the complex is only 5% or less of that of Eu (X-1), and the weak light emission is difficult to carry out applications such as immunoassay and the like.

It can be seen that the complexing problem and the effect of the present invention can be solved not only by one-sided pursuit of chelating ability. Therefore, it is a creative work to develop organic ligands with strong chelating ability and efficiently form luminescent complexes with rare earth ions, and a skillful design and a great deal of research and study are required.

Example 17

Luminescent microspheres are prepared using rare earth luminescent complexes.

Taking the simplest time-resolved luminescent microsphere as an example, the time-resolved luminescent microsphere comprises a composition of a carrier microsphere and a rare earth luminescent complex. The main core component of the time-resolved luminescent microsphere is a rare earth luminescent complex, and the service life of the common rare earth luminescent complex can reach the level of microsecond to millisecond, so that luminescent signals can be acquired by using a time-resolved technology. Therefore, when the time resolution technology is not used, the time resolution luminescent microsphere can also be used for direct luminescent intensity test and analysis, and has the functions of common fluorescent or phosphorescent luminescent microspheres.

Wherein the carrier microsphere is selected from hydrogel microspheres, styrene polymer microspheres, microspheres formed by protein, silicon nano-microspheres, polymethyl methacrylate microspheres and at least one of the hydrogel microspheres, the styrene polymer microspheres and the microspheres; preferably, the carrier microsphere is a styrene polymer microsphere, and further preferably, the carrier microsphere is a styrene polymer microsphere with an amino group, a carboxyl group, an amido group and/or an aldehyde group on the surface.

The time-resolved luminescent microsphere can be prepared according to the following preparation method:

1) providing a carrier microsphere, and a rare earth luminescent complex LnX_j；

wherein, the rare earth luminescent complex LnX can be prepared_jRespectively, in a suitable solvent such as liquid paraffin, a mixture of benzyl alcohol-ethylene glycol and water, a mixture of mesitylene and ethanol, tetrahydrofuran, dichloromethane, etc. Forming the substances into stable solution or dispersion, adding carrier microspheres or dispersion of the carrier microspheres, and performing swelling adsorption, emulsion polymerization, miniemulsion polymerization, microemulsion polymerization and other methods to obtain the rare earth luminescent complex LnX_jAnd adsorbing or coating to prepare the time-resolved luminescent microsphere. Preferably, water or other suitable solvent may be used to disperse the carrier microspheres, for exampleDeionized water, Phosphate Buffered Saline (PBS), Borate Buffered Saline (BBS), and the like.

According to the method, in this example, a Polystyrene (PS) microsphere with a particle size of 200nm is used as a carrier microsphere, and Eu (X-1) is used as a composition of the rare earth luminescent complex, so as to prepare a time-resolved luminescent microsphere (for convenience of differentiation, the microsphere is named as PS-Eu-X1, and an electron micrograph of the PS-Eu-X1 microsphere is shown in fig. 8, wherein the mass fraction of the rare earth luminescent complex in the luminescent microsphere is 2%); then, using the same method, Eu (X-13) is used as the composition of the rare earth luminescent complex, and a time-resolved luminescent microsphere (named PS-Eu-X13 for the convenience of differentiation) is prepared.

PS-Eu-X13 microspheres and PS-Eu-X1 microspheres are respectively added into two cuvettes filled with immunoassay diluent with the same volume, wherein the adding amount of the two microspheres is 5mg, and the diluent contains 10mmol/L PBS buffer reagent and 1mmol/L surfactant SDBS. Then, the light emission was continued for 4 hours after the test. As shown in FIG. 9, both PS-Eu-X1 and PS-Eu-X13 microspheres exhibited stable and efficient luminescence under the same test conditions using the Edinburgh FS-5 instrument, and PS-Eu-X13 was superior in luminescence intensity and stability.

