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WO2020034938A1 - 一种用于化学发光分析的微球组合物及其应用 - Google Patents

一种用于化学发光分析的微球组合物及其应用 Download PDF

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Publication number
WO2020034938A1
WO2020034938A1 PCT/CN2019/100340 CN2019100340W WO2020034938A1 WO 2020034938 A1 WO2020034938 A1 WO 2020034938A1 CN 2019100340 W CN2019100340 W CN 2019100340W WO 2020034938 A1 WO2020034938 A1 WO 2020034938A1
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Prior art keywords
microsphere composition
composition according
microsphere
microspheres
group
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PCT/CN2019/100340
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English (en)
French (fr)
Inventor
杨阳
康蔡俊
赵卫国
刘宇卉
李临
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博阳生物科技（上海）有限公司
北京科美生物技术有限公司
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Priority to EP19850656.0A priority Critical patent/EP3839485B1/en
Publication of WO2020034938A1 publication Critical patent/WO2020034938A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles

Definitions

  • the invention belongs to the technical field of chemiluminescence analysis, and particularly relates to a microsphere composition for chemiluminescence analysis and application thereof.
  • Chemiluminescence analysis is a method that uses light waves emitted by chemiluminescent substances for detection.
  • Chemiluminescent substances are used as markers in nucleic acid detection and immunoassay.
  • a molecule in a specific binding pair can be combined with a luminescent substance through a variety of ways to form a luminescent microsphere composition.
  • the microsphere composition can react with the test object (another molecule in the specific binding pair) in the sample, and is distributed in the solid phase and the liquid phase, and the distribution ratio is related to the amount of the test object. By measuring the amount of luminescence in the solid or liquid phase, the corresponding concentration of the detected substance in the sample can be obtained.
  • the technical problem to be solved by the present invention is to address the shortcomings of the prior art, and to provide a microsphere composition for chemiluminescence analysis.
  • the microsphere composition When used for chemiluminescence analysis and detection, it has both high sensitivity, It also has a wide detection range. In addition, the detection time can be shortened.
  • the first aspect of the present invention provides a microsphere composition for chemiluminescence analysis, which includes at least two kinds of acceptor microspheres with different particle sizes, the acceptor microspheres can react with active oxygen to produce Detected chemiluminescence signal.
  • the acceptor microsphere includes two parts: a luminescent composition and a substrate, and the luminescent composition is filled in the substrate and / or coated on the surface of the substrate.
  • the luminescent composition is capable of reacting with active oxygen to generate a detectable chemiluminescence signal, which comprises a chemiluminescent compound and a metal chelate.
  • the chemiluminescent compound is selected from olefin compounds, preferably from dimethylthiophene, dibutanedione compound, dioxane, enol ether, enamine, 9 -Alkylene xanthane, 9-alkylene-N-9,10 dihydroacridine, aryl etherene, arylimidazole, and gloss derivatives and their derivatives, more preferably selected from dimethylthiophene and Its derivatives.
  • olefin compounds preferably from dimethylthiophene, dibutanedione compound, dioxane, enol ether, enamine, 9 -Alkylene xanthane, 9-alkylene-N-9,10 dihydroacridine, aryl etherene, arylimidazole, and gloss derivatives and their derivatives, more preferably selected from dimethylthiophene and Its derivatives.
  • the metal of the metal chelate is a rare earth metal or a Group VIII metal, preferably selected from the group consisting of rhenium, osmium, osmium, osmium, osmium, and ruthenium, and more preferably selected from osmium.
  • the metal chelate comprises a chelating agent selected from the following: MTTA, NHA, BHHT, BHHCT, DPP, TTA, NPTTA, NTA, TOPO, TPPO, BFTA, 2, 2 -Dimethyl-4-perfluorobutyryl-3-butanone (fod), 2,2'-bipyridyl (bpy), bipyridylcarboxylic acid, aza crown ether, aza-hole ligand and Octylphosphine oxide and their derivatives.
