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 赵卫国 刘宇卉 李临 于 2019-07-18 设计创作，主要内容包括：本发明涉及一种均相化学发光检测试剂盒及其应用。所述试剂盒包括：供体试剂,其包括供体微球,所述供体微球能够在激发状态下生成活性氧,且所述供体微球表面包被有标记物；和,受体试剂,其包括受体微球,所述受体微球能够与活性氧反应产生可检测的化学发光信号,且所述受体微球表面包被有生物分子,所述生物分子能够与待测目标分子特异性结合；其中,所述供体微球的粒径大于所述受体微球的粒径。试剂盒中的供体微球的粒径大于受体微球的粒径,提高了该试剂盒的精密性和灵敏度。(The invention relates to homogeneous phase chemiluminescence detection kits and applications thereof, wherein the kits comprise a donor reagent and an acceptor reagent, the donor reagent comprises donor microspheres, the donor microspheres can generate active oxygen under an excitation state, labels are coated on the surfaces of the donor microspheres, the acceptor reagent comprises acceptor microspheres, the acceptor microspheres can react with the active oxygen to generate detectable chemiluminescence signals, biomolecules are coated on the surfaces of the acceptor microspheres and can be specifically combined with target molecules to be detected, the particle size of the donor microspheres is larger than that of the acceptor microspheres, the particle size of the donor microspheres in the kit is larger than that of the acceptor microspheres, and the precision and the sensitivity of the kit are improved.)

1, homogeneous chemiluminescent assay kit comprising:

a donor reagent comprising donor microspheres capable of generating reactive oxygen species in an excited state and having a label coated on the surface thereof; and the combination of (a) and (b),

the receptor reagent comprises a receptor microsphere, wherein the receptor microsphere can react with active oxygen to generate a detectable chemiluminescent signal, and the surface of the receptor microsphere is coated with a biomolecule which can be specifically combined with a target molecule to be detected;

wherein the particle size of the donor microspheres is larger than the particle size of the acceptor microspheres.

2. The kit of claim 1, wherein the donor microspheres have an average particle size of 100nm to 400nm, the acceptor microspheres have an average particle size of 100nm to 350nm, and the ratio of the average particle size of the donor microspheres to the average particle size of the acceptor microspheres is 1.1 to 4.0;

preferably, the average particle size of the donor microspheres is 190nm-250nm, the average particle size of the acceptor microspheres is 180nm-240nm, and the ratio of the average particle size of the donor microspheres to the average particle size of the acceptor microspheres is 1.2-3.0.

3. The kit of claim 2, wherein the donor microspheres have an average particle size of 150nm, the acceptor microspheres have an average particle size of 100nm, and the ratio of the average particle size of the donor microspheres to the average particle size of the acceptor microspheres is 1.5.

4. The kit of any of claims 1-3, wherein the donor microsphere comprises a th carrier, wherein the interior of the th carrier is filled with a sensitizer, and wherein the th carrier has a label chemically bonded to its surface.

5. The kit of claim 4, wherein the surface of the th carrier is not coated or linked with a polysaccharide substance that is directly chemically bonded to the label.

6. The kit of any of claims 1-5, wherein the label is avidin, preferably the avidin is selected from the group consisting of ovalbumin, streptavidin, vitellin, neutravidin, and avidin-like, and further preferably is selected from the group consisting of neutravidin and streptavidin.

7. The kit of any of claim 6, wherein the avidin is chemically bonded to the surface of the -th carrier by reacting an amino group with an aldehyde group on the surface of the -th carrier to form a schiff base.

8. The kit of any of claims 5-7, wherein the th carrier has a surface with binding functionalities for chemically binding a label to the surface of the th carrier.

9. The kit according to claim 8, wherein the bonding functional group is selected from an amine group, an amide group, a hydroxyl group, an aldehyde group, a carboxyl group, a maleimide group and a thiol group, preferably from an aldehyde group and/or a carboxyl group, and preferably the content of the bonding functional group on the th carrier surface is 100-500 nmol/mg, preferably 200-400 nmol/mg.

10. The kit of claim 4, wherein the surface of the th carrier is coated with hydrophilic aldehyde dextran, and the aldehyde group of the aldehyde dextran is chemically bonded with a label.

11. The kit of any of , wherein the photosensitizer is selected from the group consisting of methylene blue, rose bengal, porphyrin and phthalocyanine.

12. The kit of any , wherein the acceptor microsphere comprises a second support, the second support filled with the luminescent composition, the second support having a surface coated with at least layers of polysaccharide, the polysaccharide layer having biomolecules attached to the surface.

13. The kit of claim 12, wherein the surface of the second carrier is coated with hydrophilic carboxydextran.

14. The kit of claim 12 or 13, wherein the luminescent composition comprises a europium complex, and further , wherein the europium complex is MTTA-EU³⁺。

15. The kit according to , wherein the th vector and/or the second vector is made of agarose, cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polystyrene, polyethylene, polypropylene, poly (4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate, nylon, polyethylene butyrate or polyacrylate, preferably polystyrene, polypropylene, poly (4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate or polyacrylate.