Based on the above results, it is demonstrated that the luminescent microspheres containing rare earth complexes all have excellent stability and luminescent intensity. Further analysis shows that, as shown in FIG. 9, the PS-Eu-X13 microspheres have better performance: in the aspect of initial luminous intensity, the PS-Eu-X13 microsphere is more than 30% higher than the PS-Eu-X1 microsphere; in terms of stability, after being soaked in the immunoassay diluent for 4 hours, the luminescence intensity of the PS-Eu-X1 microsphere is reduced by 4% compared with the initial state, and the luminescence intensity of the PS-Eu-X13 microsphere is reduced by less than 0.5%. The structure determines the performance, and the advantage of the luminescent performance is mainly attributed to the reason of the molecular structure design, especially the ingenious design of the organic ligand. For improving the luminous intensity and stability of the rare earth complex, the organic ligand of the invention is superior to the traditional organic ligand, mainly because: the polydentate organic ligand has strong chelating and coordinating capacity with the rare earth, and the organic ligand can be effectively subjected to energy level matching with the rare earth, so that the rare earth is firmly fixed on the organic ligand, and an efficient rare earth luminescent complex is formed. In this respect, the organic ligand X-13 has superior properties to X-1, mainly because of the more unique structural design: the organic ligands X-1 and X-13 are all in a trigeminal structure, the trigeminal structure is intersected with a central C atom, three chains containing beta-diketone correspond to the trigeminal structure, and the length, rigidity and steric hindrance of the trigeminal structure are regulated and controlled by the values of m, n and k in the formula (X). Except that in X-1 the beta-diketones are at the same distance from the central C atom (m, n, k are the same and all are 1), while in X-13 the beta-diketones in one chain are at a different distance from the central C atom than in the other two chains (m, n are both 1, but k is 3). When the organic ligand is coordinated with rare earth to form a complex, the beta-diketone on the trifurcate is mainly chelated and coordinated with the rare earth ions. Compared with the X-1 ligand, the X-13 ligand skillfully reduces the steric hindrance between the three trigeminals due to the adoption of the molecular design of lengthening the third trigeminal to ensure that the length of the third trigeminal is different from that of the other two trigeminals, the beta-diketone of the third trigeminal can extend to the vicinity of the beta-diketone of the other two trigeminals from the direction with smaller steric hindrance, the rigidity of the three-dimensional configuration formed by the coordination of the beta-diketone and the rare earth is reduced, and the chelation coordination of the beta-diketone on the trigeminal and a rare earth ion is facilitated, so that the stability and the luminous intensity of the formed rare earth luminous complex Eu-X35.

In this example, the immunoassay diluent used above was replaced with deionized water and the experiment was repeated. It was found that the initial luminescence intensity of the two microspheres in aqueous solution did not change significantly and remained stable compared to the immunoassay dilution.

Based on the strategy of the invention, the strong coordination effect between the ligand and the rare earth ions ensures that the complex is very stable and is not easily interfered by the external environment, so that the PS-Eu-X1 and PS-Eu-X13 microspheres can still have stable and efficient luminescence in the diluent containing the buffer reagent and the surfactant. The method has high feasibility in constructing the microspheres of the high-performance rare earth luminescent complex, particularly ensures the stability of the microspheres in a diluent containing a buffer reagent and a surfactant, and has high universality and universality.

Comparative example 2

In addition, in a similar manner to example 17, Polystyrene (PS) microspheres having a particle size of 200nm were used as carrier microspheres, and Eu (TTA)₃Phen is a composition of comparative rare earth luminescent complexes, and another comparative luminescent microsphere (named PS-Eu-M for easy differentiation) was also prepared.

5mg of PS-Eu-M microspheres were added to a cuvette containing the same volume of immunoassay diluent as in example 17, which also contained 10mmol/L of PBS buffer and 1mmol/L of surfactant SDBS. Then, the change in luminescence was measured within 2 hours. As shown in FIG. 10, the PS-Eu-M microspheres started to emit relatively weak light under the same test conditions as in example 17 using the Edinburgh FS-5 instrument, and did not emit light at all after 2 hours. The luminescence of the PS-Eu-M microspheres of this comparative example was severely quenched compared to example 13.

To clearly illustrate the cause of severe quenching, in this comparative example, the immunoassay diluent used above was replaced with deionized water and the experiment was repeated. Experiments show that the PS-Eu-M microspheres have weak initial luminescence in aqueous solution, but the luminescence is not obviously quenched after 2 hours.

Thus, in comparison with example 17, the PS-Eu-M microspheres of this comparative example were severely quenched in the immunoassay dilution, mainly due to the quenching effect caused by the complexation of the rare earth complex by the buffer reagent and the surfactant in the dilution.

Based on the strategy of the invention, the strong coordination effect of the ligand and the rare earth ensures that the complex is very stable and is not easily interfered by the external environment, so that the PS-Eu-X1 and PS-Eu-X13 microspheres can still have bright luminescence in the diluent containing the buffer reagent and the surfactant. The patent shows that the preparation method has obvious advantages in constructing the microspheres of the high-performance rare earth luminescent complex, and particularly improves the stability of the microspheres in common reagents for immunoassay.

Example 18

A rare earth complex Eu (X-20) was prepared in accordance with the method of example 8. The microsphere preparation method of example 17 was repeated to prepare PS-Eu-X20 microspheres from Eu (X-20) complex. The PS-Eu-X20 microspheres were then tested according to the same test method as in example 17.

