Microsphere composition for chemiluminescence analysis and application thereof

文档序号：1463558 发布日期：2020-02-21 浏览：2次中文

阅读说明：本技术 一种用于化学发光分析的微球组合物及其应用 (Microsphere composition for chemiluminescence analysis and application thereof ) 是由杨阳康蔡俊赵卫国刘宇卉李临于 2019-08-13 设计创作，主要内容包括：本发明涉及化学发光分析技术领域的一种用于用于化学发光分析的微球组合物及其应用。所述微球组合物,其包括至少两种不同粒径的受体微球,所述受体微球能够与活性氧反应产生可检测的化学发光信号。本发明所述微球组合物的既有超高的灵敏度,又有很宽的检测量程。另外,由于小粒径受体微球的反应速度快,因此采用本发明所述受体微球组合物进行检测时还能缩短检测时间,反应速度得到提升。(The invention relates to a microsphere composition for chemiluminescence analysis and application thereof, belonging to the technical field of chemiluminescence analysis. The microsphere composition comprises at least two acceptor microspheres with different particle sizes, and the acceptor microspheres can react with active oxygen to generate detectable chemiluminescent signals. The microsphere composition has ultrahigh sensitivity and wide detection range. In addition, the small-particle-size receptor microsphere has high reaction speed, so that the detection time can be shortened when the receptor microsphere composition is used for detection, and the reaction speed is improved.)

1. A microsphere composition for use in chemiluminescent assays comprising at least two receptor microspheres of different particle size capable of reacting with reactive oxygen species to produce a detectable chemiluminescent signal.

2. The microsphere composition according to claim 1, wherein the acceptor microsphere comprises two parts of a luminescent composition and a matrix, and the luminescent composition is filled in the matrix and/or coated on the surface of the matrix.

3. The microsphere composition of claim 2, wherein said luminescent composition is capable of reacting with active oxygen to produce a detectable chemiluminescent signal comprising a chemiluminescent compound and a metal chelate.

4. A microsphere composition according to claim 3, wherein said chemiluminescent compound is selected from the group consisting of olefinic compounds, preferably from the group consisting of dimethylthiophene, dibutyldiketone compounds, dioxines, enol ethers, enamines, 9-alkylidenexanthanes, 9-alkylene-N-9, 10 dihydroacridines, arylethyletherenes, arylimidazoles and lucigenins and their derivatives, more preferably from the group consisting of dimethylthiophene and its derivatives.

5. A microsphere composition according to claim 3 or 4, wherein the metal of said metal chelate is a rare earth metal or a group VIII metal, preferably selected from europium, terbium, dysprosium, samarium osmium and ruthenium, more preferably europium.

6. A microsphere composition according to any one of claims 3 to 5, wherein said metal chelate comprises a chelating agent selected from the group consisting of: MTTA, NHA, BHHT, BHHCT, DPP, TTA, NPPTA, NTA, TOPO, TPPO, BFTA, 2-dimethyl-4-perfluorobutanoyl-3-butanone (fod), 2' -bipyridine (bpy), bipyridylcarboxylic acid, azacrown ether, azacryptand trioctylphosphine oxide and derivatives thereof.

7. A microsphere composition according to any one of claims 3 to 6, wherein said luminescent compound is a derivative of dimethylthiophene and said metal chelate is a europium chelate.

8. A microsphere composition according to any one of claims 1 to 7, wherein said matrix is selected from the group consisting of tape, sheet, rod, tube, well, microtiter plate, bead, particle and microsphere; beads and microspheres are preferred.

9. A microsphere composition according to any one of claims 1 to 8, wherein said matrix is magnetic or non-magnetic particles.

10. A microsphere composition according to any one of claims 1 to 9, wherein the matrix of the acceptor microspheres of different particle sizes is of the same or different material.

11. A microsphere composition according to any one of claims 1 to 10, wherein said matrix material is selected from natural, synthetic or modified naturally occurring polymers selected from agarose, cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polystyrene, polyethylene, polypropylene, poly (4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate, nylon, polyvinyl butyrate or polyacrylate; preferably, the matrix is polystyrene latex microspheres, and more preferably carboxyl and/or aldehyde-based polystyrene latex microspheres.

12. A microsphere composition according to any one of claims 1 to 11, wherein a biologically active substance is directly attached to the surface of said matrix, said biologically active substance being capable of specifically binding to the target molecule to be detected.