16. The kit of any of , wherein the donor microspheres have a coefficient of variation of particle size distribution C.V value of 5% or more in the donor agent and/or,

the variation coefficient C.V value of the particle size distribution of the receptor microsphere in the receptor reagent is more than or equal to 5%.

17. The kit of any of claims 1-16, wherein the reactive oxygen species is singlet oxygen.

18. The kit of any of claims 1-17, wherein the kit specifically comprises:

the receptor reagent comprises a receptor microsphere combined with th antigen, wherein the th antigen epitope can be specifically combined with th binding site of the epitope of a target antibody to be detected;

reagent comprising a second antigen binding to biotin, the second antigen being capable of specifically binding to an epitope second binding site of a test target antibody, and the epitope binding site and the epitope second binding site of the test target antibody do not overlap,

a donor reagent comprising donor microspheres bound to avidin; the donor microsphere is capable of generating singlet oxygen in an excited state;

wherein the particle size of the donor microspheres is larger than the particle size of the acceptor microspheres.

19. The kit of any of claims 1-17, wherein the kit specifically comprises:

the receptor reagent comprises a receptor microsphere combined with th antibody, wherein the th antibody can be specifically combined with an antigenic determinant of a target antigen to be detected;

reagent comprising a second antibody that binds to biotin, the second antibody being capable of specifically binding to an epitope of a target antigen to be detected, and,

a donor reagent comprising donor microspheres bound to avidin; the donor microsphere is capable of generating singlet oxygen in an excited state;

wherein the particle size of the donor microspheres is larger than the particle size of the acceptor microspheres.

20. The kit of any of claims 1-17, wherein the kit specifically comprises:

a receptor reagent comprising a receptor microsphere bound to an antigen, the antigen being capable of specifically binding to an antibody of interest, the receptor microsphere being capable of reacting with singlet oxygen to produce a detectable chemiluminescent signal;

an th reagent comprising a second antibody that binds to biotin, the second antibody being capable of specifically binding to the antigen, and

a donor reagent comprising a donor microsphere bound to avidin, the donor microsphere capable of generating singlet oxygen in an excited state;

wherein the particle size of the donor microspheres is larger than the particle size of the acceptor microspheres.

21. The kit of any of claims 1-17, wherein the kit specifically comprises:

an th reagent comprising a th antigen that binds biotin, the th antigen being capable of specifically binding to an epitope binding site of an antibody of interest;

a receptor reagent comprising a receptor microsphere bound to an anti-immune complex antibody, the anti-immune complex antibody being capable of specifically binding to a target antibody in an immune complex formed between the th antigen and the target antibody, the receptor microsphere being capable of reacting with singlet oxygen to produce a detectable chemiluminescent signal, and,

a donor reagent comprising a donor microsphere bound to avidin, the donor microsphere capable of generating singlet oxygen in an excited state;

wherein the particle size of the donor microspheres is larger than the particle size of the acceptor microspheres.

22. The kit of any of claims 1-17, wherein the kit specifically comprises:

a receptor reagent comprising a receptor microsphere bound to th antigen, the th antigen being capable of specifically binding to an antibody of interest, the receptor microsphere being capable of reacting with singlet oxygen to produce a detectable chemiluminescent signal;

an th reagent comprising an anti-immune complex antibody that binds to biotin, the anti-immune complex antibody being capable of specifically binding to a target antibody in an immune complex formed between a th antigen and the target antibody;

a donor reagent comprising a donor microsphere bound to avidin, the donor microsphere being capable of generating singlet oxygen in an excited state.

23. The kit of claim 21 or 22, wherein the anti-immune complex antibody does not bind to the target antibody free or bound to antigen.

24, homogeneous chemiluminescent assay method for detecting a target molecule to be detected in a sample to be detected using the kit of any one of claims 1-23, .

25, homogeneous chemiluminescent assay device for detecting a target molecule to be detected in a sample to be detected using the kit of any of claims 1-23 or the method of claim 24.

26. The apparatus of claim 25, wherein the apparatus is a POCT point-of-care testing apparatus.

Technical Field

The invention belongs to the technical field of chemiluminescence, and particularly relates to homogeneous phase chemiluminescence detection kits and application thereof.

Background

The evolution process is mainly determined by the increasing demands for sensitivity, accuracy and simplicity of operation of detection methods.

For example, one molecule of a specific binding pair can be combined with the luminescent material in a variety of ways to form a luminescent complex, which can react with the analyte (the other molecule of the specific binding pair) in the sample, partition into solid and liquid phases, and the partition ratio is related to the amount of the analyte.

To increase the efficiency of the emission of the donor and/or acceptor microspheres, methods to increase the efficiency of the light exposure of the dye in the donor microsphere and/or the efficiency and efficiency of the emission of the light-emitting compound in the acceptor microsphere are commonly used in the art .