5mg of PS-Eu-X20 microspheres were added to a cuvette containing the same volume of immunoassay diluent as in example 17, which contained 10mmol/L of PBS buffer and 1mmol/L of surfactant SDBS. Then, the change in luminescence was measured within 2 hours. The initial emission intensity and the emission intensity data after 2 hours are shown in table 1.

Example 19

The experiment of example 18 was repeated except that the dilution was replaced with an aqueous solution containing 10mmol/L of PBS buffer without the surfactant SDBS. The initial emission intensity and the emission intensity data after 2 hours are shown in table 1.

Example 20

The experiment of example 18 was repeated except that the dilution was replaced with an aqueous solution containing only 1mmol/L of the surfactant SDBS without PBS buffer. The initial emission intensity and the emission intensity data after 2 hours are shown in table 1.

Example 21

The experiment of example 18 was repeated except that the dilution was replaced with an aqueous solution containing only 1mmol/L of surfactant SDS without PBS buffer. The initial emission intensity and the emission intensity data after 2 hours are shown in table 1.

Example 22

The experiment of example 18 was repeated, except that the dilution contained only 10mmol/L PB buffer, i.e. only PB buffer was added to deionized water. The initial emission intensity and the emission intensity data after 2 hours are shown in table 1.

Example 23

The experiment of example 18 was repeated, except that only 10mmol/L MES buffer was contained in the dilution, i.e., only MES buffer was added to deionized water. The initial emission intensity and the emission intensity data after 2 hours are shown in table 1.

Example 24

The experiment of example 18 was repeated, except that only 10mmol/L of BBS buffer was contained in the dilution, i.e. only BBS buffer was added to deionized water. The initial emission intensity and the emission intensity data after 2 hours are shown in table 1.

Example 25

The experiment of example 18 was repeated except that only 1mmol/L of CTAB surfactant was contained in the dilution, i.e. only CTAB surfactant was added to deionized water. The initial emission intensity and the emission intensity data after 2 hours are shown in table 1.

Comparative example 3

A rare earth complex Eu (M-1) was prepared according to the method of example 8, using M-1 as a ligand. The microsphere preparation method of example 17 was repeated to prepare PS-Eu-M1 microspheres from Eu (M-1) complex. The PS-Eu-M1 microspheres were then tested according to the same test method as in example 17.

5mg of PS-Eu-M1 microspheres were added to a cuvette containing the same volume of immunoassay diluent as in example 17, which contained 10mmol/L of PBS buffer and 1mmol/L of surfactant SDBS. Then, the change in luminescence was measured within 2 hours. The initial emission intensity and the emission intensity data after 2 hours are shown in table 1.

Comparative example 4

A rare earth complex Eu (M-2) was prepared according to the method of example 8, using M-2 as a ligand. The microsphere preparation method of example 17 was repeated to prepare PS-Eu-M2 microspheres using Eu (M-2) complex. The PS-Eu-M2 microspheres were then tested according to the same test method as in example 17.

5mg of PS-Eu-M2 microspheres were added to a cuvette containing the same volume of immunoassay diluent as in example 17, which contained 10mmol/L of PBS buffer and 1mmol/L of surfactant SDBS. Then, the change in luminescence was measured within 2 hours. The initial emission intensity and the emission intensity data after 2 hours are shown in table 1.

Comparative example 5

Comparative example 3 was repeated except that PBS/SDBS in the dilution was changed to BBS/SDBS. The initial emission intensity and the emission intensity data after 2 hours are shown in table 1.

Comparative example 6

The procedure in example 17 is repeated for the preparation of microspheres of Eu (TTA)₃The Phen complex is used for preparing PS-Eu-M microspheres. The PS-Eu-M microspheres were then subjected to the test in accordance with the same test method as in example 17.

5mg of PS-Eu-M microspheres were added to a cuvette containing the same volume of immunoassay diluent as in example 17, the diluent containing 10mmol/L MES buffer and 1mmol/L CTAB surfactant. Then, the change in luminescence was measured within 2 hours. The initial emission intensity and the emission intensity data after 2 hours are shown in table 1. The initial emission intensity and the emission intensity data after 2 hours are shown in table 1.

Table 1: variation of luminescence intensity of several kinds of microspheres

Example 26

The PS-Eu-X13 has long luminescence life, and tau is 1 ms. Based on the excellent light-emitting brightness and stability characteristics demonstrated in the above embodiments, it can be used in a time-resolved immunoassay method to perform highly sensitive accurate detection on a target.

