Homogeneous phase chemiluminescence detection kit and application thereof

文档序号：1463560 发布日期：2020-02-21 浏览：2次中文

阅读说明：本技术 一种均相化学发光检测试剂盒及其应用 (Homogeneous phase chemiluminescence detection kit 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技术领域的一种均相化学发光检测试剂盒及其应用。该试剂盒包括：受体试剂,所述受体试剂包括至少两种不同粒径的受体微球,所述受体微球能够与单线态氧活性氧反应产生可检测的化学发光信号；供体试剂,所述供体试剂包括供体微球,所述供体微球能够在激发状态下生成单线态氧活性氧。本发明所述试剂盒既有超高的灵敏度,又有很宽的检测量程。另外,由于小粒径发光微球的反应速度快,因此采用本发明所述免疫分析方法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 homogeneous phase chemiluminescence detection kit and application thereof in the technical field of chemiluminescence. The kit comprises: an acceptor reagent comprising at least two acceptor microspheres of different particle sizes capable of reacting with singlet oxygen reactive oxygen species to produce a detectable chemiluminescent signal; a donor agent comprising donor microspheres capable of generating singlet oxygen reactive oxygen species in an excited state. The kit has ultrahigh sensitivity and wide detection range. In addition, the reaction speed of the small-particle-size luminescent microspheres is high, so that the detection time can be shortened when the immunoassay method is used for detection, and the reaction speed is improved.)

1. A homogeneous chemiluminescent assay kit comprising:

an acceptor reagent comprising at least two acceptor microspheres of different particle sizes capable of reacting with reactive oxygen species to produce a detectable chemiluminescent signal;

a donor agent comprising donor microspheres capable of generating reactive oxygen species in an excited state.

2. The kit of claim 1, wherein the receptor reagent comprises receptor microspheres of two different particle sizes.

3. The kit according to claim 2, wherein the difference between the particle sizes of the two acceptor microspheres with different particle sizes is not less than 100 nm; preferably not less than 150 nm; more preferably not less than 200 nm.

4. The kit of claim 2 or 3, wherein the ratio of the two acceptor microspheres with different particle sizes is selected from 1 (1.1-10); preferably selected from 1 (2-8); more preferably from 1 (3-6).

5. The kit of any one of claims 1 to 4, wherein the concentration of the acceptor microsphere in the acceptor reagent is from 1ug/mL to 1000 ug/mL; preferably 10ug/mL to 500ug/mL, more preferably 10ug/mL to 250 ug/mL.

6. The kit of claim 1, wherein the receptor reagent comprises receptor microspheres of three different particle sizes.

7. The kit according to any one of claims 1 to 6, wherein the acceptor microsphere comprises a luminescent composition and a matrix, and the luminescent composition is filled in the matrix and/or coated on the surface of the matrix.

8. The kit of claim 7, wherein the luminescent composition is capable of reacting with reactive oxygen species to produce a detectable chemiluminescent signal and comprises a chemiluminescent compound and a metal chelate.

9. Kit according to claim 8, characterized in that the chemiluminescent compound is chosen from olefinic compounds, preferably from dimethylthiophene, dibutyldione compounds, dioxines, enol ethers, enamines, 9-alkylidenexanthanes, 9-alkylene-N-9, 10 dihydroacridines, arylethyletherenes, arylimidazoles and lucigenins and their derivatives, more preferably from dimethylthiophene and its derivatives.

10. Kit according to claim 8 or 9, characterized in that 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.

11. The kit of any one of claims 8-10, wherein the metal chelate comprises a chelating agent selected from the group consisting of: NHA, BHHT, BHHCT, DPP, TTA, NPPTA, NTA, TOPO, TPPO, BFTA, 2-dimethyl-4-perfluorobutanoyl-3-butanone (fod), 2' -bipyridine (bpy), bipyridylcarboxylic acids, azacrown ethers, azacryptands and trioctylphosphine oxides and derivatives thereof.

12. The kit of any one of claims 8 to 11, wherein the luminescent compound is a derivative of dimethylthiophene and the metal chelate is a europium chelate.