Although the detection sensitivity of the chemiluminescence detection method can be improved in degree range by adopting the method in the field, the detection range or linear range is narrower, therefore, kits which can improve the luminous efficiency in steps based on the prior art are needed to be developed, and not only have higher sensitivity, but also have wider detection range.

Disclosure of Invention

The invention provides kits for homogeneous phase chemiluminescence detection aiming at the defects of the prior art, and the kit has higher sensitivity and wider detection range when being used for homogeneous phase chemiluminescence detection

To this end, the aspect of the invention provides homogeneous chemiluminescent assay kits comprising:

a donor reagent comprising donor microspheres capable of generating reactive oxygen species in an excited state and having a label coated on the surface thereof; and the combination of (a) and (b),

the receptor reagent comprises a receptor microsphere, wherein the receptor microsphere can react with active oxygen to generate a detectable chemiluminescent signal, and the surface of the receptor microsphere is coated with a biomolecule which can be specifically combined with a target molecule to be detected;

wherein the particle size of the donor microspheres is larger than the particle size of the acceptor microspheres.

In embodiments of the present invention, the donor microspheres have an average particle size of 100nm to 400nm, the acceptor microspheres have an average particle size of 100nm to 350nm, and the ratio of the average particle size of the donor microspheres to the average particle size of the acceptor microspheres is 1.1 to 4.0;

preferably, the average particle size of the donor microspheres is 190nm-250nm, the average particle size of the acceptor microspheres is 180nm-240nm, and the ratio of the average particle size of the donor microspheres to the average particle size of the acceptor microspheres is 1.2-3.0;

, the average grain size of the donor microballoon is 100nm-200nm, the average grain size of the acceptor microballoon is 50nm-150nm, and the ratio of the average grain size of the donor microballoon to the average grain size of the acceptor microballoon is 1.3-2.0;

and , preferably, the average particle size of the donor microsphere is 150nm-200nm, the average particle size of the acceptor microsphere is 100nm-140nm, and the ratio of the average particle size of the donor microsphere to the average particle size of the acceptor microsphere is 1.4-1.6.

In particularly preferred embodiments of the present invention, the donor microspheres have an average particle size of 150nm, the acceptor microspheres have an average particle size of 100nm, and the ratio of the average particle size of the donor microspheres to the average particle size of the acceptor microspheres is 1.5.

In , the donor microsphere includes a carrier, a carrier filled with a sensitizer, and a carrier with a chemically bonded label at its surface.

In still other embodiments of the invention, the th vector has no polysaccharide moiety coated or attached to its surface, which is directly chemically bonded to the label.

In embodiments of the invention, the label is avidin.

In still further embodiments of the invention, the avidin is selected from the group consisting of ovalbumin, streptavidin, vitellin, neutravidin, and avidin-like, preferably selected from the group consisting of neutravidin and streptavidin.

In embodiments of the present invention, the avidin is chemically bonded to the surface of the th carrier by reacting an amino group with an aldehyde group on the surface of the th carrier to form a schiff base.

In other embodiments of the present invention, the th carrier has a bonding functional group on its surface for chemically bonding a label to the th carrier surface.

In the embodiments of the present invention, the bonding functional group is selected from amine group, amide group, hydroxyl group, aldehyde group, carboxyl group, maleimide group and thiol group, preferably selected from aldehyde group and/or carboxyl group.

In another embodiments of the present invention, the bonding functional group on the th carrier surface is 100 to 500nmol/mg, preferably 200 to 400 nmol/mg.

In of the embodiments of the present invention, the surface of carrier is coated with hydrophilic aldehyde dextran, and the aldehyde group of the aldehyde dextran is chemically bonded with the label.

In preferred embodiments of the present invention, the photosensitizer is selected from of methylene blue, rose bengal, porphyrin and phthalocyanine.

In still other embodiments of the present invention, the acceptor microsphere includes a second support filled with the luminescent composition, the second support having a surface coated with at least polysaccharide layers, the polysaccharide layers having biomolecules attached to the surface.

In embodiments of the invention, the surface of the second carrier is coated with hydrophilic carboxydextran.

In other embodiments of the present invention, the light emitting composition comprises a europium complex, and preferably the europium complex is MTTA-EU³⁺。

In the embodiments of the present invention, the material of the th carrier and/or the second carrier is selected from agarose, cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polystyrene, polyethylene, polypropylene, poly (4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate, nylon, polyethylene butyrate or polyacrylate, preferably selected from polystyrene, polypropylene, poly (4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate or polyacrylate.

In still other embodiments of the invention, the donor microspheres have a coefficient of variation of particle size distribution C.V value of 5% or more in the donor reagent and/or,

the variation coefficient C.V value of the particle size distribution of the receptor microsphere in the receptor reagent is more than or equal to 5%.