Example 27

Preparing a homogeneous chemiluminescence detection reagent composition and a kit.

The method for preparing the light absorption donor microsphere, the light emitting acceptor microsphere and the kit for immunoassay comprises the following steps:

light-absorbing donor microspheres comprising a support microsphere and a light absorber;

a luminescent receptor microsphere comprising a composition of a support microsphere, a photochemical buffering agent and the rare earth luminescent complex of the second aspect of the invention.

The photochemical buffering agent is a compound shown in the following structural formula:

g and T are heteroatoms selected from O, S, Se and N;

2) Dispersing or adsorbing a light absorbing agent into carrier microspheres in dispersion liquid or solution, and preparing light absorbing donor microspheres by methods of swelling adsorption, emulsion polymerization, miniemulsion polymerization, microemulsion polymerization and the like;

3) in dispersion or solution, a photochemical buffer agent and a rare earth luminescent complex LnX_jDispersing or adsorbing the carrier microspheres, and preparing luminescent receptor microspheres by methods such as swelling adsorption, emulsion polymerization, miniemulsion polymerization, microemulsion polymerization and the like;

wherein the light absorbing agent, photochemical buffering agent and rare earth luminescent complex LnX can be added_jRespectively, in a suitable solvent such as liquid paraffin, a mixture of benzyl alcohol-ethylene glycol and water, a mixture of mesitylene and ethanol, tetrahydrofuran, dichloromethane, etc. Forming the above materials into stable solution or dispersion, adding carrier microsphere or dispersion of carrier microsphere, and performing swelling adsorption, emulsion polymerization, miniemulsion polymerization, and microemulsion polymerization to obtain light absorbent, photochemical buffer agent and rare earth luminescent complex LnX_jAbsorbing or coating to prepare light absorption donor microspheres or light-emitting acceptor microspheres. Preferably, water or other suitable solvent may be used to disperse the carrier microspheres, such as deionized water, Phosphate Buffered Saline (PBS), Borate Buffered Saline (BBS), and the like.

In the embodiment, palladium phthalocyanine (PdPpc) is used as a light absorbent, and Polystyrene (PS) microspheres with the particle size of 200nm are used as carrier microspheres to prepare light absorption donor microspheres; an electron microscope image of the luminescence acceptor microsphere is shown in fig. 11, wherein organic small molecule NSOF is used as a photochemical buffering agent, Eu (X-1) is used as a composition of the rare earth luminescence complex, and Polystyrene (PS) microsphere with the particle size of 200nm is used as a carrier microsphere, so that the luminescence acceptor microsphere is prepared, and the electron microscope image of the luminescence acceptor microsphere is shown in fig. 11, (wherein the mass fraction of the rare earth luminescence complex in the luminescence acceptor microsphere is 5%, and the mass fraction of the chemical buffering agent NSOF in the luminescence acceptor microsphere is 5%). The Polystyrene (PS) microspheres are formed by polymerizing styrene and acrylic acid through monomers, so that the surfaces of the PS microspheres contain carboxyl groups, and the PS microspheres can be used for subsequent bioconjugation.

For microsphere conjugation to the biomarker-specific binding counterpart, this example schematically shows a simple method of bioconjugation in Procalcitonin (PCT) immunoassays as follows:

centrifuging the light-absorbing donor microspheres prepared in the embodiment at a high speed, washing the microspheres for 3 times by using BBS buffer solution, finally fixing the volume to 0.5 wt% of solid content, and performing ultrasonic treatment to uniformly disperse the microspheres; mu.L of the above dispersion was added to 500. mu.L of MES and 10mg of carbodiimide (EDC), and reacted at room temperature for 2 hours. After completion of the reaction, the reaction mixture was centrifuged and washed, and each of the solutions was redissolved in 500. mu.L of PBS buffer, to which 0.01mg of PCT-Ab1 monoclonal antibody was added, and reacted at room temperature for 24 hours. After the reaction was completed, the mixture was stirred at 16000 rpm, washed by centrifugation, and redissolved in 500. mu.L of PBS buffer. 10mg of BSA was added thereto, and the reaction was carried out at room temperature for 8 hours. After the reaction, the microspheres were washed by centrifugation to obtain light-absorbing donor microspheres coated with PCT-Ab1 antibody, and weighed 1 mg. This was redissolved in 3.0mL BBS buffer and stored at 4 ℃ until use.