  • a chelating agent selected from the following: MTTA, NHA, BHHT, BHHCT, DPP, TTA, NPTTA, NTA, TOPO, TPPO, BFTA, 2, 2 -Dimethyl-4-perfluorobutyryl-3-butanone (fod), 2,2'-bipyridyl (bpy), bipyridylcarboxylic acid, aza crown ether, aza-hole
  • the light-emitting compound is a derivative of dimethylthiophene, and the metal chelate is a europium chelate.
  • the matrix is selected from the group consisting of tapes, sheets, rods, tubes, wells, microtiter plates, beads, particles and microspheres; preferably beads and microspheres.
  • the matrix is magnetic or non-magnetic particles.
  • the matrix material is selected from the group consisting of natural, synthetic, or modified naturally occurring polymers, including but not limited to: agarose, cellulose, nitrocellulose, cellulose acetate, polymer Vinyl chloride, polystyrene, polyethylene, polypropylene, poly (4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate, nylon, polyethylene butyrate or Polyacrylate.
  • natural, synthetic, or modified naturally occurring polymers including but not limited to: agarose, cellulose, nitrocellulose, cellulose acetate, polymer Vinyl chloride, polystyrene, polyethylene, polypropylene, poly (4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate, nylon, polyethylene butyrate or Polyacrylate.
  • the matrix is an aldehyde-based latex microsphere; preferably an aldehyde-based polystyrene latex microsphere.
  • a biologically active substance is directly connected to the surface of the matrix, and the biologically active substance can specifically bind to a target molecule to be detected.
  • the surface of the substrate is coated with a coating layer, and the surface of the coating layer is connected with a biologically active substance, and the biologically active substance can specifically bind to a target molecule to be detected.
  • the coating in the coating layer is selected from a polysaccharide, a polymer, or a biomacromolecule, and is preferably a polysaccharide.
  • the surface of the matrix is coated with a coating of at least two continuous polysaccharide layers, wherein the first polysaccharide layer is spontaneously associated with the second polysaccharide layer.
  • each of the continuous polysaccharide layers is spontaneously associated with each of the previous polysaccharide layers.
  • the polysaccharide has a side group functional group, and the functional group of the continuous polysaccharide layer has an opposite charge to the functional group of the previous polysaccharide layer.
  • the polysaccharide has a pendant functional group, and the continuous layer of the polysaccharide is reacted with the functional group of the continuous layer and the functional group of the previous layer by The former polysaccharide layer is covalently linked.
  • the functional group of the continuous polysaccharide layer alternates between an amine functional group and an amine-reactive functional group.
  • the amine-reactive functional group is an aldehyde group or a carboxyl group.
  • the first polysaccharide layer is spontaneously associated with the carrier.
  • the outermost polysaccharide layer of the coating has at least one pendant functional group.
  • the side functional group of the outermost polysaccharide layer of the coating is selected from at least one of an aldehyde group, a carboxyl group, a thiol group, an amino group, a hydroxyl group, and a maleamine group; From aldehyde and / or carboxyl.
  • the side group functional group of the outermost polysaccharide layer of the coating is directly or indirectly connected to the bioactive substance.
  • the polysaccharide is selected from a carbohydrate containing three or more unmodified or modified monosaccharide units; preferably selected from dextran, starch, glycogen, inulin, fruit Glycans, mannans, agaroses, galactans, carboxydextran and aminodextran; more preferably selected from dextran, starch, glycogen and polyribose.
  • the matrix diameters of the receptor microspheres with different particle diameters are the same.
  • the size of the matrix of the receptor microspheres with different particle sizes is different.
  • the active oxygen is singlet oxygen.
  • the microsphere composition includes two types of acceptor microspheres with different particle sizes.
  • the difference between the particle sizes of the two acceptor microspheres with different particle sizes is not less than 100 nm; preferably not less than 150 nm; more preferably not less than 200 nm.
  • the particle size ratio of the two acceptor microspheres with different particle sizes is selected from 1: (1.1-10); preferably selected from 1: (2-8); more preferably Select from 1: (3-6).
  • the microsphere composition is used at a concentration of 1 ug / mL-1000 ug / mL; preferably 10 ug / mL-500 ug / mL, and more preferably 10 ug / mL-250 ug / mL.
  • the microsphere composition includes at least three receptor microspheres of different particle sizes.
  • a second aspect of the present invention provides a receptor agent comprising the microsphere composition according to the first aspect of the present invention.