13. The microsphere composition according to any one of claims 1 to 11, wherein the surface of the substrate is coated with a coating layer, and the surface of the coating layer is connected with a bioactive substance, wherein the bioactive substance can be specifically bound with a target molecule to be detected.

14. Microsphere composition according to claim 13, wherein the coating in the coating layer is selected from polysaccharides, high molecular polymers or biological macromolecules, preferably polysaccharides.

15. The microsphere composition of claim 14, wherein the surface of the substrate is coated with at least two successive polysaccharide layers, wherein a first polysaccharide layer is associated spontaneously with a second polysaccharide layer.

16. The microsphere composition of claim 15, wherein each of said successive polysaccharide layers is spontaneously associated with each of the previous polysaccharide layers.

17. The microsphere composition of any one of claims 14 to 16, wherein said polysaccharide has pendant functional groups and said functional groups of said continuous polysaccharide layer are oppositely charged to said functional groups of said previous polysaccharide layer.

18. The microsphere composition of any one of claims 14 to 16, wherein said polysaccharide has pendant functional groups and said continuous layer of polysaccharide is covalently linked to said previous polysaccharide layer by a reaction between said functional groups of said continuous layer and said functional groups of said previous layer.

19. The microsphere composition of claim 18, wherein the functional groups of the continuous polysaccharide layer alternate between amine functional groups and amine reactive functional groups.

20. The microsphere composition of claim 19, wherein said amine reactive functional group is an aldehyde group or a carboxyl group.

21. The microsphere composition of any one of claims 15 to 20, wherein said first polysaccharide layer is spontaneously associated with said support.

22. A microsphere composition according to any one of claims 15 to 21, wherein the outermost polysaccharide layer of said coating layer has at least one pendant functional group.

23. A microsphere composition according to any one of claims 15 to 22, wherein the pendant functional groups of the outermost polysaccharide layer of said coating are selected from at least one of aldehyde, carboxyl, thiol, amino, hydroxyl and maleic groups; preferably selected from aldehyde groups and/or carboxyl groups.

24. The microsphere composition of claim 22 or 23, wherein the pendant functional groups of the outermost polysaccharide layer of the coating are directly or indirectly attached to a biologically active material.

25. A microsphere composition according to any one of claims 14 to 24, wherein said polysaccharide is selected from the group consisting of carbohydrates comprising three or more unmodified or modified monosaccharide units; preferably selected from the group consisting of dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably selected from dextran, starch, glycogen and polyribose.

26. The microsphere composition of any one of claims 1 to 25, wherein said receptor microspheres of different particle size have the same matrix particle size.

27. The microsphere composition of any one of claims 1 to 25, wherein said acceptor microspheres of different particle sizes have different matrix particle sizes.

28. A microsphere composition according to any one of claims 1 to 27, wherein said active oxygen is singlet oxygen.

29. A microsphere composition according to any one of claims 1 to 28, wherein said microsphere composition comprises receptor microspheres of two different particle sizes.

30. The microsphere composition of claim 29, wherein the difference between the particle sizes of said two different particle size acceptor microspheres is not less than 50 nm; preferably not less than 100 nm; more preferably not less than 150 nm.

31. The microsphere composition of claim 29 or 30, wherein the ratio of the two acceptor microspheres with different particle sizes is selected from the group consisting of 1 (1.1-10); preferably selected from 1 (2-8); more preferably from 1 (3-6).

32. A microsphere composition according to any one of claims 1 to 31, wherein said microsphere composition is used at a concentration of from 1ug/mL to 1000 ug/mL; preferably 10ug/mL to 500ug/mL, more preferably 10ug/mL to 250 ug/mL.

33. A microsphere composition according to any one of claims 1 to 28, wherein said microsphere composition comprises at least three acceptor microspheres of different particle size.

34. An acceptor reagent comprising a microsphere composition according to any one of claims 1 to 33.

35. A chemiluminescent assay kit comprising the microsphere composition of any one of claims 1 to 33 or the receptor reagent of claim 34.

36. A chemiluminescent assay method comprising detecting the presence of a target molecule to be detected in a sample to be tested and/or the concentration of the target molecule to be detected in the sample to be tested using the microsphere composition of any one of claims 1 to 33 or the receptor reagent of claim 34 or the kit of claim 35.

