阅读说明：本技术 一种化学发光分析方法及其应用 (Chemiluminescence analysis method 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化学发光分析方法,其通过检测待测样品中的至少两种不同粒径的受体微球与活性氧反应产生的化学发光信号强度来分析判断待测样品中是否包含待测目标分子和/或待测目标分子的浓度。本发明所述方法既有超高的灵敏度,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 chemiluminescence analysis method in the technical field of chemiluminescence analysis, which analyzes and judges whether a sample to be detected contains target molecules to be detected and/or the concentration of the target molecules to be detected by detecting the intensity of chemiluminescence signals generated by the reaction of at least two receptor microspheres with different particle sizes in the sample to be detected and active oxygen. The method of the invention has ultra-high 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 chemiluminescence analysis method analyzes and judges whether a sample to be detected contains target molecules to be detected and/or the concentration of the target molecules to be detected by detecting the intensity of chemiluminescence signals generated by the reaction of at least two receptor microspheres with different particle sizes in the sample to be detected and active oxygen.

2. The method according to claim 1, wherein the method is characterized in that the method analyzes and judges whether the sample to be detected contains the target molecule to be detected and/or the concentration of the target molecule to be detected by detecting the intensity of a chemiluminescent signal generated by the reaction of two acceptor microspheres with different particle sizes in the sample to be detected and active oxygen.

3. The method of claim 2, wherein the difference in particle size between the two different sized acceptor microspheres is no less than 100 nm; preferably not less than 150 nm; more preferably not less than 200 nm.

4. The method of claim 2 or 3, wherein the ratio of the two different receptor microspheres is selected from the group consisting of 1 (1.1-10); preferably selected from 1 (2-8); more preferably from 1 (3-6).

5. The method of any one of claims 1 to 4, wherein the receptor microsphere 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.

6. The method of any one of claims 1-5, wherein the sample to be tested further comprises donor microspheres capable of generating reactive oxygen species in an excited state.

7. The method of any one of claims 1 to 6, wherein the method is a homogeneous chemiluminescent assay.

8. The method according to claim 7, characterized in that it comprises the steps of:

s1, mixing a sample to be detected with a reagent a containing at least two acceptor microspheres with different particle sizes, and then mixing with a reagent b containing donor microspheres to obtain a sample to be detected;

s2, contacting the sample to be tested obtained in the step S1 with energy or active chemical, and exciting the donor to generate active oxygen;

s3, analyzing and judging whether the sample to be detected contains the target molecules to be detected and/or the concentration of the target molecules to be detected by detecting the intensity of chemiluminescence signals generated by the reaction of at least two receptor microspheres with different particle sizes in the sample to be detected and active oxygen.

9. The method of claim 8, wherein the sample to be tested is diluted with a diluent and then reacted.

10. The method according to claim 1, wherein the method is characterized in that the method analyzes and judges whether the sample to be detected contains the target molecule to be detected and/or the concentration of the target molecule to be detected by detecting the intensity of chemiluminescence signals generated by the reaction of three receptor microspheres with different particle sizes and active oxygen in the sample to be detected.

11. The method according to any one of claims 1 to 10, wherein the detection wavelength of the chemiluminescent signal is 520 to 620 nm.

12. The method according to any one of claims 8 to 11, wherein the laser irradiation is performed with red excitation light of 600 to 700 nm.

13. The method according to any one of claims 1 to 12, 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.

14. The method of claim 13, 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.

15. The method according to claim 14, wherein the chemiluminescent compound is selected from the group consisting of olefinic compounds, preferably from the group consisting of dimethylthiophene, dibutyldione compounds, dioxines, enol ethers, enamines, 9-alkylidenexanthanes, 9-alkylene-N-9, 10 dihydroacridines, arylethyletherenes, arylimidazoles, and lucigenins and derivatives thereof, more preferably from the group consisting of dimethylthiophene and its derivatives.

16. The process according to claim 14 or 15, 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.

17. The method of any one of claims 14-16, wherein 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.

18. The method of any one of claims 14-17, wherein the luminescent compound is a derivative of dimethylthiophene and the metal chelate is a europium chelate.