The coupling method of the luminescence receptor microsphere is similar, and the PCT-Ab2 monoclonal antibody is coupled on the luminescence receptor microsphere prepared in the embodiment to obtain the luminescence receptor microsphere coated with the PCT-Ab2 antibody. 1mg of this was redissolved in 3.0mL of BBS buffer and stored at 4 ℃ until use.

Diluting the microspheres to solid content of 0.01%, mixing the diluted two microspheres with 100mM BBS buffer solution (containing 0.5% PEG, 0.1% SDS, 0.9% NaCl, 0.1% KCl, 0.5% BSA, pH 7.0-pH 7.6) according to a volume ratio of 1:1, filling the mixture into a reagent bottle to obtain the kit for PCT homogeneous phase chemiluminescence immunoassay, and storing the kit in a dark place at the temperature of 2-8 ℃.

Example 28

A homogeneous chemiluminescence-based detection reagent composition and a detection application of the kit.

When the kit is prepared, the skilled person can carry out conventional homogeneous assays using means common in the art. Example 27 was repeated to prepare a kit for PCT homogeneous chemiluminescent immunoassay PCT-1. The kit is used for detecting the PCT, wherein the detection limit of the PCT-1 kit on the PCT is 15pg/mL, and the detection limit can meet the test requirement of clinical PCT.

The above operation uses a conventional homogeneous chemiluminescent detection method, and the present inventors have found that in the homogeneous detection, in order to further simplify the test instrument and equipment and obtain an optimized detection effect, a long afterglow technique, i.e., signal acquisition after complete shut-off of excitation light, can be preferably introduced. The long afterglow technology can avoid the scattering of exciting light and the interference of autofluorescence, avoid the complicated instrument problem caused by using optical filter and other equipment elements, and lower the requirement and cost of equipment. In addition, experiments show that the detection limit of the homogeneous detection improved by the long afterglow technology is further reduced, and the requirement of high-sensitivity detection is better met.

Example 29

When the kit is prepared, routine homogeneous assays can be performed by those skilled in the art according to well-known techniques. Kit PCT-2 for PCT homogeneous chemiluminescent immunoassay was prepared according to example 27, replacing Eu (X-1) with Eu (X-13) under otherwise identical conditions. PCT was detected using the kit according to the method of example 28, wherein PCT-2 kit had a detection limit of 10pg/mL, which met the clinical PCT testing requirements.

The above operation uses a conventional homogeneous chemiluminescent detection method, and the present inventors have found that in this homogeneous detection, in order to further simplify the test instrument and equipment and obtain an optimized detection effect, the present invention may preferably incorporate a long afterglow technique, i.e., signal acquisition after the excitation light is completely turned off. The long afterglow technology can avoid the scattering of exciting light and the interference of autofluorescence, avoid the complicated instrument problem caused by using optical filter and other equipment elements, and lower the requirement and cost of equipment. In addition, experiments show that the detection limit of the homogeneous detection improved by the long afterglow technology is further reduced to 5pg/mL, so that the requirement of high-sensitivity detection is better met.

Comparative example 7

Replacement of Eu (X-1) by Eu (TTA) according to example 27₃Phen, the other conditions are consistent, and a kit PCT-3 for PCT homogeneous phase chemiluminescence immunoassay is prepared. The PCT is respectively detected by using the kit according to the conventional detection method in the example 27, and the result shows that the detection limit of the PCT-3 kit on the PCT is 5ng/mL, so that the test requirement of clinical PCT is difficult to meet.

Comparative example 8

According to example 27, the kit PCT-4 for PCT homogeneous chemiluminescent immunoassay was prepared by replacing Eu (X-1) with Eu (M-2) under otherwise identical conditions. The PCT is respectively detected by using the kit according to the conventional detection method in the example 27, and the result shows that the detection limit of the PCT-4 kit on the PCT is 1ng/mL, so that the test requirement of clinical PCT is difficult to meet.

Example 30

Preparing the long-afterglow lateral immunochromatography detection reagent composition and the test strip.

The long-afterglow luminescent microsphere comprises a carrier microsphere, a light absorbing agent, a photochemical buffering agent and the composition of the rare earth luminescent complex of the second aspect of the invention.