13. The kit of any one of claims 7 to 12, wherein the matrix 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.

14. The kit according to any one of claims 7 to 13, wherein the matrix is magnetic or non-magnetic particles.

15. The kit according to any one of claims 7 to 14, wherein the matrix of the acceptor microspheres with different particle sizes is made of the same or different materials.

16. The kit of any one of claims 7 to 15, wherein 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.

17. The kit of claim 16, wherein the matrix is an aldehydized latex microsphere; preferably, aldehyde polystyrene latex microspheres.

18. The kit according to any one of claims 7 to 17, wherein a biologically active substance capable of specifically binding to a target molecule to be detected is directly attached to the surface of the substrate.

19. The kit according to any one of claims 7 to 17, wherein the surface of the substrate is coated with a coating layer, and a bioactive substance is attached to the surface of the coating layer, and the bioactive substance can specifically bind to a target molecule to be detected.

20. Kit according to claim 19, wherein the coating in the coating is selected from the group consisting of polysaccharides, high molecular polymers or biological macromolecules, preferably polysaccharides.

21. The kit of claim 20, wherein the surface of the substrate is coated with at least two successive polysaccharide layers, wherein a first polysaccharide layer is spontaneously associated with a second polysaccharide layer.

22. The kit of claim 21, wherein each of the successive polysaccharide layers is spontaneously associated with each of the previous polysaccharide layers.

23. The kit of any one of claims 20-22, wherein said polysaccharide has pendant functional groups, said functional groups of said continuous polysaccharide layer being oppositely charged from said functional groups of said previous polysaccharide layer.

24. The kit of any one of claims 20-23, wherein 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.

25. The kit of claim 24, wherein the functional groups of the continuous polysaccharide layer alternate between amine functional groups and amine-reactive functional groups.

26. The kit of claim 25, wherein the amine-reactive functional group is an aldehyde group or a carboxyl group.

27. The kit of any one of claims 21-26, wherein the first polysaccharide layer is spontaneously associated with the support.

28. The kit of any one of claims 21-27, wherein the outermost polysaccharide layer of the coating has at least one pendant functional group.

29. The kit of any one of claims 21-28, wherein the pendant functional groups of the outermost polysaccharide layer of the coating are selected from at least one of aldehyde groups, carboxyl groups, thiol groups, amino groups, hydroxyl groups, and maleic groups; preferably selected from aldehyde groups and/or carboxyl groups.

30. The kit of claim 28 or 29, wherein the pendant functional groups of the outermost polysaccharide layer of the coating are directly or indirectly attached to the biologically active substance.

31. The kit according to any one of claims 20 to 30, wherein 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.

32. The kit of any one of claims 7-31, wherein the matrix particle sizes of the different sized acceptor microspheres are the same.

33. The kit of any one of claims 7-31, wherein the receptor microspheres of different particle sizes have different matrix particle sizes.

34. The kit according to any one of claims 1 to 33, wherein the kit specifically comprises:

component a1, comprising at least two receptor microspheres of different particle size that bind to a first antigen, the epitope of which is capable of specifically binding to a first binding site of an epitope of a target antibody to be detected; the receptor microsphere is capable of reacting with active oxygen to generate a detectable chemiluminescent signal;

component b1 comprising a second antigen that binds to one of the members of the specific binding pair, said second antigen being capable of specifically binding to an epitope second binding site of a test target antibody, and the epitope first binding site and the epitope second binding site of the test target antibody do not overlap;

component c1 comprising a donor microsphere bound to the other member of the specific binding pair member; the donor microspheres are capable of generating reactive oxygen species in an excited state.

35. The kit according to any one of claims 1 to 34, wherein the kit specifically comprises:

component a2 comprising at least two different particle size receptor microspheres bound to a first antibody capable of specifically binding to an antigenic determinant of a target antigen to be detected; the receptor microsphere is capable of reacting with active oxygen to generate a detectable chemiluminescent signal;

component b2 comprising a second antibody that binds to one of the members of the specific binding pair, said second antibody being capable of specifically binding to an epitope of the target antigen to be detected;

component c2 comprising a donor microsphere bound to the other member of the specific binding pair member; the donor microspheres are capable of generating reactive oxygen species in an excited state.