37. A chemiluminescence analyzer, wherein the microsphere composition according to any one of claims 1 to 33 or the receptor reagent according to claim 34 or the kit according to claim 35 and/or the method according to claim 36 is used to detect the presence and/or concentration of a target molecule to be detected in a sample to be detected.

38. A chemiluminescence analyzer according to claim 37, comprising at least the following:

the incubation module is used for providing a proper temperature environment for a chemiluminescent reaction between a sample to be detected and a microsphere composition, and the microsphere composition comprises at least two receptor microspheres with different particle sizes;

the detection module is used for detecting a chemiluminescent signal generated by the reaction of the receptor microsphere and active oxygen;

and the processor is used for judging whether the target molecules to be detected exist in the sample to be detected and/or the concentration of the target molecules to be detected in the sample to be detected according to the condition of the chemiluminescence signal detected by the detection module.

Technical Field

The invention belongs to the technical field of chemiluminescence analysis, and particularly relates to a microsphere composition for chemiluminescence analysis and application thereof.

Background

Chemiluminescence analysis is a method of detection using the light waves emitted by chemiluminescent substances. The chemiluminescent substance is used as a label for nucleic acid detection and immunoassay. For example, one of the molecules of the specific binding pair can be combined with the luminescent material in a variety of ways to form a luminescent microsphere composition. The microsphere composition can react with a detected object (another molecule in the specific binding pair) in a sample, and is distributed in a solid phase and a liquid phase, and the distribution ratio is related to the amount of the detected object. The corresponding concentration of the test substance in the sample can be obtained by measuring the luminescence intensity in the solid phase or the liquid phase.

At present, with the progress of the detection industry, the demand for a hypersensitive reagent is more and more, the requirement on sensitivity is extremely high, the linear range is very wide, and the existing microsphere composition is difficult to meet the detection conditions.

Therefore, there is a need to develop a microsphere composition for chemiluminescence analysis that can meet both the sensitivity requirement and the linear range requirement.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a microsphere composition for chemiluminescence analysis, aiming at the defects of the prior art, and when the microsphere composition is used for chemiluminescence analysis detection, the microsphere composition has ultrahigh sensitivity and wide detection range. In addition, the detection time can be shortened.

To this end, a first aspect of the invention provides a microsphere composition for use in chemiluminescent assays comprising at least two receptor microspheres of different particle size, said receptor microspheres being capable of reacting with reactive oxygen species to produce a detectable chemiluminescent signal.

In some embodiments of the present invention, the acceptor microsphere includes two parts, namely, a luminescent composition and a matrix, wherein the luminescent composition is filled in the matrix and/or coated on the surface of the matrix.

In some embodiments of the invention, the luminescent composition is capable of reacting with reactive oxygen species to produce a detectable chemiluminescent signal comprising a chemiluminescent compound and a metal chelate.

In other embodiments of the present invention, the chemiluminescent compound is selected from the group consisting of olefinic compounds, preferably from the group consisting of dimethylthiophene, dibutyldione compounds, dioxins, enol ethers, enamines, 9-alkylidenexanthanes, 9-alkylene-N-9, 10 dihydroacridines, arylethyletherenes, arylimidazoles, and lucigenins and derivatives thereof, and more preferably from the group consisting of dimethylthiophene and derivatives thereof.

In some embodiments of the invention, the metal of the metal chelate is a rare earth metal or a group VIII metal, preferably selected from europium, terbium, dysprosium, samarium osmium and ruthenium, more preferably from europium.

In other embodiments of the present invention, the metal chelate comprises a chelating agent selected from the group consisting of: MTTA, NHA, BHHT, BHHCT, DPP, TTA, NPPTA, NTA, TOPO, TPPO, BFTA, 2-dimethyl-4-perfluorobutanoyl-3-butanone (fod), 2' -bipyridine (bpy), bipyridylcarboxylic acid, azacrown ether, azacryptand trioctylphosphine oxide and derivatives thereof.

In some embodiments of the invention, the luminescent compound is a derivative of dimethylthiophene and the metal chelate is a europium chelate.

In some embodiments of the invention, the substrate is selected from the group consisting of a tape, a sheet, a rod, a tube, a well, a microtiter plate, a bead, a particle, and a microsphere; beads and microspheres are preferred.

In other embodiments of the present invention, the matrix is a magnetic or non-magnetic particle.