The long afterglow lateral immunochromatography is an improved technology of fluorescence lateral immunochromatography, long afterglow luminescence is used for replacing traditional luminescence (fluorescence and phosphorescence), luminescence signals can be collected after exciting light is turned off, interference caused by background fluorescence in the immunoassay process can be effectively avoided, the detection sensitivity is improved, the detection limit is reduced, and accurate and high-sensitivity immunoassay is favorably realized.

g and T are heteroatoms selected from O, S, Se and N;

The long-afterglow luminescent microsphere can be prepared by the following method:

2) In dispersion or solution, light-absorbing agent, photochemical buffer agent and rare earth luminescent complex LnX_jDispersing or adsorbing the long-afterglow luminescent microspheres into carrier microspheres, and preparing the long-afterglow luminescent microspheres by methods such as swelling adsorption, emulsion polymerization, miniemulsion polymerization, microemulsion polymerization and the like;

wherein the light absorbing agent, photochemical buffering agent and rare earth luminescent complex LnX can be added_jRespectively, in a suitable solvent such as liquid paraffin, a mixture of benzyl alcohol-ethylene glycol and water, a mixture of mesitylene and ethanol, tetrahydrofuran, dichloromethane, etc. Forming the above materials into stable solution or dispersion, adding carrier microsphere or dispersion of carrier microsphere to obtain light absorbent, photochemical buffer agent and rare earth luminescent complex LnX_jAnd adsorbing or coating to prepare the long-afterglow luminescent microsphere. Preferably, water or other suitable solvent may be used to disperse the carrier microspheres, such as deionized water, Phosphate Buffered Saline (PBS), Borate Buffered Saline (BBS), and the like.

In this embodiment, palladium phthalocyanine (PdPc) is used as a light absorber, organic small molecule NSOF is used as a photochemical buffering agent, Eu (X-1) is used as a composition of the rare earth luminescent complex, and Polystyrene (PS) microspheres with a particle size of 200nm are used as carrier microspheres to prepare long-afterglow luminescent microspheres (wherein the mass fraction of the rare earth luminescent complex in the luminescent acceptor microspheres is 5%, the mass fraction of the chemical buffering agent NSOF in the luminescent acceptor microspheres is 5%, and the mass fraction of the light absorber PdPc in the luminescent acceptor microspheres is 0.01%). The Polystyrene (PS) microspheres are formed by polymerizing styrene and acrylic acid through monomers, so that the surfaces of the PS microspheres contain carboxyl groups, and the PS microspheres can be used for subsequent bioconjugation.

The microspheres are conjugated to a counterpart that specifically binds to the biomarker and then used for immunochromatographic detection. This example schematically shows a simple method for preparing a bioconjugate and immunochromatographic test strip for C-reactive protein (CRP) immunoassay as follows:

the long afterglow luminescent microsphere prepared in the embodiment is centrifuged at high speed, then is evenly dispersed to PBS buffer solution with pH of 7.35-7.44, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide sulfonic acid sodium salt are added for activation, and the intermediate product after the activation reaction and CRP-Ab are reacted₁Coupling the monoclonal antibody to prepare a probe for detecting the C-reactive protein; dissolving the prepared probe, spraying the dissolved probe on a probe sample loading line, and drying for later use; CRP-Ab is respectively coated on the test line and the quality control line of the NC membrane₂And a secondary antibody; fixing the prepared NC membrane in the middle of the bottom plate, and overlapping the sample pad and the absorbent paper on two sides of the NC membrane in a staggered manner respectively to prepare the immunochromatographic test strip for detecting the C-reactive protein.

An immunochromatographic test strip for detecting C-reactive protein, comprising a base plate; the top of the bottom plate is provided with a sample area, a detection area and an adsorption area which are sequentially connected; the detection area is provided with a test line and a quality control line; the sample area is provided with a probe for detecting C-reactive protein; the probe consists of an organic long afterglow luminescent material and a detection antibody; the detection antibody or the aptamer can be specifically combined with the C-reactive protein; the sample area is provided with a sample loading position; the probe is located and is close to detection zone one side, the sample position of getting ready is located and is kept away from detection zone one side.

Example 31

The detection reagent composition and the detection application of the test strip are based on the long afterglow lateral immunochromatography.

A method for detecting C-reactive protein using the immunochromatographic test strip prepared in example 30, comprising the steps of:

firstly, diluting a C-reactive protein antigen stock solution into serum C-reactive protein antigen solutions with different concentrations, and then loading the serum C-reactive protein antigen solutions onto an immunochromatography test strip; after the immunochromatographic test strip is irradiated by exciting light, the exciting light is turned off; measuring the long afterglow luminous intensity of the test line and the quality control line, calculating the ratio of the intensities, and establishing a standard curve according to the corresponding relation between the ratio and the antigen concentration;

then, loading a serum sample to be detected onto an immunochromatography test strip; after the immunochromatographic test strip is irradiated by exciting light, the exciting light is turned off; and measuring the long afterglow luminous intensity of the test line and the quality control line, calculating the ratio of the intensities, and calculating the concentration of the C-reactive protein in the sample by combining the ratio with the drawn standard curve.