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

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

the receptor reagent comprises a receptor microsphere combined with th antigen, wherein the th antigen epitope can be specifically combined with th binding site of the epitope of a target antibody to be detected;

reagent comprising a second antigen binding to biotin, the second antigen being capable of specifically binding to an epitope second binding site of a test target antibody, and the epitope binding site and the epitope second binding site of the test target antibody do not overlap,

a donor reagent comprising donor microspheres bound to avidin; the donor microsphere is capable of generating singlet oxygen in an excited state;

wherein the particle size of the donor microspheres is larger than the particle size of the acceptor microspheres.

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

the receptor reagent comprises a receptor microsphere combined with th antibody, wherein the th antibody can be specifically combined with an antigenic determinant of a target antigen to be detected;

reagent comprising a second antibody that binds to biotin, the second antibody being capable of specifically binding to an epitope of a target antigen to be detected, and,

a donor reagent comprising donor microspheres bound to avidin; the donor microsphere is capable of generating singlet oxygen in an excited state;

wherein the particle size of the donor microspheres is larger than the particle size of the acceptor microspheres.

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

a receptor reagent comprising a receptor microsphere bound to an antigen, the antigen being capable of specifically binding to an antibody of interest, the receptor microsphere being capable of reacting with singlet oxygen to produce a detectable chemiluminescent signal;

an th reagent comprising a second antibody that binds to biotin, the second antibody being capable of specifically binding to the antigen, and

a donor reagent comprising a donor microsphere bound to avidin, the donor microsphere capable of generating singlet oxygen in an excited state;

wherein the particle size of the donor microspheres is larger than the particle size of the acceptor microspheres.

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

an th reagent comprising a th antigen that binds biotin, the th antigen being capable of specifically binding to an epitope binding site of an antibody of interest;

a receptor reagent comprising a receptor microsphere bound to an anti-immune complex antibody, the anti-immune complex antibody being capable of specifically binding to a target antibody in an immune complex formed between the th antigen and the target antibody, the receptor microsphere being capable of reacting with singlet oxygen to produce a detectable chemiluminescent signal, and,

a donor reagent comprising a donor microsphere bound to avidin, the donor microsphere capable of generating singlet oxygen in an excited state;

wherein the particle size of the donor microspheres is larger than the particle size of the acceptor microspheres.

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

a receptor reagent comprising a receptor microsphere bound to th antigen, the th antigen being capable of specifically binding to an antibody of interest, the receptor microsphere being capable of reacting with singlet oxygen to produce a detectable chemiluminescent signal;

an th reagent comprising an anti-immune complex antibody that binds to biotin, the anti-immune complex antibody being capable of specifically binding to a target antibody in an immune complex formed between a th antigen and the target antibody;

a donor reagent comprising a donor microsphere bound to avidin, the donor microsphere being capable of generating singlet oxygen in an excited state.

According to the present invention, the anti-immune complex antibody does not bind to the target antibody which is free or not bound to the th antigen.

In a second aspect, the invention provides homogeneous phase chemiluminescence detection methods, which utilize the kit according to the aspect of the invention to detect a target molecule to be detected in a sample to be detected.

In a third aspect, the present invention provides homogeneous chemiluminescent assay devices for detecting a target molecule to be detected in a sample to be detected using the kit according to the aspect of the present invention or the method according to the second aspect of the present invention.

In preferred embodiments of the present invention, the device is a POCT point of care device.

The invention has the beneficial effects that: by controlling the matrixes and the grain sizes of the acceptor microspheres and the donor microspheres, the kit improves the luminous efficiency of detection when being used for chemiluminescence detection, and has good detection sensitivity. In addition, hydrophilic carboxyl glucan is coated on the surface of the acceptor microsphere, hydrophilic aldehyde glucan is coated on the surface of the donor microsphere, so that nonspecific adsorption is greatly reduced, the influence of other environmental factors outside a system such as pH value and electrolyte is reduced, and the detection accuracy can be improved.

Drawings

The invention will now be described in further detail with reference to the drawings.

FIG. 1 is a Gaussian distribution diagram of aldehyde-based polystyrene latex microspheres prepared in example 7.

FIG. 2 is a Nicomp distribution plot of aldehyde-based polystyrene latex microspheres prepared in example 7.

FIG. 3 is a Gaussian distribution plot of donor microspheres prepared in example 7.

FIG. 4 is a Gaussian distribution plot of dextran-coated microspheres prepared in example 8

FIG. 5 is a Gaussian distribution plot of donor microspheres prepared in example 8.

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 a stated range of upper and lower limits and any other stated or intervening value in that stated range is encompassed within the invention, that the upper and lower limits of such smaller ranges may independently be included in the smaller ranges, and that there is also included in the invention, subject to any specifically excluded limit in the stated range, in the event that a stated range includes or two limits, any range or both excluding those included limits is also encompassed within 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 "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.

In the present invention, the "donor microsphere" can be a polymeric microparticle coated on a carrier via a functional group to form a photosensitizer filled polymer microsphere capable of generating active oxygen (e.g., singlet oxygen) upon photoexcitation, in which case the donor microsphere can also be referred to as a photosensitive microsphere or photosensitive microparticle, the photosensitizer is filled inside the donor microsphere, the photosensitizer can be a photosensitizer 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, porphyrin, and phthalocyanine, and derivatives of these compounds having 1 to 50 atom substituents that are used to render these compounds more lipophilic or hydrophilic and/or as a linking group to a specific binding partner.