In some embodiments of the invention, the matrix material is selected from natural, synthetic or modified naturally occurring polymers including, but not limited to: agarose, cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polystyrene, polyethylene, polypropylene, poly (4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate, nylon, polyvinyl butyrate, or polyacrylate.

In other embodiments of the present invention, the matrix is an aldehydized latex microsphere; preferably, aldehyde polystyrene latex microspheres.

In some embodiments of the invention, the surface of the substrate is directly linked to a bioactive substance that is capable of specifically binding to the target molecule to be detected.

In some embodiments of the invention, the coating in the coating is selected from polysaccharides, high molecular polymers or biological macromolecules, preferably polysaccharides.

In other embodiments of the invention, the surface of the substrate is coated with at least two successive polysaccharide layers, wherein the first polysaccharide layer is spontaneously associated with the second polysaccharide layer.

In some embodiments of the invention, each of the successive polysaccharide layers is spontaneously associated with each of the previous polysaccharide layers.

In other embodiments of the present invention, the polysaccharide has pendant functional groups, and the functional groups of the continuous polysaccharide layer are oppositely charged from the functional groups of the previous polysaccharide layer.

In some embodiments of the invention, the polysaccharide has pendant functional groups, and the continuous layer of polysaccharide is covalently linked to the previous polysaccharide layer by a reaction between the functional groups of the continuous layer and the functional groups of the previous layer.

In other embodiments of the present invention, the functional groups of the continuous polysaccharide layer alternate between amine functional groups and amine reactive functional groups.

In some embodiments of the invention, the amine-reactive functional group is an aldehyde group or a carboxyl group.

In other embodiments of the present invention, the first polysaccharide layer is spontaneously associated with the support.

In some embodiments of the invention, the outermost polysaccharide layer of the coating has at least one pendant functional group.

In other embodiments of the present invention, the pendant functional groups of the outermost polysaccharide layer of the coating are selected from at least one of aldehyde, carboxyl, thiol, amino, hydroxyl, and maleic groups; preferably selected from aldehyde groups and/or carboxyl groups.

In some embodiments of the invention, the pendant functional groups of the outermost polysaccharide layer of the coating are directly or indirectly attached to the biologically active material.

In other embodiments of the invention, the polysaccharide is selected from the group consisting of carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from the group consisting of dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably selected from dextran, starch, glycogen and polyribose.

In some embodiments of the invention, the substrate particle sizes of the different particle size acceptor microspheres are the same.

In some embodiments of the invention, the receptor microspheres of different particle sizes have different matrix particle sizes.

In some preferred embodiments of the present invention, the active oxygen is singlet oxygen.

In some embodiments of the invention, the microsphere composition comprises two acceptor microspheres of different particle sizes.

In some embodiments of the invention, the microsphere composition is used at a concentration of 1ug/mL to 1000 ug/mL; preferably 10ug/mL to 500ug/mL, more preferably 10ug/mL to 250 ug/mL.

In some embodiments of the invention, the microsphere composition comprises at least three acceptor microspheres of different particle sizes.

In a second aspect, the invention provides an acceptor agent comprising a microsphere composition according to the first aspect of the invention.

In a third aspect, the present invention provides a chemiluminescent detection kit comprising a microsphere composition according to the first aspect of the present invention or a receptor reagent according to the second aspect of the present invention.

In a fourth aspect, the present invention provides a chemiluminescence analysis method, which comprises detecting the presence of a target molecule to be detected in a sample to be detected and/or the concentration of the target molecule to be detected in the sample to be detected by using the microsphere composition according to the first aspect, the receptor reagent according to the second aspect, or the kit according to the third aspect.

In a fifth aspect, the present invention provides a chemiluminescence analyzer, wherein the microsphere composition according to the first aspect, the receptor reagent according to the second aspect, the kit according to the third aspect, and/or the method according to the fourth aspect of the present invention are/is used for detecting the presence and/or concentration of the target molecule to be detected in the sample to be detected.

In some preferred embodiments of the present invention, the chemiluminescent analyzer comprises at least the following:

the detection module is used for detecting a chemiluminescent signal generated by the reaction of the receptor microsphere and active oxygen;

The invention has the beneficial effects that: the microsphere composition for chemiluminescence analysis simultaneously comprises at least two acceptor microspheres with different particle sizes. The small-particle-size receptor microsphere can widen the detection range, and the large-particle-size receptor microsphere can improve the detection sensitivity, so that the microsphere composition disclosed by the invention has greatly improved performance compared with the prior art, and not only has ultrahigh sensitivity, but also has a very wide range. In addition, the reaction speed of the small-particle-size receptor microspheres is high, so that the detection time can be shortened when the microsphere composition is used for detection, and the reaction speed is improved.