  • a third aspect of the present invention provides a chemiluminescence detection kit, comprising the microsphere composition according to the first aspect of the present invention or the acceptor reagent according to the second aspect of the present invention.
  • a fourth aspect of the present invention provides a chemiluminescence analysis method, which comprises using the microsphere composition according to the first aspect of the present invention or the acceptor reagent according to the second aspect of the present invention or the third aspect of the present invention.
  • the kit detects the presence of the target molecule and / or the concentration of the target molecule in the sample.
  • a fifth aspect of the present invention provides a chemiluminescence analyzer using the microsphere composition according to the first aspect of the present invention or the acceptor reagent according to the second aspect of the present invention or the third aspect of the present invention.
  • the kit and / or the method according to the fourth aspect of the present invention detects whether the target molecule is present in the sample to be tested and / or the concentration of the target molecule in the sample to be tested is present.
  • the chemiluminescence analyzer includes at least the following parts:
  • An incubation module configured to provide a suitable temperature environment for a chemiluminescent reaction between a sample to be measured and a microsphere composition, the microsphere composition comprising at least two acceptor microspheres of different particle sizes;
  • a processor which determines whether a target molecule to be tested exists in the sample to be tested and / or a concentration of the target molecule to be tested in the sample to be tested according to the situation of the chemiluminescence signal detected by the detection module.
  • the beneficial effect of the present invention is that the microsphere composition for chemiluminescence analysis according to the present invention includes at least two kinds of acceptor microspheres with different particle sizes. Because the small size of the acceptor microspheres can widen the detection range, and the large size of the acceptor microspheres can improve the detection sensitivity, the performance of the microsphere composition of the present invention is greatly improved compared with the prior art, which has an ultra high The sensitivity has a wide range. In addition, since the response speed of the small-particle-size acceptor microspheres is fast, when the microsphere composition of the present invention is used for detection, the detection time can also be shortened, and the reaction speed can be improved.
  • FIG. 1 is a Gaussian distribution curve diagram of the aldehyde-based polystyrene latex microspheres prepared in Example 3.
  • FIG. 1 is a Gaussian distribution curve diagram of the aldehyde-based polystyrene latex microspheres prepared in Example 3.
  • FIG. 2 is a Gaussian distribution curve diagram of the aldehyde-based polystyrene latex microspheres filled with the luminescent composition prepared in Example 3.
  • FIG. 2 is a Gaussian distribution curve diagram of the aldehyde-based polystyrene latex microspheres filled with the luminescent composition prepared in Example 3.
  • FIG. 3 is a Gaussian distribution diagram of the dextran-coated aldehyde-containing polystyrene latex microspheres coated with a luminescent composition prepared in Example 3.
  • Example 4 is a Gaussian distribution diagram of the acceptor microspheres having an average particle diameter of about 250 nm prepared in Example 3.
  • FIG. 5 is a Gaussian distribution diagram of the acceptor microspheres having a particle size of about 110 nm prepared in Example 3.
  • FIG. 5 is a Gaussian distribution diagram of the acceptor microspheres having a particle size of about 110 nm prepared in Example 3.
  • FIG. 6 is a Nicomp distribution diagram of the acceptor microspheres having a particle size of about 110 nm prepared in Example 3.
  • FIG. 6 is a Nicomp distribution diagram of the acceptor microspheres having a particle size of about 110 nm prepared in Example 3.
  • FIG. 7 is a Gaussian distribution diagram of the acceptor microspheres having a particle size of about 350 nm prepared in Example 3.
  • FIG. 7 is a Gaussian distribution diagram of the acceptor microspheres having a particle size of about 350 nm prepared in Example 3.
  • FIG. 8 is a Nicomp distribution diagram of the acceptor microspheres having a particle diameter of about 350 nm prepared in Example 3.
  • FIG. 8 is a Nicomp distribution diagram of the acceptor microspheres having a particle diameter of about 350 nm prepared in Example 3.
  • FIG. 9 is a Gaussian distribution diagram of the particle size distribution of the mixed acceptor microspheres in Example 4.
  • FIG. 9 is a Gaussian distribution diagram of the particle size distribution of the mixed acceptor microspheres in Example 4.