And a smart phone, a luminescence imaging system and/or a professional long afterglow luminescence detection device are/is used for measuring the long afterglow luminescence intensity.

In the embodiment, the detection limit of CRP obtained by using the intelligent mobile phone for photographing and image recognition is 1ng/mL, and the detection limit can meet the test requirement of clinical CRP.

Example 32

The immunochromatographic test strip prepared in example 30 was repeated except that Eu (X-1) was replaced with Eu (X-13), and the conditions were otherwise the same. According to the method of the embodiment 31, the detection limit of CRP is 0.5ng/mL by using the smartphone for photographing and image recognition, and the detection limit can meet the test requirement of clinical CRP.

Comparative example 9

Example 30 was repeated to prepare an immunochromatographic test strip except that Eu (X-1) was replaced with Eu (TTA)₃Phen, otherwise identical. According to the method of the embodiment 31, the detection limit of CRP is 100ng/mL by using the photographing and picture recognition intensity of the smart phone, and the detection limit cannot meet the test requirement of clinical CRP.

Comparative example 10

The immunochromatographic test strip prepared in example 30 was repeated except that Eu (X-1) was replaced with Eu (M-2) and the conditions were otherwise the same. According to the method of the embodiment 31, the detection limit of CRP is 50ng/mL by using the photographing and picture recognition intensity of the smart phone, and the detection limit cannot meet the test requirement of clinical CRP.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to the above-described embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A multidentate β -diketone ligand characterized in that the multidentate β -diketone ligand has the structural formula:

2. A multidentate β -diketone ligand according to claim 1, wherein m, n and k have the same value;

R₂、R₃and R₄The same;

3. A multidentate β -diketone ligand according to claim 1, wherein m, n, and k are not all the same;

preferably, R₂、R₃And R₄Are not identical;

preferably, m, n and k are different from each other;

preferably, R₂、R₃And R₄Are different from each other;

4. A multidentate β -diketone ligand according to any one of claims 1 to 3, wherein R is₁Is hydrogen or hydroxy;

preferably, R₁Is an unbranched hydrocarbon oxy group;

preferably, R₁Is a branched hydrocarbonoxy group;

preferably, R₁Is an unbranched alkoxy group;

preferably, R₁Is an alkoxy group with a branched chain.

5. The multidentate β -diketone ligand according to claim 1, wherein said multidentate β -diketone ligand is selected from any one of the following compounds:

6. the multidentate β -diketone ligand according to claim 1, wherein said multidentate β -diketone ligand is selected from any one of the following compounds:

7. the rare earth complex is characterized in that the general formula of the rare earth complex is LnX_jWherein X is a multidentate β -diketone ligand as defined in any one of claims 1 to 6;

j is an integer selected from 1 to 10; preferably, j is 1;

ln is selected from Eu (europium), Tb (terbium) and Sm (samarium); preferably, Ln is Eu (europium).

8. A rare earth complex with stable luminescence is characterized in that the general formula of the rare earth complex is LnX_jWherein X is a multidentate β -diketone ligand as defined in any one of claims 1 to 6;

j is an integer selected from 1 to 10; preferably, j is 1;

ln is selected from Eu (europium), Tb (terbium) and Sm (samarium); preferably, Ln is Eu (europium);

after the rare earth complex with stable luminescence is mixed with the immunodetection diluent for a period of time, the luminescence intensity of the rare earth complex is greater than or equal to 50% of the initial luminescence intensity;

preferably, the buffer reagent comprises at least one of PBS, PB, BBS, MES;

preferably, the surfactant comprises at least one of SDBS, SDS, CTAB.

9. A luminescent microsphere comprising a support microsphere and the rare earth complex of claim 7, or comprising a support microsphere and the luminescence-stable rare earth complex of claim 8;

the carrier microsphere is at least one selected from hydrogel microspheres, styrene polymer microspheres, microspheres formed by protein, silicon nano microspheres and polymethyl methacrylate microspheres;

preferably, the carrier microspheres are styrene polymer microspheres;

further preferably, the carrier microspheres are selected from styrene polymer microspheres with amino, carboxyl, amido and/or aldehyde groups on the surface.

10. The luminescent microsphere of claim 9, wherein the mass of the rare earth complex is 0.01 to 50 percent of the mass of the carrier microsphere;

11. The luminescent microsphere of claim 9, wherein the luminescent intensity of the luminescent microsphere is greater than or equal to 50% of the initial luminescent intensity for a period of time after the luminescent microsphere is mixed with the immunoassay diluent;

preferably, the buffer reagent comprises at least one of PBS, PB, BBS, MES;

preferably, the surfactant comprises at least one of SDBS, SDS, CTAB.