36. The kit of claim 34 or 35, wherein the specific binding pair member is selected from the group consisting of an antibody, an antibody fragment, a ligand, an oligonucleotide binding protein, a lectin, a hapten, an antigen, an immunoglobulin binding protein, avidin, or biotin; preferably, the specific binding pair member is avidin-biotin; further preferably, the avidin is selected from the group consisting of ovalbumin, streptavidin, vitellin, neutravidin and avidin-like, preferably neutravidin and/or streptavidin.

37. The kit according to any one of claims 1 to 36, wherein the reactive oxygen species is singlet oxygen.

38. A homogeneous chemiluminescent assay for detecting a target molecule of interest using the kit of any one of claims 1-37.

39. Use of a kit according to any one of claims 1 to 37 and/or a method of detection according to claim 38 for detecting markers of myocardial injury including cTnI and/or inflammatory markers including procalcitonin.

Technical Field

The invention belongs to the technical field of chemiluminescence, and particularly relates to a homogeneous phase chemiluminescence detection kit 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 kit is difficult to meet the detection conditions.

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

Disclosure of Invention

The invention aims to solve the technical problem of the prior art and provides a kit for homogeneous phase chemiluminescence analysis. In addition, the detection time can be shortened.

To this end, the present invention provides in a first aspect a homogeneous chemiluminescent assay kit comprising:

an acceptor reagent comprising at least two acceptor microspheres of different particle sizes capable of reacting with reactive oxygen species to produce a detectable chemiluminescent signal;

a donor agent comprising donor microspheres capable of generating reactive oxygen species in an excited state.

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

In some embodiments of the invention, the concentration of the acceptor microsphere in the acceptor agent is from 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 acceptor agent comprises acceptor microspheres of three different particle sizes.

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

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

In some embodiments of the 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 other embodiments of the present 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 selected from europium.

In some preferred embodiments of the present invention, the metal chelate comprises a chelating agent selected from the group consisting of: NHA, BHHT, BHHCT, DPP, TTA, NPPTA, NTA, TOPO, TPPO, BFTA, 2-dimethyl-4-perfluorobutanoyl-3-butanone (fod), 2' -bipyridine (bpy), bipyridylcarboxylic acids, azacrown ethers, azacryptands and trioctylphosphine oxides 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 present invention, the materials of the matrices of the acceptor microspheres with different particle sizes are the same or different.

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 some preferred embodiments of the 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 other embodiments of the present invention, the receptor microspheres of different particle sizes have different matrix particle sizes.

In some embodiments of the invention, the kit specifically comprises:

In other embodiments of the present invention, the kit specifically comprises:

In some embodiments of the invention, the specific binding pair member is selected from the group consisting of an antibody, an antibody fragment, a ligand, an oligonucleotide binding protein, a lectin, a hapten, an antigen, an immunoglobulin binding protein, avidin, or biotin; preferably, the specific binding pair member is avidin-biotin; further preferably, the avidin is selected from the group consisting of ovalbumin, streptavidin, vitellin, neutravidin and avidin-like, preferably neutravidin and/or streptavidin.

In other embodiments of the present invention, the reactive oxygen species is singlet oxygen.

In a second aspect, the invention provides a homogeneous chemiluminescent assay for detecting a target molecule to be detected using the kit of the first aspect of the invention.

In a third aspect of the invention there is provided a kit according to the first aspect of the invention and/or a method of detection according to the second aspect of the invention for use in detecting a marker of myocardial injury including cTnI and/or a marker of inflammation including procalcitonin.

The invention has the beneficial effects that: in the kit for homogeneous chemiluminescence analysis, the receptor reagent simultaneously comprises at least two receptor microspheres with different particle sizes. The small-particle-size receptor microspheres can widen the detection range, and the large-particle-size receptor microspheres can improve the detection sensitivity, so that the performance of the kit disclosed by the invention is greatly improved compared with the prior art, and the kit has ultrahigh sensitivity and very wide range. In addition, the small-particle-size receptor microspheres have high reaction speed, so that the detection time can be shortened when the kit 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 refers to a general term for a substance which is composed of oxygen, contains oxygen, and is active in nature, and is mainly an excited oxygen molecule, including superoxide anion (O) which is an electron reduction product 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 microspheres 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 "sample to be tested" refers to a multi-component mixed liquid to be tested, which contains a sample to be tested, the acceptor microspheres and the donor microspheres before being tested and analyzed on a computer.