19. The method of any one of claims 13-18, wherein 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.

20. The method of any one of claims 13 to 19, wherein the matrix is magnetic or non-magnetic particles.

21. The method of any one of claims 13 to 20, wherein the matrices of the acceptor microspheres of different particle sizes are of the same or different materials.

22. The method of any one of claims 13-21, 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; preferably, the matrix is polystyrene latex microspheres; more preferably, the polystyrene latex microspheres are carboxyl and/or aldehyde groups.

23. The method according to any one of claims 13 to 22, wherein a biologically active substance capable of specifically binding to the target molecule to be detected is directly attached to the surface of the substrate.

24. The method according to any one of claims 13 to 23, wherein the surface of the substrate is coated with a coating layer, and the surface of the coating layer is linked with a bioactive substance capable of specifically binding to the target molecule to be detected.

25. The method according to claim 24, wherein the coating in the coating layer is selected from the group consisting of polysaccharides, high molecular polymers or biological macromolecules, preferably polysaccharides.

26. The method of claim 25, 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.

27. The method of claim 26, wherein each of the successive polysaccharide layers is spontaneously associated with each of the previous polysaccharide layers.

28. The method of any one of claims 25-27, wherein said polysaccharide has pendant functional groups, and wherein said functional groups of said continuous polysaccharide layer are oppositely charged from said functional groups of said previous polysaccharide layer.

29. The method of any one of claims 25-28, 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.

30. The method of claim 29, wherein the functional groups of the continuous polysaccharide layer alternate between amine functional groups and amine-reactive functional groups.

31. The method of claim 30, wherein the amine-reactive functional group is an aldehyde group or a carboxyl group.

32. The method of any one of claims 26-31, wherein the first polysaccharide layer spontaneously associates with the support.

33. The method of any one of claims 26-32, wherein the outermost polysaccharide layer of the coating has at least one pendant functional group.

34. The method of any one of claims 26-33, 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.

35. The method of claim 33 or 34, wherein the pendant functional groups of the outermost polysaccharide layer of the coating are directly or indirectly attached to the biologically active substance.

36. The method according to any one of claims 25 to 35, wherein the 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.

37. The method of any one of claims 13-36, wherein the substrate particles of the different size receptor microspheres are the same size.

38. The method of any one of claims 13-36, wherein the different sized acceptor microspheres have different substrate particle sizes.

39. The method according to any one of claims 1 to 38, wherein the reactive oxygen species is singlet oxygen.

40. A chemiluminescence analyzer for detecting the presence and/or concentration of a target molecule to be detected in a sample to be detected by the method of any one of claims 1 to 39.

41. A chemiluminescence analyzer according to claim 40, comprising at least the following:

the incubation module is used for providing a proper temperature environment for a chemiluminescent reaction after a sample to be tested and at least two receptor microspheres with different particle sizes are mixed;

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.

42. Use of a method according to any one of claims 1 to 39 and/or a chemiluminescent analyzer according to claim 40 or 41 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 analysis, and particularly relates to a chemiluminescence analysis method 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 chemiluminescence analysis method is difficult to meet the detection conditions.

Therefore, it is highly desirable to develop a chemiluminescence analysis method that can satisfy 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 chemiluminescence analysis method aiming at the defects of the prior art, wherein the method has ultrahigh sensitivity and wide detection range. In addition, the method can shorten the detection time.

Therefore, the invention provides a chemiluminescence analysis method, which analyzes and judges whether a sample to be detected contains target molecules to be detected and/or the concentration of the target molecules to be detected by detecting the intensity of chemiluminescence signals generated by the reaction of at least two receptor microspheres with different particle sizes in the sample to be detected and active oxygen.

In some embodiments of the present invention, the method analyzes and determines whether the sample to be tested contains the target molecule to be tested and/or the concentration of the target molecule to be tested by detecting the intensity of the chemiluminescent signal generated by the reaction of two acceptor microspheres with different particle sizes in the sample to be tested and active oxygen.

In some embodiments of the present invention, the difference in particle size between the two different sized acceptor microspheres is no less than 100 nm; preferably not less than 150 nm; more preferably not less than 200 nm.