The "acceptor microsphere" may be a polymer particle coated with a functional group on a carrier to form a light-emitting compound, which may be referred to as a light-emitting microsphere or a light-emitting particle, and the light-emitting microsphere may have hydrophilic carboxyl dextran on the surface and a light-emitting composition capable of reacting with active oxygen (e.g., singlet oxygen) filled therein³⁺。

The "carrier" according to the present invention is selected from the group consisting of strips, sheets, rods, tubes, wells, microtiter plates, beads, particles and microspheres, which may be microspheres or microparticles known to those skilled in the art, which may be of any size, which may be organic or inorganic, which may be expandable or non-expandable, which may be porous or non-porous, which may be magnetic or non-magnetic, which has any density, but preferably has a density close to that of water, preferably capable of floating in water, and which are composed of transparent, partially transparent or opaque materials.

The term "test sample" as used herein refers to mixtures that may contain test target molecules including, but not limited to, proteins, hormones, antibodies or antigens, typical test samples that can be used in the methods disclosed herein include body fluids such as whole blood, serum, plasma, saliva, urine, etc. the test sample can be diluted with a diluent as necessary before use.

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 "antibody" as used herein is used in its 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.

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 "biotin" as used herein is broadly applicable to animal and plant tissues, which have two cyclic structures on the molecule, namely, an imidazolone ring and a thiophene ring, wherein the imidazolone ring is the main site for binding with streptavidin, the activated biotin can be coupled with almost all known biological macromolecules including proteins, nucleic acids, polysaccharides, lipids, etc., under the mediation of a protein cross-linking agent, and "streptavidin" is a protein secreted by streptomyces, and the "streptavidin" molecule with a molecular weight of 65 kD. consists of 4 identical peptide chains, each of which can bind biotin.

The term "particle size" as used herein refers to the average particle size of the microspheres, as measured by conventional particle sizers.

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%.

Compared with a Gaussian unimodal algorithm, the Nicomp multimodal algorithm has unique advantages on the analysis of a multi-component liquid dispersion system with nonuniform particle size distribution and the stability analysis of a colloid system.

Detailed description of the preferred embodiments

The present invention will be described in more detail below.

The chemiluminescent homogeneous assay kit of aspect of the present invention comprises:

a donor reagent comprising donor microspheres capable of generating reactive oxygen species in an excited state and having a label coated on the surface thereof; and the combination of (a) and (b),

the receptor reagent comprises a receptor microsphere, wherein the receptor microsphere can react with active oxygen to generate a detectable chemiluminescent signal, and the surface of the receptor microsphere is coated with a biomolecule which can be specifically combined with a target molecule to be detected;

wherein the particle size of the donor microspheres is larger than the particle size of the acceptor microspheres.

In embodiments of the present invention, the donor microspheres have an average particle size of 100nm to 400nm, the acceptor microspheres have an average particle size of 100nm to 350nm, and the ratio of the average particle size of the donor microspheres to the average particle size of the acceptor microspheres is 1.1 to 4.0;

preferably, the average particle size of the donor microspheres is 190nm-250nm, the average particle size of the acceptor microspheres is 180nm-240nm, and the ratio of the average particle size of the donor microspheres to the average particle size of the acceptor microspheres is 1.2-3.0;

preferably, the ratio of the average particle size of the donor microspheres to the average particle size of the acceptor microspheres can be 1.3-2.0;

more preferably in step , the ratio of the average particle size of the donor microspheres to the average particle size of the acceptor microspheres may be from 1.4 to 1.6.

For example, in some embodiments of the invention, the donor microspheres may have an average particle size of 50nm, 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, and 400nm and the corresponding acceptor microspheres may have an average particle size of 20nm, 50nm, 100nm, 150nm, 200nm, 250nm, 300nm, and 350 nm.

In preferred embodiments of the present invention, the average particle size of the donor microspheres is 150nm, the average particle size of the acceptor microspheres is 100nm, and the ratio of the average particle size of the donor microspheres to the average particle size of the acceptor microspheres is 1.5.

In , the donor microsphere includes a carrier, a carrier filled with a sensitizer, and a carrier with a chemically bonded label at its surface.

In still other embodiments of the invention, the th vector has no polysaccharide moiety coated or attached to its surface, which is directly chemically bonded to the label.

In embodiments of the invention, the label is avidin.

In still further embodiments of the invention, the avidin is selected from the group consisting of ovalbumin, streptavidin, vitellin, neutravidin, and avidin-like, preferably selected from the group consisting of neutravidin and streptavidin.

In embodiments of the present invention, the avidin is chemically bonded to the surface of the th carrier by reacting an amino group with an aldehyde group on the surface of the th carrier to form a schiff base.