12. A homogeneous chemiluminescent assay reagent comprising a light absorbing donor microsphere and a light emitting acceptor microsphere;

wherein the light absorbing donor microspheres comprise a support microsphere and a light absorber;

the luminescence receptor microsphere comprises a carrier microsphere, a photochemical buffer agent and the rare earth complex of claim 7, or the luminescence receptor microsphere comprises a carrier microsphere, a photochemical buffer agent and the luminescence-stable rare earth complex of claim 8;

preferably, the carrier microsphere is at least one selected from hydrogel microspheres, styrene polymer microspheres, microspheres formed by protein, silicon nano-microspheres and polymethyl methacrylate microspheres;

preferably, the carrier microspheres are styrene polymer microspheres;

further preferably, the carrier microspheres are selected from styrene polymer microspheres with amino, carboxyl, amido and/or aldehyde groups on the surfaces;

the light absorber is selected from porphyrin dyes and phthalocyanine dyes, transition metal complexes, Quantum Dots (QDs), and derivatives or copolymers of the compounds;

preferably, the photochemical buffering agent comprises a substance represented by the following structural formula:

wherein G and T are heteroatoms selected from O, S, Se and N;

R₁' and R₂' and R₄' to R₈' are each independently selected from H, hydroxyl, carboxyl, amino, mercapto, ester, nitro, sulfonic, halogen, amide, or alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, aryl, aralkyl, heteroaryl or heteroaralkyl having N, O or S, or combinations thereof, having 1 to 50, preferably 1 to 24, carbon atoms, such as 2 to 14, wherein the aryl, aralkyl, heteroaryl or heteroaralkyl optionally has one or more substituents L;

wherein L is selected from hydroxyl, carboxyl, amino, mercapto, ester, nitro, sulfonic, halogen, amide, or alkyl, alkenyl, alkynyl, alkoxy, and alkylamino groups having 1 to 50, preferably 1 to 24, such as 2 to 14 or 6 to 12 carbon atoms, or combinations thereof;

preferably, R₃' is an electron withdrawing group or an aryl group comprising an electron withdrawing group.

13. The homogeneous chemiluminescent detection reagent of claim 12 wherein the amount of the rare earth complex is 0.01% to 50% of the amount of the carrier microsphere;

14. A fluorescence lateral immunochromatography detection reagent, comprising a long-afterglow luminescent microsphere, wherein the long-afterglow luminescent microsphere comprises a carrier microsphere, a light absorbing agent, a photochemical buffering agent and the rare earth complex of claim 7; or the long persistence luminescent microsphere comprises a support microsphere, a light absorber, a photochemical buffering agent and the luminescence-stable rare earth complex of claim 8;

preferably, the carrier microspheres are styrene polymer microspheres;

preferably, the light absorbers are selected from the group consisting of porphyrins and phthalocyanines, transition metal complexes, Quantum Dots (QDs), and derivatives or copolymers of these compounds;

wherein G and T are heteroatoms selected from O, S, Se and N;

15. The fluorescence lateral immunochromatography detection reagent according to claim 14, wherein the mass of the rare earth complex is 0.01% to 50% of the mass of the carrier microsphere;

16. An immunoassay kit comprising standards and diluents suitable for immunoassay and a reagent composition, wherein the reagent composition is selected from at least one of the luminescent microspheres of any one of claims 9 to 11, the homogeneous chemiluminescent detection reagent of claim 12 or 13, the fluorescent lateral immunochromatography detection reagent of claim 14 or 15;

preferably, the diluent comprises a buffer, a surfactant and a preservative;

preferably, the buffer comprises at least one of Phosphate Buffered Saline (PBS), phosphate buffered saline (PB), Borate Buffered Saline (BBS), 2- (N-morpholino) ethanesulfonic acid buffer (MES);

preferably, the surfactant includes at least one of sodium dodecylbenzene sulfonate (SDBS), Sodium Dodecyl Sulfate (SDS), cetyltrimethylammonium bromide (CTAB).

17. The multidentate β -diketone ligand according to any one of claims 1 to 6, the rare earth complex according to claim 7, the luminescence-stabilized rare earth complex according to claim 8, the luminescent microsphere according to any one of claims 9 to 11, the homogeneous chemiluminescent detection reagent according to claim 12 or 13, the fluorescent lateral immunochromatography detection reagent according to claim 14 or 15, or the immunoassay kit according to claim 16 for use in immunoassays.