In other embodiments of the present invention, the ratio of the two different receptor microspheres 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 receptor microsphere is used at a concentration of 1ug/mL to 1000 ug/mL; preferably 10ug/mL to 500 ug/mL; more preferably from 10ug/mL to 250 ug/mL.

In some embodiments of the present invention, the sample to be tested further comprises donor microspheres, and the donor microspheres are capable of generating active oxygen in an excited state.

In other embodiments of the present invention, the method is a homogeneous chemiluminescent assay method.

In some preferred embodiments of the invention, the method comprises the steps of:

s1, mixing a sample to be detected with a reagent a containing at least two acceptor microspheres with different particle sizes, and then mixing with a reagent b containing donor microspheres to obtain a sample to be detected;

s2, contacting the sample to be tested obtained in the step S1 with energy or active chemical, and exciting the donor to generate active oxygen;

s3, analyzing and judging whether the sample to be detected contains the target molecules to be detected and/or the concentration of the target molecules to be detected by detecting the intensity of chemiluminescence signals generated by the reaction of at least two receptor microspheres with different particle sizes in the sample to be detected and active oxygen.

In some embodiments of the present invention, the sample to be tested is diluted with a diluent and then subjected to a reaction.

In some embodiments of the present invention, the method analyzes and determines whether the sample to be tested contains the target molecule to be tested and/or the concentration of the target molecule to be tested by detecting the intensity of the chemiluminescent signal generated by the reaction of the three acceptor microspheres with different particle sizes in the sample to be tested and the active oxygen.

In some embodiments of the present invention, the detection wavelength of the chemiluminescent signal is 520-620 nm.

In other embodiments of the present invention, the laser irradiation is performed using red excitation light of 600 to 700 nm.

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: 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 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 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.

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 reactive oxygen species is singlet oxygen.

In a second aspect, the present invention provides a chemiluminescent analyzer for detecting the presence and/or concentration of a target molecule to be detected in a sample to be detected using the method of the first aspect of the present invention.

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

the incubation module is used for providing a proper temperature environment for a chemiluminescent reaction after a sample to be tested and at least two receptor microspheres with different particle sizes are mixed;

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.

In a third aspect the invention provides the use of a method according to the first aspect of the invention and/or a chemiluminescence analyzer according to the second aspect of the invention to detect markers of myocardial injury including cTnI and/or markers of inflammation including procalcitonin.

The invention has the beneficial effects that: according to the chemiluminescence analysis method, the receptor microspheres (small-particle-size receptor microspheres and large-particle-size receptor microspheres) with at least two different particle sizes are added into a sample to be detected, and 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 detection performance of the method is greatly improved compared with the prior art, and the chemiluminescence analysis method has ultrahigh sensitivity and very wide detection range. In addition, the small-particle-size receptor microspheres have high reaction speed, so that the detection time can be shortened when the method 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 "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 luminescent microspheres, as measured by 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.

Description of the preferred embodiments

The present invention will be described in more detail below.

The first aspect of the present invention relates to a chemiluminescence analysis method, wherein the chemiluminescence analysis method is used for analyzing and judging whether a sample to be detected contains a target molecule to be detected and/or the concentration of the target molecule to be detected by detecting the intensity of chemiluminescence signals generated by the reaction of at least two receptor microspheres with different particle sizes in the sample to be detected and active oxygen.

In some embodiments of the present invention, the method analyzes and determines whether the sample to be tested contains the target molecule to be tested and/or the concentration of the target molecule to be tested by detecting the intensity of the chemiluminescent signal generated by the reaction of two acceptor microspheres with different particle sizes in the sample to be tested and active oxygen.

In some embodiments of the present invention, the difference in particle size between the two different sized acceptor microspheres is no 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 other embodiments of the present invention, the ratio of the two different receptor microspheres 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 a further preferred embodiment 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 a still further embodiment 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.