In other embodiments of the present invention, the th carrier has a bonding functional group on its surface for chemically bonding a label to the th carrier surface.

In the embodiments of the present invention, the bonding functional group is selected from amine group, amide group, hydroxyl group, aldehyde group, carboxyl group, maleimide group and thiol group, preferably selected from aldehyde group and/or carboxyl group.

In another embodiments of the present invention, the bonding functional group on the th carrier surface is 100 to 500nmol/mg, preferably 200 to 400 nmol/mg.

In of the embodiments of the present invention, the surface of carrier is coated with hydrophilic aldehyde dextran, and the aldehyde group of the aldehyde dextran is chemically bonded with the label.

In preferred embodiments of the present invention, the photosensitizer is selected from of methylene blue, rose bengal, porphyrin and phthalocyanine.

In still other embodiments of the present invention, the acceptor microsphere includes a second support filled with the luminescent composition, the second support having a surface coated with at least polysaccharide layers, the polysaccharide layers having biomolecules attached to the surface.

In embodiments of the invention, the surface of the second carrier is coated with hydrophilic carboxydextran.

When the microsphere containing the carrier is used for detection, nonspecific adsorption can be greatly reduced, and the influence of other environmental factors outside a system, such as pH value, electrolyte and the like, is reduced, so that the detection accuracy is improved.

In other embodiments of the present invention, the light emitting composition comprises a europium complex, and preferably the europium complex is MTTA-EU³⁺Europium complexes filled in the polystyrene microspheres interact with the polystyrene microspheres, and the step is further carried outIn a further preferred embodiment of the present invention at step , the europium complex is MTTA-EU³⁺The complex can directly capture singlet oxygen generated by phthalocyanine dye in the photosensitive microsphere and then emit red light with europium ion characteristic wavelength of 614-615 nm.

MTTA: [4 ' - (10-methyl-9-anthryl) -2,2 ': 6 ' 2 ' -bipyridine-6, 6 ' -dimethylamine ] tetraacetic acid has a structural formula shown in a formula I, and the synthesis is referred to CN 200510130851.9.

Europium complex MTTA-EU³⁺The synthesis of the (europium (III) complex) is as follows:

(1) a500 mL three-necked flask was charged with 732mg of MTTA (1mmoL) and 366mg of EuCl₃·6H₂O (1mmoL) was dissolved in 100mL of methanol and refluxed at 70 ℃ for 2 hours with stirring.

(2) The solvent was distilled off under reduced pressure.

(3) To the resultant was added 50mL of diethyl ether, and the cake was collected by filtration and washed three times with acetone.

(4) Vacuum drying to obtain 830mg MTTA-EU³⁺。

In specific embodiments of the present invention, the donor and acceptor microspheres are both polystyrene microspheres.

In the embodiments of the present invention, the material of the th carrier and/or the second carrier is selected from agarose, cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polystyrene, polyethylene, polypropylene, poly (4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate, nylon, polyethylene butyrate or polyacrylate, preferably selected from polystyrene, polypropylene, poly (4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate or polyacrylate.

In the embodiments of the present invention, the biomolecule is selected from the group consisting of protein molecules, nucleic acid molecules, polysaccharide molecules and lipid molecules, preferably protein molecules, however, the biomolecule is not limited to protein molecules, nucleic acid molecules, polysaccharide molecules and lipid molecules, and any substance that can be designed to satisfy the above conditions can be used as the biomolecule in the present invention, as long as the biomolecule is combined with the prior art under the technical idea disclosed in the present invention, and thus, the details thereof are not repeated.

In the preferred embodiments of the present invention, the protein molecule is an antigen and/or an antibody, wherein the antigen is an immunogenic material and the antibody is an immunoglobulin produced by the body that recognizes a specific foreign substance.

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

In addition, the more uniform the particle size of the microsphere is, the more the better the performance of homogeneous chemiluminescence detection by using the microsphere is, so that the current research on microspheres used in homogeneous chemiluminescence tends to obtain microspheres with more uniform particle size of .

Thus, in embodiments of the invention, the donor microspheres have a coefficient of variation of particle size distribution C.V value of 5% or more in the donor agent.

In still other embodiments of the present invention, the donor microspheres have a coefficient of variation of particle size distribution C.V value ≥ 8% in the donor reagent, and preferably have a coefficient of variation of particle size distribution C.V value ≥ 10% in the donor reagent.

In embodiments of the present invention, the donor microspheres have a coefficient of variation of particle size distribution C.V value of 40% or less in the donor reagent, and more preferably has a coefficient of variation of particle size distribution C.V value of 20% or less in the donor reagent.

In some embodiments of , the donor microsphere may have a coefficient of variation of particle size distribution C.V value of 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 25%, 30%, 35%, 40%, etc. in the recipient reagent.

It should be noted that the C.V value of the variation coefficient of the particle size distribution of the donor microspheres in the present invention refers to C.V value of the variation coefficient of the particle size distribution of the donor microspheres coated with the desired material.