  • FIG. 10 is a Nicomp distribution diagram of the particle size distribution of the mixed acceptor microspheres in Example 4.
  • FIG. 10 is a Nicomp distribution diagram of the particle size distribution of the mixed acceptor microspheres in Example 4.
  • active oxygen in the present invention refers to a general term for a substance composed of oxygen in the body or in the natural environment, which contains oxygen and is active in nature. It is mainly an excited oxygen molecule, including superoxide, an electron reduction product of oxygen. Anions (O 2 ⁇ -), two-electron reduction products, hydrogen peroxide (H 2 O 2 ), three-electron reduction products, hydroxyl radicals ( ⁇ OH), nitric oxide, and singlet oxygen (1O 2 ).
  • the term "receptor microsphere” refers to a nanomicrosphere capable of reacting with active oxygen to generate a detectable chemiluminescence signal, which may also be referred to as an oxygen-receiving microsphere or a light-emitting microsphere.
  • the acceptor microspheres may be polymer particles filled with a functional group to form a polymer particle filled with a light emitting composition, and the light emitting composition includes a chemiluminescent compound capable of reacting with active oxygen.
  • the chemiluminescent compound undergoes a chemical reaction with active oxygen to form an unstable metastable intermediate, which can be decomposed and emit light simultaneously or subsequently.
  • Typical examples of these materials include, but are not limited to, enol ethers, enamines, 9-alkylidene xanthan gum, 9-alkylidene-N-alkylacridine, aryl etherene, dioxyethylene, dimethyl ether Thiophene, aryl imidazole or gloss fine.
  • the "chemiluminescent compound” is a compound called a label, which can undergo a chemical reaction to cause light emission, for example, by being converted into another compound formed in an electronically excited state.
  • the excited state can be a singlet state or a triplet excited state.
  • the excited state can relax to the ground state and emit light directly, or it can restore itself to the ground state by transmitting the excitation energy to the emitting energy acceptor. During this process, the energy acceptor microspheres will be transitioned to an excited state and emit light.
  • the chemiluminescent compound can be bound to a specific binding partner member, which can directly or indirectly bind to a target molecule or a test component under test, and the concentration of the test component is affected by the presence of the target molecule to be tested.
  • a specific binding partner member which can directly or indirectly bind to a target molecule or a test component under test, and the concentration of the test component is affected by the presence of the target molecule to be tested.
  • “capable of binding directly or indirectly” means that the specified entity can specifically bind to the entity (directly), or the specified entity can specifically bind to a specific binding pair member, or has two Or more (indirectly) a complex of a specific binding partner capable of binding other entities.
  • the "specific binding pair member" of the present invention is selected from (1) a small molecule and a binding partner for the small molecule, and (2) a large molecule and a binding partner for the large molecule.
  • the active oxygen may be provided by a “donor microsphere”, and the donor microsphere is a nanomicrosphere capable of generating active oxygen in an excited state.
  • the donor microspheres can be coated with functional groups to form polymer particles filled with a photosensitive compound, which can generate singlet oxygen under light excitation.
  • the photosensitive microspheres can also be called Oxygen-supplying microspheres or photosensitive microspheres.
  • the surface of the donor microsphere may have a hydrophilic aldehyde dextran, and the interior is filled with a photosensitizer.
  • the photosensitizer may be a photosensitizer known in the art, preferably a compound that is relatively light stable and does not effectively react with singlet oxygen, and non-limiting examples thereof include compounds such as methylene blue, rose red, porphyrin, and phthalocyanine, And derivatives of these compounds with 1-50 atomic substituents, which are used to make these compounds more lipophilic or more hydrophilic, and / or as linkers attached to specific binding pair members group.
  • the surface of the donor microspheres may also be filled with other sensitizers. Non-limiting examples are certain compounds that catalyze the conversion of hydrogen peroxide to singlet oxygen and water.
  • Examples of other donors include: 1,4-dicarboxyethyl-1,4-naphthalene endoxide, 9,10-diphenylanthracene-9,10-endoperoxide, etc., heating these compounds or These compounds directly absorb light and release reactive oxygen species, such as singlet oxygen.