Drawings

The invention will be further explained with reference to the drawings.

FIG. 1 is a graph showing a Gaussian distribution of aldehyde-based polystyrene latex microspheres prepared in example 3.

Fig. 2 is a graph showing a Gaussian distribution of aldehyde-based polystyrene latex microspheres embedded with a light-emitting composition prepared in example 3.

FIG. 3 is a Gaussian distribution diagram of aldehyde-based polystyrene latex microspheres with embedded luminescent composition coated with dextran prepared in example 3

FIG. 4 is a Gaussian distribution diagram of acceptor microspheres prepared in example 3 with an average particle size around 250 nm.

FIG. 5 is a Gaussian distribution diagram of acceptor microspheres prepared in example 3 with a particle size around 110 nm.

FIG. 6 is a Nicomp distribution plot of acceptor microspheres prepared in example 3 with a particle size around 110 nm.

FIG. 7 is a Gaussian distribution diagram of acceptor microspheres prepared in example 3 with a particle size around 350 nm.

FIG. 8 is a Nicomp distribution plot of acceptor microspheres prepared in example 3 with a particle size around 350 nm.

FIG. 9 is a Gaussian distribution diagram of the particle size distribution of the mixed acceptor microspheres of example 4.

FIG. 10 is a Nicomp distribution plot of the particle size distribution of the mixed acceptor microspheres from example 4.

Detailed Description

In order that the invention may be readily understood, a detailed description of the invention is provided below. However, before the invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

Where a range of values is provided, it is understood that each intervening value, to the extent that there is no stated or intervening value in that stated range, to the extent that there is no such intervening value, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where a specified range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

Term (I)

The term "active oxygen" as used herein means in vivo or in vitroThe general term for oxygen-containing and active materials, which consist of oxygen in the environment, is primarily an excited oxygen molecule, including superoxide anion (O) which is the product of the reduction of an electron of oxygen₂(-) and the two-electron reduction product hydrogen peroxide (H)₂O₂) The three-electron reduction product hydroxyl radical (. OH) and nitric oxide and singlet oxygen (1O)₂) And the like.

In the present invention, the term "acceptor microsphere" refers to a nanoparticle capable of reacting with active oxygen to generate a detectable chemiluminescent signal, which may also be referred to as an oxygen-accepting microsphere or a luminescent microsphere. Preferably, the acceptor microsphere may be a polymer microparticle filled in a matrix through a functional group to form a luminescent composition filled in the matrix, wherein the luminescent composition comprises a chemiluminescent compound capable of reacting with active oxygen. In some embodiments of the invention, the chemiluminescent compound undergoes a chemical reaction with reactive oxygen species to form an unstable metastable intermediate that can decompose with or subsequently luminesce. Typical examples of such substances include, but are not limited to: enol ethers, enamines, 9-alkylidene xanthan gums, 9-alkylidene-N-alkyl acridines, aryl ethylether alkenes, diepoxides, dimethylthiophenes, aryl imidazoles, or lucigenins.

In the present invention, the "chemiluminescent compound", i.e., a compound referred to as a label, may undergo a chemical reaction to cause luminescence, such as by being converted to another compound formed in an electronically excited state. The excited state may be a singlet state or a triplet excited state. The excited state may relax to the ground state to emit light directly, or may return to the ground state itself by transferring excitation energy to an emission energy acceptor. In this process, the energy-acceptor microsphere will be transitioned to an excited state to emit light.

The chemiluminescent compound may be bound to a specific binding partner member that is capable of binding, directly or indirectly, to the target molecule to be tested or to a test component whose concentration is affected by the presence of the target molecule to be tested. By "capable of binding, directly or indirectly," it is meant that the specified entity is capable of specifically binding to the entity (directly), or that the specified entity is capable of specifically binding to a specific binding pair member, or a complex having two or more specific binding partners capable of binding to other entities (indirectly).