In , the acceptor microspheres have a coefficient of variation of particle size distribution C.V value of 5% or more in the acceptor reagent.

In embodiments of the present invention, the acceptor microspheres have a variation coefficient of particle size distribution C.V value of 8% or more in the acceptor reagent, and preferably have a variation coefficient of particle size distribution C.V value of 10% or more in the acceptor reagent.

In still other embodiments of the present invention, the acceptor microspheres have a coefficient of variation of particle size distribution C.V value of 40% or less in the acceptor reagent, and more preferably a coefficient of variation of particle size distribution C.V value of 20% or less in the acceptor reagent, .

In some embodiments of the present invention, the acceptor microsphere may have a coefficient of variation of particle size distribution C.V value of 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 25%, 30%, 35%, 40%, etc. in the acceptor 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 embodiments of the present invention, the value of the coefficient of variation C.V of the particle size distribution is calculated from a Gaussian distribution.

In the embodiments of the present invention, the kit further includes reagent, it should be noted that the reagent of the present invention does not refer to a reagent, and the reagent is added to ensure the successful or optimized performance of certain specific reaction-based detection methods.

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

the receptor reagent comprises a receptor microsphere combined with th antigen, wherein the th antigen epitope can be specifically combined with th binding site of the epitope of a target antibody to be detected;

reagent comprising a second antigen binding to biotin, the second antigen being capable of specifically binding to an epitope second binding site of a test target antibody, and the epitope binding site and the epitope second binding site of the test target antibody do not overlap,

a donor reagent comprising donor microspheres bound to avidin; the donor microsphere is capable of generating singlet oxygen in an excited state;

wherein the particle size of the donor microspheres is larger than the particle size of the acceptor microspheres.

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

the receptor reagent comprises a receptor microsphere combined with th antibody, wherein the th antibody can be specifically combined with an antigenic determinant of a target antigen to be detected;

reagent comprising a second antibody that binds to biotin, the second antibody being capable of specifically binding to an epitope of a target antigen to be detected, and,

a donor reagent comprising donor microspheres bound to avidin; the donor microsphere is capable of generating singlet oxygen in an excited state;

wherein the particle size of the donor microspheres is larger than the particle size of the acceptor microspheres.

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

a receptor reagent comprising a receptor microsphere bound to an antigen, the antigen being capable of specifically binding to an antibody of interest, the receptor microsphere being capable of reacting with singlet oxygen to produce a detectable chemiluminescent signal;

an th reagent comprising a second antibody that binds to biotin, the second antibody being capable of specifically binding to the antigen, and

a donor reagent comprising a donor microsphere bound to avidin, the donor microsphere capable of generating singlet oxygen in an excited state;

wherein the particle size of the donor microspheres is larger than the particle size of the acceptor microspheres.

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

an th reagent comprising a th antigen that binds biotin, the th antigen being capable of specifically binding to an epitope binding site of an antibody of interest;

a receptor reagent comprising a receptor microsphere bound to an anti-immune complex antibody, the anti-immune complex antibody being capable of specifically binding to a target antibody in an immune complex formed between the th antigen and the target antibody, the receptor microsphere being capable of reacting with singlet oxygen to produce a detectable chemiluminescent signal, and,

a donor reagent comprising a donor microsphere bound to avidin, the donor microsphere capable of generating singlet oxygen in an excited state;

wherein the particle size of the donor microspheres is larger than the particle size of the acceptor microspheres.

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

a receptor reagent comprising a receptor microsphere bound to th antigen, the th antigen being capable of specifically binding to an antibody of interest, the receptor microsphere being capable of reacting with singlet oxygen to produce a detectable chemiluminescent signal;

an th reagent comprising an anti-immune complex antibody that binds to biotin, the anti-immune complex antibody being capable of specifically binding to a target antibody in an immune complex formed between a th antigen and the target antibody;

a donor reagent comprising a donor microsphere bound to avidin, the donor microsphere being capable of generating singlet oxygen in an excited state.

According to the present invention, the anti-immune complex antibody does not bind to the target antibody which is free or not bound to the th antigen.

The second aspect of the present invention relates to homogeneous phase chemiluminescence detection methods, which uses the kit according to the invention, aspect , to detect target molecules to be detected in a sample to be detected.

In embodiments of the present invention, the method includes the steps of:

s1, mixing the sample to be tested with the acceptor reagent and the th reagent, and then mixing the mixture with the donor reagent to obtain a mixture to be tested;

s2, performing laser irradiation on the mixture to be detected obtained in the step S1, and exciting the donor to generate singlet oxygen;

and 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 a chemiluminescence signal generated by the reaction of the receptor microsphere in the mixture to be detected and singlet oxygen.

In the preferred embodiments of the present invention, in step S1, the sample to be tested is diluted with a diluent, and then mixed with the receptor reagent and the donor reagent to form a mixture to be tested.

In another embodiments of the present invention, in step S2, the laser irradiation is performed by using red excitation of 600-700 nm.