  • the sensitizer is a compound that, when excited or induced, can cause a chemical reaction in another compound or substance.
  • Sensitizers include photosensitizers that can be induced by light irradiation to form an activated excited state.
  • Sensitizers also include compounds that can undergo chemical reactions to produce metastable active oxygen species such as singlet oxygen.
  • the photosensitizer generally activates a chemiluminescent compound by irradiating a medium containing the above-mentioned reactant.
  • the medium must be irradiated with light having a certain wavelength and sufficient energy to convert the photosensitizer to an excited state, so that it can activate molecular oxygen to singlet oxygen.
  • the excited state of a photosensitizer capable of exciting molecular oxygen is generally in the triplet state, which is about 20 Kcal / mol, and usually at least 23 Kcal / mol, higher than the energy of the photosensitizer in the ground state.
  • the medium can be irradiate with light having a wavelength of approximately 450-950 nm.
  • the light produced can be measured in any conventional manner, such as photography, visual inspection, photometer, etc., in order to determine its amount related to the amount of analyte in the medium.
  • the photosensitizer is preferably relatively non-polar to ensure solubility in lipophilic members.
  • the "matrix” according to the present invention can be any size, it can be organic or inorganic, it can be expandable or non-expandable, it can be porous or non-porous, it can have any density, However, it preferably has a density close to that of water, and is preferably capable of floating in water and composed of a transparent, partially transparent or opaque material.
  • the matrix may or may not be electrically charged, and when electrically charged, it is preferably negatively charged.
  • the matrix can be a solid (e.g., polymer, metal, glass, organic and inorganic materials such as minerals, salts, and diatoms), small oil droplets (e.g., hydrocarbon, fluorocarbon, siliceous fluid), vesicles (e.g., Synthetic such as phospholipids, or natural such as cells, and cellular organs).
  • the matrix can be latex particles or other particles containing organic or inorganic polymers, lipid bilayers such as liposomes, phospholipid vesicles, small oil droplets, silicon particles, metal sols, cells and microcrystalline dyes.
  • Matrices are generally versatile or capable of binding to a donor or acceptor through specific or non-specific covalent or non-covalent interactions.
  • Typical functional groups include carboxylic acid, acetaldehyde, amino, cyano, vinyl, hydroxyl, mercapto, and the like.
  • a matrix suitable for use in the present invention is aldehyde-based polystyrene latex microspheres.
  • the photosensitizer and / or chemiluminescent compound can be selected to be dissolved, or non-covalently bound to the surface of the particle.
  • these compounds are preferably hydrophobic to reduce their ability to dissociate from the particles so that both compounds can bind to the same particles.
  • the term “particle diameter” in the present invention refers to the average particle diameter of the acceptor microspheres, which is measured by a conventional particle size meter.
  • the "receptor microspheres" of the present invention include at least a matrix, a luminescent composition, and a bioactive molecule, and preferably also a coating layer; the luminescent composition may be filled in the matrix and / or coated on the surface of the matrix.
  • the receptor microsphere does not include a coating, the bioactive substance is directly connected to the surface of the substrate.
  • the receptor microsphere includes a coating layer, the coating layer is coated on the surface of the substrate, and the outermost layer of the coating layer is connected with a bioactive active substance.
  • the "average particle diameter of the acceptor microspheres" in the present invention refers to the average particle diameter of the acceptor microspheres after being connected and / or coated with the corresponding substance.
  • the particle diameter of the matrix in the receptor microspheres of different particle diameters may be the same or different, as long as the particle diameters of the receptor microspheres finally formed are different.
  • the most preferred technical solution of the present invention is that the receptors of different particle diameters
  • the particle size of the matrix in the bulk microspheres is also different.
  • sample to be tested refers to a mixture containing the target molecule to be tested or suspected of containing the target molecule to be tested.
  • Test samples that can be used in the present disclosure include body fluids, such as blood (which can be anticoagulated commonly seen in collected blood samples), plasma, serum, urine, semen, saliva, cell culture, tissue extraction Things.
  • Other types of samples to be tested include solvents, seawater, industrial water samples, food samples, environmental samples such as soil or water, plant materials, eukaryotic cells, bacteria, plasmids, viruses, fungi, and cells from prokaryotes.