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 term "homogeneous" as used herein is defined in english as "homogeneous" and means that the bound antigen-antibody complex and the remaining free antigen or antibody are detected without separation.

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 receptor reagent, 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.

The homogeneous phase chemiluminescence detection kit provided by the invention comprises:

an acceptor reagent comprising at least two acceptor microspheres of different particle sizes capable of reacting with reactive oxygen species to produce a detectable chemiluminescent signal;

a donor agent comprising donor microspheres capable of generating reactive oxygen species in an excited state.

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

In other embodiments of the present invention, the difference in particle size between the two acceptor microspheres of different particle size is not less than 100 nm; preferably not less than 150 nm; more preferably not less than 200 nm. In some embodiments of the invention, the difference between the particle sizes of the two acceptor microspheres with different particle sizes is no less than 100nm, 130nm, 150nm, 170nm, 190nm, 200nm, 220nm, 240nm or 250 nm.

In some embodiments of the invention, the ratio of the two different receptor microspheres in size is selected from the group consisting of 1 (1.1-10); preferably selected from 1 (2-8); more preferably from 1 (3-6). In some embodiments of the invention, the ratio of the two different receptor microspheres in size is selected from 1:1.5, 1:2, 1:2.7, 1:3, 1:3.2, 1:3.75, 1:4, 1:5, or 1: 6.

In some preferred embodiments of the present invention, one of the acceptor microspheres has a particle size selected from the range of 50nm to 300nm and the other acceptor microsphere has a particle size selected from the range of 200nm to 400 nm. For example, one of the acceptor microspheres has a particle size selected from 50nm, 80nm, 110nm, 140nm, 170nm, 200nm, or 300nm, and the other acceptor microsphere has a particle size selected from 200nm, 250nm, 300nm, 350nm, or 400 nm. In some more preferred embodiments of the present invention, one of the acceptor microspheres has a particle size selected from the range of 50nm to 200nm and the other acceptor microsphere has a particle size selected from the range of 200nm to 350 nm.

In some further preferred embodiments of the present invention, one of the acceptor microspheres has a particle size selected from 80nm to 150nm and the other acceptor microsphere has a particle size selected from 220nm to 350 nm. In the present invention, the size of the acceptor microsphere should be such that a uniform and stable emulsion solution can be produced, and the size of the acceptor microsphere generally meeting the requirement should be in the nanometer range. Therefore, the upper limit of the particle size of the large-particle-size acceptor microsphere is such that a stable emulsion solution can be formed, and about 300nm is generally preferred. Meanwhile, the coating and washing of the acceptor microspheres are carried out under the existing technical conditions so as to meet the production requirement of the reagent.

In some embodiments of the invention, the concentration of the acceptor microsphere in the acceptor agent is from 1ug/mL to 1000 ug/mL; preferably 10ug/mL to 500 ug/mL; more preferably 50ug/mL to 250 ug/mL. In the invention, the use concentration of the receptor microsphere is determined by the concentration of different target molecules to be detected in blood and the characteristics of the target molecules to be detected.

In some embodiments of the invention, the acceptor agent comprises acceptor microspheres of three different particle sizes.

In some embodiments of the present invention, the composition and chemical structure of the acceptor microspheres with different particle sizes may be the same or different. For example, the respective luminescent compositions and/or matrices of the differently sized acceptor microspheres may be the same or different, provided that they are capable of reacting with singlet oxygen to produce a detectable chemiluminescent signal.

In some embodiments of the invention, the recipient microsphere may have a coefficient of variation of particle size distribution C.V value of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, etc. in the recipient agent.

It should be noted that the value of C.V for the variation coefficient of particle size distribution of the acceptor microspheres in the present invention refers to the value of C.V for the variation coefficient of particle size distribution after the acceptor microspheres are coated with the desired substance.

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

In some embodiments of the invention, the chemiluminescent compound is selected from 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 selected from polymeric microspheres, preferably latex microspheres, more preferably polystyrene latex microspheres.

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 some preferred embodiments of the 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, and 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.