The third aspect of the present invention relates to homogeneous chemiluminescent detection devices for detecting a target molecule to be detected in a sample to be detected using the kit according to the aspect of the present invention or the method according to the second aspect of the present invention.

In preferred embodiments of the present invention, the device is a POCT point of care device.

In embodiments of the invention, the apparatus comprises:

a. the reagent cup strip is provided with a plurality of hole sites for containing reagents, and the hole sites at least comprise:

a sample hole site to be detected for containing a sample to be detected containing target molecules to be detected;

a reagent well site for holding a donor reagent comprising donor microspheres capable of generating reactive oxygen species in an excited state;

a second reagent well site for holding an acceptor reagent comprising acceptor microspheres capable of reacting with reactive oxygen species to produce a chemiluminescent signal, the donor microspheres having a particle size greater than that of the acceptor microspheres;

b. the sample adding mechanism is used for mutually moving the reagents contained in the hole sites among the hole sites; the mass transferred by the sample adding mechanism is 1-500 mu L each time;

c. and the detection mechanism is electrically connected with the sample adding mechanism and is used for detecting a chemiluminescent signal generated by the reaction of the receptor microsphere and the active oxygen.

In other embodiments of the present invention, the sample well to be tested, the donor reagent well and the acceptor reagent well are coated to seal the opening of the well, so as to prevent the contamination of the substances therein.

In order to conveniently identify and read the information of the sample to be tested, the preferable technical scheme is that the side surface of the reagent cup strip along the width direction is provided with a bar code area, and the bar code area contains the information of the reagent cup strip, wherein the bar code can be -dimensional or two-dimensional.

Correspondingly, the POCT device also comprises a bar code scanning module, and the bar code scanning module is used for identifying and reading information in the bar code.

The bar code scanning module supports IC card scanning and bar code medium (paper or reagent card) printing scanning, and the information reading adopts contact scanning or non-contact scanning in a mode of infrared or radio frequency and the like; the information includes, but is not limited to, assay project name, standard curve, reagent composition, lot number, expiration date, manufacturer information.

In order to improve the accuracy of the final detection result and the stability of the sample to be detected, in embodiments of the present invention, the reagent cup strip is further provided with a diluent hole site, and the diluent hole site is used for containing a diluent.

In embodiments of the present invention, the reagent cup strip further comprises an additional reagent well for containing an additional reagent (reagent ), and the additional reagent well is coated to close the opening.

In preferred embodiments of the present invention, the sample application mechanism comprises:

a pipetting assembly for aspirating or discharging a liquid;

the liquid transfer assembly is arranged on the vertical moving assembly, and the vertical moving assembly is used for driving the liquid transfer assembly to vertically move;

the vertical moving assembly is arranged on the horizontal moving assembly, and the horizontal moving assembly is used for driving the liquid transfer assembly to move horizontally.

In preferred embodiments of the present invention, the detection mechanism comprises:

a base for carrying the reagent cup strips;

the driving assembly is used for driving the base to rotate around the center of the base and driving the reagent cup strips to rotate;

the detection component is used for detecting a chemiluminescent signal generated by the reaction of the receptor microsphere in the reagent cup strip and active oxygen.

In the specific embodiments of the invention, the detection assembly comprises an exciter capable of emitting 600-700 nm red excitation light.

In , the detection wavelength of the chemiluminescent signal generated by the reaction of the acceptor microsphere and active oxygen is 450-650 nm.

In preferred embodiments of the present invention, the liquid transfer assembly includes a piston mechanism, a connector and a pipette sequentially arranged from top to bottom, the piston mechanism is connected to the connector, the pipette is arranged at the edge of the end face of the base, when liquid transfer is required, the connector descends and is connected to the pipette, and the piston mechanism can move up and down to drive the pipette to suck or discharge liquid.

In , the device further comprises an incubation module for providing a suitable ambient temperature for the chemiluminescent reaction, wherein the temperature of the reagent cup strip and the contents of the reagent cup strip is 20-50 ℃ by means of a metal bath, water bath or oil bath.

In still other embodiments of the present invention, the sample well site to be tested, the donor reagent well site, and the acceptor reagent well site have cross-sections with different shapes.

The using process of the device comprises the steps of respectively containing a sample to be detected, a donor reagent hole and an acceptor reagent hole in the sample hole to be detected, placing the reagent card in the POCT analyzer, taking the sample to be detected with the corresponding volume by using a sample adding mechanism, adding the sample to be detected into an reagent hole, reacting for time, continuously taking mixed liquid with the fixed volume, adding the mixed liquid into a second reagent hole, irradiating laser to the second reagent hole by using an exciter in the detection assembly, reacting for time, detecting a chemiluminescence signal generated by the reaction of acceptor microspheres and active oxygen by using the detection mechanism, and calculating the concentration of procalcitonin in the sample to be detected.

Example III

In order that the invention may be more readily understood, the invention is now described in further detail at with reference to the following examples, which are intended to be illustrative only and are not intended to limit the scope of the invention.