  • the sample to be tested can be diluted with diluent as needed before use. For example, in order to avoid the HOOK effect, you can use a diluent to dilute the sample to be tested before testing on the machine.
  • target molecule to be detected refers to a substance in a sample to be detected during detection.
  • One or more substances with specific binding affinity to the target molecule to be detected will be used to detect the target molecule.
  • the target molecule to be detected may be a protein, a peptide, an antibody, or a hapten capable of binding to the antibody.
  • the target molecule to be detected may be a nucleic acid or an oligonucleotide bound to a complementary nucleic acid or an oligonucleotide.
  • the target molecule to be detected may be any other substance that can form a specific binding pair member.
  • Examples of other typical target molecules to be tested include: drugs such as steroids, hormones, proteins, glycoproteins, mucins, nucleoproteins, phosphoproteins, drugs of abuse, vitamins, antibacterials, antifungals, antivirals, Purines, antitumor agents, amphetamines, aza compounds, nucleic acids and prostaglandins, and metabolites of any of these drugs; pesticides and their metabolites; and receptors.
  • Analytes also include cells, viruses, bacteria, and fungi.
  • antibody as used in the present invention is used in the broadest meaning and includes antibodies of any isotype, antibody fragments that retain specific binding to the antigen, including but not limited to Fab, Fv, scFv, and Fd fragments, and chimeric antibodies , A humanized antibody, a single chain antibody, a bispecific antibody, and a fusion protein comprising an antigen-binding portion of the antibody and a non-antibody protein.
  • the antibody can be further associated with other parts, such as specific binding pair members, such as biotin or streptavidin (a member of the biotin-streptavidin specific binding pair member), etc. Conjugation.
  • antigen in the present invention refers to a substance capable of stimulating the body to produce an immune response, and capable of binding to immune response product antibodies and sensitized lymphocytes in vivo and in vitro to generate an immune effect.
  • binding refers to a direct association between two molecules caused by interactions such as covalent, electrostatic, hydrophobic, ionic, and / or hydrogen bonding, including, but not limited to, interactions such as salt bridges and water bridges. .
  • the term “specific binding” in the present invention refers to the mutual discrimination and selective binding reaction between two substances, and from the perspective of the three-dimensional structure, it is the conformational correspondence between the corresponding reactants.
  • the detection method of the specific binding reaction includes, but is not limited to, a double antibody sandwich method, a competition method, a neutralization competition method, an indirect method or a capture method.
  • C.V value of the particle size distribution variation coefficient in the present invention refers to the variation coefficient of the particle size in the Gaussian distribution in the detection result of the nanometer particle size analyzer.
  • Nicomp distribution in the present invention refers to an algorithmic distribution in the American PSS nano particle size analyzer NICOMP. Compared with the Gaussian unimodal algorithm, the Nicomp multimodal algorithm has unique advantages for the analysis of multi-component, liquid dispersion systems with uneven particle size distribution and the stability analysis of colloidal systems.
  • the invention controls the particle size of the receptor microspheres in the microsphere composition, thereby controlling the amount of bioactive substances (such as antibodies / antigens) on the surface of each receptor microsphere (small particle size microspheres have large specific surface area and unit mass The number of molecules reported on the surface of the microsphere is large, the specific surface area of the microparticles with large particle size is small, and the number of molecules reported on the surface of the unit mass of the microsphere is small), thereby improving the detection sensitivity and widening the detection range.
  • the small diameter of the acceptor microspheres with small particle diameter the activation efficiency of the single-line oxygen generated by the donor microspheres is improved, and the luminous efficiency of the acceptor microspheres can also be improved.
  • the first aspect of the present invention relates to a microsphere composition for chemiluminescence analysis, which includes at least two kinds of acceptor microspheres with different particle diameters, and the acceptor microspheres can react with active oxygen to generate a detectable chemiluminescence signal.
  • the acceptor microsphere includes two parts: a luminescent composition and a substrate, and the luminescent composition is filled in the substrate and / or coated on the surface of the substrate.
  • the luminescent composition is capable of reacting with active oxygen to generate a detectable chemiluminescence signal, which comprises a chemiluminescent compound and a metal chelate.