The method controls the particle size of the receptor microsphere in the used microsphere composition, and further controls the amount of bioactive substances (such as antibodies/antigens) on the surface of each receptor microsphere (the small-particle-size microsphere has large specific surface area, the reporter molecules on the surface of the unit-mass microsphere have more amount, the large-particle-size microsphere has small specific surface area, and the reporter molecules on the surface of the unit-mass microsphere have less amount), so that the detection sensitivity is improved, and the detection range is widened. 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 some embodiments of the invention, the receptor microsphere is used at a concentration of 1ug/mL to 1000 ug/mL; preferably 10ug/mL to 500ug/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 present invention, the sample to be tested further comprises donor microspheres, and the donor microspheres are capable of generating active oxygen in an excited state.

In other embodiments of the present invention, the method is a homogeneous chemiluminescent assay method.

In some preferred embodiments of the invention, the method comprises the steps of:

s1, mixing a sample to be detected with a reagent a containing at least two acceptor microspheres with different particle sizes, and then mixing with a reagent b containing donor microspheres to obtain a sample to be detected;

s2, contacting the sample to be tested obtained in the step S1 with energy or active chemical, and exciting the donor to generate active oxygen;

s3, analyzing and judging whether the sample to be detected contains the target molecules to be detected and/or the concentration of the target molecules to be detected by detecting the intensity of chemiluminescence signals generated by the reaction of at least two receptor microspheres with different particle sizes in the sample to be detected and active oxygen.

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

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, in step S1, the sample to be tested is mixed with the first reagent, and then mixed with the reagent a containing at least two acceptor microspheres with different particle sizes. It should be noted that the first reagent described in the present invention does not refer to a certain kind of reagent, and the first reagent is added to ensure the successful or optimized performance of certain detection methods based on specific reactions, and the first reagent includes but is not limited to: biotinylated antigen or antibody.

In some preferred embodiments of the invention, the detection method of the specific reaction is a sandwich method. For example, the immune complex pattern is: donor microsphere-streptavidin-biotin-antibody 1-antigen-antibody 2-acceptor microsphere, in which case the first reagent is a biotinylated antigen or antibody; the donor microsphere is coupled with a streptavidin donor microsphere, and the receptor microsphere is coupled with an antigen or an antibody.

In order to further improve the accuracy of the final detection result and the stability of the sample to be detected, in some preferred embodiments of the present invention, in step S1, the sample to be detected is diluted with a diluent, and then mixed with the first reagent, and then mixed with the donor microsphere reagent.

In some embodiments of the present invention, the method analyzes and determines whether the sample to be tested contains the target molecule to be tested and/or the concentration of the target molecule to be tested by detecting the intensity of the chemiluminescent signal generated by the reaction of the three acceptor microspheres with different particle sizes in the sample to be tested and the active oxygen.

In some embodiments of the present invention, the detection wavelength of the chemiluminescent signal is 520-620 nm, preferably 610-620nm, and more preferably 615 nm.

In other embodiments of the present invention, the laser irradiation is performed with 600 to 700nm red excitation light; preferably, red exciting light with 640-680nm is adopted for laser irradiation; more preferably, the laser irradiation is performed with 660nm red excitation light.

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 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 some preferred embodiments of the 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 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 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.

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

In the present invention, the compositions and chemical structures of the acceptor microspheres with different particle sizes may be the same or different. For example, the luminescent composition and/or matrix of each of the receptor microspheres of different particle sizes may be the same or different, provided that they react with reactive oxygen species to produce a detectable chemiluminescent signal.

A second aspect of the present invention relates to a chemiluminescent analyzer for detecting the presence and/or concentration of a target molecule of interest in a test sample using the method of the first aspect of the present invention.

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

the incubation module is used for providing a proper temperature environment for a chemiluminescent reaction after a sample to be tested and at least two receptor microspheres with different particle sizes are mixed;

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.

A third aspect of the invention relates to the use of a method according to the first aspect of the invention and/or a chemiluminescent analyzer according to the second aspect of the invention to detect markers of myocardial injury including cTnI and/or markers of inflammation including procalcitonin.

Detailed description of the preferred embodiments

In order that the present invention may be more readily understood, the following detailed description will proceed with reference being made to examples, which are intended to be illustrative only and are not intended to limit the scope of the invention. The starting materials or components used in the present invention may be commercially or conventionally prepared unless otherwise specified.