The "specific binding pair member" of the present invention is selected from the group consisting of (1) a small molecule and a binding partner for the small molecule, and (2) a macromolecule and a binding partner for the macromolecule

In the present invention, the active oxygen may be provided by "donor microspheres," which are nanospheres capable of generating active oxygen in an excited state. Preferably, the donor microsphere is a polymer microparticle which is formed by coating a functional group on a substrate and is filled with a photosensitive compound, and can generate singlet oxygen under the excitation of light, and in this case, the photosensitive microsphere can also be called an oxygen supply microsphere or a photosensitive microsphere. The surface of the donor microsphere can be provided with hydrophilic aldehyde dextran, and the inside of the donor microsphere is filled with a photosensitizer. The photosensitizer may be one known in the art, preferably a compound that is relatively light stable and does not react efficiently with singlet oxygen, non-limiting examples of which include compounds such as methylene blue, rose bengal, porphyrins, and phthalocyanines, and derivatives of these compounds having 1-50 atom substituents that serve to render these compounds more lipophilic or more hydrophilic and/or as a linker for attachment to a member of a specific binding pair. The donor microsphere surface may also be filled with other sensitizers, non-limiting examples of which are certain compounds that catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Other examples of donors include: 1, 4-dicarboxyethyl-1, 4-naphthalene endoperoxide, 9, 10-diphenylanthracene-9, 10-endoperoxide, etc., which are heated or which absorb light directly to release active oxygen, such as singlet oxygen.

The sensitizer is a compound that, when excited or induced to react, causes a chemical reaction of another compound or substance. Sensitizers include photosensitizers that are capable of being induced by light irradiation to form an activated excited state. Sensitizers also include compounds that chemically react to produce a metastable reactive oxygen species, such as singlet oxygen.

Photosensitizers generally activate chemiluminescent compounds by irradiating the medium containing the reactant. The medium must be irradiated with light having a wavelength and an energy sufficient to convert the photosensitizer to an excited state, thereby enabling it to activate molecular oxygen to singlet oxygen. The excited state of a photosensitizer capable of exciting molecular oxygen is generally in the triplet state, which is about 20Kcal/mol, usually at least 23Kcal/mol higher than the energy of the photosensitizer in the ground state. Preferably, the medium is irradiated with light having a wavelength of about 450 and 950nm, although shorter wavelengths, such as 230 and 950nm, may be used. The light generated can be measured in any conventional manner, such as by photography, visual inspection, photometer, etc., to determine its amount relative to the amount of analyte in the medium. The photosensitizer is preferably relatively non-polar to ensure solubility into the lipophilic member.

The "substrate" according to the invention, which may be of any size, organic or inorganic, may be swellable or non-swellable, may be porous or non-porous, has any density, but preferably has a density close to that of water, is preferably capable of floating in water, and is composed of a transparent, partially transparent or opaque material. The substrate may or may not have a charge, and when charged, is preferably negatively charged. The matrix may be a solid (e.g., polymers, metals, glass, organic and inorganic materials such as minerals, salts, and diatoms), oil droplets (e.g., hydrocarbons, fluorocarbons, siliceous fluids), vesicles (e.g., synthetic such as phospholipids, or natural such as cells, and organelles). The matrix may be latex particles or other particles containing organic or inorganic polymers, lipid bilayers such as liposomes, phospholipid vesicles, oil droplets, silica particles, metal sols, cells, and microcrystalline dyes. The matrix is generally multifunctional or capable of binding to a donor or recipient by specific or non-specific covalent or non-covalent interactions. Many functional groups are available or incorporated. Typical functional groups include carboxylic acid, acetaldehyde, amino, cyano, vinyl, hydroxy, mercapto, and the like. One non-limiting example of a suitable matrix for use in the present invention is aldehyde based polystyrene latex microspheres.

The photosensitizer and/or chemiluminescent compound may be selected to be dissolved in, or non-covalently bound to, the surface of the particle. In this case, the compounds are preferably hydrophobic to reduce their ability to dissociate from the particles, thereby allowing both compounds to bind to the same particle.

The term "particle size" as used herein refers to the average particle size of the acceptor microspheres, as measured using a conventional particle sizer. The receptor microsphere at least comprises a substrate, a luminous composition and a bioactive molecule, and preferably further comprises a coating layer; the luminescent composition may be filled in the matrix and/or coated on the surface of the matrix. When the receptor microsphere does not include a coating, the biologically active substance is directly attached to the surface of the substrate. When the acceptor microspheres include a coating, the coating coats the surface of the substrate, and the outermost layer of the coating connects the bioactive agent.