  • the chemiluminescent compound is selected from an olefin compound, and the olefin compound is a compound capable of reacting with active oxygen (such as singlet oxygen).
  • active oxygen such as singlet oxygen.
  • suitable electron-rich olefinic compounds are listed in US Patent No. 5,709,994, which is incorporated herein by reference.
  • the olefin compound is selected from the group consisting of dimethylthiophene, dibutanedione compound, dioxane, enol ether, enamine, 9-alkylene xanthene , 9-alkylene-N-9,10 dihydroacridine, aryl etherene, arylimidazole, and gloss derivatives and their derivatives, more preferably selected from dimethylthiophene and its derivatives.
  • the chemiluminescent compounds include complexes (metal chelates) of a metal and one or more chelating agents.
  • the metal of the metal chelate is a rare earth metal or a Group VIII metal, preferably selected from the group consisting of osmium, osmium, osmium, osmium, osmium, and ruthenium, and more preferably selected from osmium.
  • the metal chelate comprises a chelating agent selected from the group consisting of 4 '-(10-methyl-9-anthryl) -2,2': 6'2 "- Bipyridine-6,6 ”-dimethylamine] tetraacetic acid (MTTA), 2- (1 ', 1', 2 ', 2', 3 ', 3'-heptafluoro-4', 6'-hexane Dione-6'-yl) -naphthalene (NHA), 4,4'-bis (2 ", 3", 3 "-heptafluoro-4", 6 "-hexanedione-6" -yl) -o -Terphenyl (BHHT), 4,4'-bis (1 ”, 1”, 1 ”, 2”, 2 ”, 3”, 3 ”-heptafluoro-4”, 6 ”-hexanedione-6” -Yl) -chlorosulfo-o-terphenyl (MTTA), 2- (1 ', 1
  • the light-emitting compound is a derivative of dimethylthiophene, and the metal chelate is a europium chelate.
  • the matrix is selected from the group consisting of tapes, sheets, rods, tubes, wells, microtiter plates, beads, particles and microspheres; preferably beads and microspheres.
  • the matrix is magnetic or non-magnetic particles.
  • the materials of the substrates of the receptor microspheres with different particle sizes are the same or different.
  • 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, polybutyrate Or polyacrylate.
  • 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, polybutyrate Or polyacrylate.
  • the matrix is an aldehyde-based latex microsphere; preferably an aldehyde-based polystyrene latex microsphere.
  • a biologically active substance is directly connected to the surface of the matrix, and the biologically active substance can specifically bind to a target molecule to be detected.
  • the surface of the substrate is coated with a coating layer, and the surface of the coating layer is connected with a biologically active substance, which can specifically bind to a target molecule to be measured;
  • a biologically active substance which can specifically bind to a target molecule to be measured
  • the ions of the biologically active substance-target molecule binding partner include antigen-antibody, hormone-hormone receptor, nucleic acid duplex, IgG-protein A, and polynucleotide pair such as DNA- DNA, DNA-RNA, etc.
  • the biologically active substance is an antigen and / or an antibody;
  • the antigen refers to a substance having immunogenicity;
  • the antibody refers to an immunity produced by the body that can recognize a specific foreign object globulin.
  • the coating in the coating layer is selected from a polysaccharide, a polymer, or a biomacromolecule, and is preferably a polysaccharide.
  • the surface of the matrix is coated with a coating of at least two continuous polysaccharide layers, wherein the first polysaccharide layer is spontaneously associated with the second polysaccharide layer.
  • each of the continuous polysaccharide layers is spontaneously associated with each of the previous polysaccharide layers.
  • the polysaccharide has a side group functional group, and the functional group of the continuous polysaccharide layer has an opposite charge to the functional group of the previous polysaccharide layer.
  • the polysaccharide has a pendant functional group, and the continuous layer of the polysaccharide is reacted with the functional group of the continuous layer and the functional group of the previous layer by The former polysaccharide layer is covalently linked.
  • the functional group of the continuous polysaccharide layer alternates between an amine functional group and an amine-reactive functional group.
  • the amine-reactive functional group is an aldehyde group or a carboxyl group.