It is noted that the "average particle size of the receptor microsphere" in the present invention refers to the average particle size of the receptor microsphere after being linked and/or coated with the corresponding substance. The particle size of the matrix in the acceptor microspheres with different particle sizes may be the same or different, as long as the particle size of the finally formed acceptor microspheres is different, and the most preferred technical scheme of the invention is that the particle size of the matrix in the acceptor microspheres with different particle sizes is different.

The term "test sample" as used herein refers to a mixture containing or suspected of containing a target molecule to be tested. Samples to be tested that can be used in the present disclosure include bodily fluids such as blood (which can be anticoagulated blood commonly seen in collected blood samples), plasma, serum, urine, semen, saliva, cell cultures, tissue extracts, and the like. Other types of samples to be tested include solvents, seawater, industrial water samples, food samples, environmental samples such as soil or water, plant material, eukaryotic cells, bacteria, plasmids, viruses, fungi, and cells from prokaryotes. The sample to be tested can be diluted with a diluent as required before use. For example, to avoid the HOOK effect, the sample to be tested may be diluted with a diluent before the on-line detection and then detected on the detection instrument.

The term "target molecule to be detected" as used herein refers to a substance in a sample to be detected during detection. One or more substances having a specific binding affinity for the target molecule to be detected will be used for the detection of the target molecule. The target molecule to be detected may be a protein, a peptide, an antibody or a hapten which allows it to bind to an antibody. The target molecule to be detected may be a nucleic acid or oligonucleotide that binds to a complementary nucleic acid or oligonucleotide. The target molecule to be detected may be any other substance that can form a member of a specific binding pair. Other examples of typical target molecules to be detected include: drugs such as steroids, hormones, proteins, glycoproteins, mucins, nucleoproteins, phosphoproteins, drugs of abuse, vitamins, antibacterial agents, antifungal agents, antiviral agents, purines, antitumor agents, amphetamines, heteroazoids, nucleic acids, and prostaglandins, and metabolites of any of these drugs; pesticides and metabolites thereof; and a receptor. Analytes also include cells, viruses, bacteria, and fungi.

The term "antibody" as used herein is used in the broadest sense and includes antibodies of any isotype, antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, bispecific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. In any case desired, the antibody may be further conjugated to other moieties, such as a specific binding pair member, e.g., biotin or streptavidin (a member of a biotin-streptavidin specific binding pair member), and the like.

The term "antigen" as used herein refers to a substance that stimulates the body to produce an immune response and that binds to the immune response product antibodies and sensitized lymphocytes in vitro and in vivo to produce an immune effect.

The term "binding" as used herein refers to direct association between two molecules due to interactions such as covalent, electrostatic, hydrophobic, ionic and/or hydrogen bonding, including but not limited to interactions such as salt and water bridges.

The term "specific binding" as used herein refers to the mutual discrimination and selective binding reaction between two substances, and is the conformation correspondence between the corresponding reactants in terms of the three-dimensional structure. Under the technical idea disclosed by the invention, the detection method of the specific binding reaction comprises but is not limited to the following steps: double antibody sandwich, competition, neutralization competition, indirect or capture.

The "variation coefficient C.V value of particle size distribution" described in the present invention refers to the variation coefficient of particle size in Gaussian distribution in the detection result of the nanometer particle size analyzer. The coefficient of variation is calculated as: C.V value (standard deviation SD/Mean) x 100%.

The term "Nicomp distribution" as used herein refers to an algorithmic distribution in the US PSS nanometer particle sizer, NICOMP. Compared with a Gaussian single-peak algorithm, the Nicomp multi-peak algorithm has unique advantages in the analysis of multi-component liquid dispersion systems with nonuniform particle size distribution and the stability analysis of colloidal systems.

Detailed description of the preferred embodiments

The present invention will be described in more detail below.

The invention controls the particle size of the receptor microsphere in the microsphere composition, and further controls the amount of bioactive substances (such as antibody/antigen) on the surface of each receptor microsphere (the small-particle-size microsphere has large specific surface area, the large-particle-size microsphere has large amount of reporter molecules on the surface of the unit-mass microsphere, the large-particle-size microsphere has small specific surface area, and the small-particle-size microsphere has small amount of reporter molecules on the surface of the unit-mass microsphere), thereby improving the detection sensitivity and widening the detection range. In addition, the acceptor microsphere with small grain size has smaller diameter, so that the activation efficiency of singlet oxygen generated by the donor microsphere is improved, and the luminous efficiency of the acceptor microsphere can also be improved.

In a first aspect, the present invention relates to a microsphere composition for use in chemiluminescent assays comprising at least two receptor microspheres of different particle size capable of reacting with reactive oxygen species to produce a detectable chemiluminescent signal.

In other embodiments of the present invention, the chemiluminescent compound is selected from the group consisting of olefinic compounds, which are compounds capable of reacting with reactive oxygen species (e.g., singlet oxygen). Examples of suitable electron rich ene compounds are listed in U.S. patent No. 5,709,994, the relevant disclosure of which is incorporated herein by reference. In some preferred embodiments of the present invention, the olefinic compound is selected from the group consisting of dimethylthiophene, dibutyldiketone compounds, dioxins, enol ethers, enamines, 9-alkylidenexanthanes, 9-alkylene-N-9, 10-dihydroacridines, arylethyletherenes, arylimidazoles, and lucigenins and derivatives thereof, and more preferably from the group consisting of dimethylthiophene and derivatives thereof.

In addition to olefinic compounds, the chemiluminescent compounds include complexes of a metal and one or more chelating agents (metal chelates). In some embodiments of the invention, the metal of the metal chelate is a rare earth metal or a group VIII metal, preferably selected from europium, terbium, dysprosium, samarium osmium and ruthenium, more preferably from europium.

In other embodiments of the present invention, the metal chelate comprises a chelating agent selected from the group consisting of: 4 ' - (10-methyl-9-anthracenyl) -2,2 ': 6 ' 2 "-bipyridine-6, 6" -dimethylamine ] tetraacetic acid (MTTA), 2- (1 ', 1 ', 2 ', 2 ', 3 ', 3 ' -heptafluoro-4 ', 6 ' -hexanedion-6 ' -yl) -Naphthalene (NHA), 4 ' -bis (2 ', 3 ', 3 "-heptafluoro-4 ', 6" -hexanedion-6 "-yl) -o-terphenyl (BHHT), 4 ' -bis (1 ', 2 ', 3 ', 3" -heptafluoro-4 ', 6 "-hexanedion-6" -yl) -chlorosulphonyl-o-terphenyl (BHHCT), 4, 7-biphenyl-1, 10-phenanthroline (DPP), 1,1, 1-trifluoroacetone (TTA), 3-naphthoyl-1, 1, 1-trifluoroacetone (NPPTA), Naphthyltrifluorobutanedione (NTA), trioctylphosphine oxide (TOPO), triphenylphosphine oxide (TPPO), 3-benzoyl-1, 1, 1-trifluoroacetone (BFTA), 2-dimethyl-4-perfluorobutanoyl-3-butanone (fod), 2' -bipyridine (bpy), bipyridylcarboxylic acid, azacrown ether, azacryptand trioctylphosphine oxide and derivatives thereof.

In some embodiments of the invention, the luminescent compound is a derivative of dimethylthiophene and the metal chelate is a europium chelate.

In other embodiments of the present invention, the matrix is a magnetic or non-magnetic particle.

In some embodiments of the present invention, the materials of the matrices of the acceptor microspheres with different particle sizes are the same or different.

In other embodiments of the present invention, the matrix material is selected from natural, synthetic or modified naturally occurring polymers including, but not limited to: agarose, cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polystyrene, polyethylene, polypropylene, poly (4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate, nylon, polyvinyl butyrate, or polyacrylate.

In other embodiments of the present invention, the matrix is an aldehydized latex microsphere; preferably, aldehyde polystyrene latex microspheres.

In some embodiments of the invention, the surface of the substrate is directly linked to a bioactive substance that is capable of specifically binding to the target molecule to be detected.

In other embodiments of the present invention, the surface of the substrate is coated with a coating layer, and the surface of the coating layer is connected with a bioactive substance, wherein the bioactive substance can be specifically combined with a target molecule to be detected; examples of biologically active substance-target molecule binding partner ions include, by way of example and not limitation, antigen-antibody, hormone-hormone receptor, nucleic acid duplex, IgG-protein A, polynucleotide pairs such as DNA-DNA, DNA-RNA, and the like.

In some preferred embodiments of the invention, the biologically active substance is an antigen and/or an antibody; the antigen refers to a substance with immunogenicity; the antibody refers to immunoglobulin which is produced by an organism and can recognize specific foreign matters.