  • the first polysaccharide layer is spontaneously associated with the carrier.
  • the outermost polysaccharide layer of the coating has at least one pendant functional group.
  • the side functional group of the outermost polysaccharide layer of the coating is selected from at least one of an aldehyde group, a carboxyl group, a thiol group, an amino group, a hydroxyl group, and a maleamine group; From aldehyde and / or carboxyl.
  • the side group functional group of the outermost polysaccharide layer of the coating is directly or indirectly connected to the bioactive substance.
  • the polysaccharide is selected from a carbohydrate containing three or more unmodified or modified monosaccharide units; preferably selected from dextran, starch, glycogen, inulin, fruit Glycans, mannans, agaroses, galactans, carboxydextran and aminodextran; more preferably selected from dextran, starch, glycogen and polyribose.
  • the matrix diameters of the receptor microspheres with different particle diameters are the same.
  • the size of the matrix of the acceptor microspheres with different particle sizes is different.
  • the detection wavelength of the chemiluminescence signal is 450-680 nm; preferably 520-620 nm, more preferably 610-620 nm, and most preferably 615 nm. Accordingly, the wavelength range of the excitation light used for the excitation is 640-680 nm, and preferably, the wavelength of the excitation light is 660 nm.
  • the active oxygen is singlet oxygen.
  • the microsphere composition includes two types of acceptor microspheres with different particle sizes.
  • the difference between the particle sizes of the two acceptor microspheres with different particle sizes is not less than 100 nm; preferably not less than 150 nm; more preferably not less than 200 nm. In some specific embodiments of the present invention, the difference between the particle diameters of the two acceptor microspheres with different particle diameters is not less than 100 nm, 130 nm, 150 nm, 170 nm, 190 nm, 200 nm, 220 nm, 240 nm, or 250 nm.
  • the particle size ratio of the two acceptor microspheres with different particle sizes is selected from 1: (1.1-10); preferably selected from 1: (2-8); more preferably Select from 1: (3-6). In some specific embodiments of the present invention, the particle size ratio of the two types of acceptor microspheres with different particle sizes 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.
  • the particle size of one of the acceptor microspheres is selected from 50 nm to 300 nm, and the particle size of the other acceptor microsphere is selected from 200 nm to 400 nm.
  • 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 400nm .
  • the particle size of one of the acceptor microspheres is selected from 50 nm to 200 nm, and the particle size of the other acceptor microsphere is selected from 200 nm to 350 nm.
  • the particle size of one of the acceptor microspheres is selected from 80 nm to 150 nm, and the particle size of the other acceptor microsphere is selected from 220 nm to 350 nm.
  • the size of the particle size of the acceptor microspheres should be able to produce a uniform and stable latex solution.
  • the particle size of the acceptor microspheres that can meet this requirement should be in the nanometer range. Therefore, the upper limit of the particle size of the large-size acceptor microspheres is to produce a stable latex solution, and it is generally appropriate to select about 300 nm.
  • the coating and cleaning of the receptor microspheres must be able to be performed under the existing technical conditions to meet the production of reagents.
  • the microsphere composition is used at a concentration of 1 ug / mL-1000 ug / mL; preferably 10 ug / mL-500 ug / mL, and more preferably 50 ug / mL-250 ug / mL.
  • the used concentration of the microsphere composition is determined by the concentration of different target molecules in the blood and the characteristics of the target molecules to be measured.
  • the microsphere composition includes at least three receptor microspheres of different particle sizes.
  • the composition and chemical structure of the acceptor microspheres with different particle diameters may be the same or different.
  • the respective light-emitting compositions and / or substrates of the acceptor microspheres with different particle diameters may be the same or different, as long as they are capable of reacting with active oxygen to generate a detectable chemiluminescence signal.
  • a second aspect of the present invention relates to a receptor agent comprising the microsphere composition according to the first aspect of the present invention.
  • the particle size distribution variation coefficient CV value of the microsphere composition in the receptor reagent may be 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35% or 40%, etc.
  • the coefficient of variation C.V of the particle size distribution of the microsphere composition according to the present invention refers to the value of the coefficient of variation C.V of the particle size distribution after coating the desired substance on the acceptor microspheres.
  • a third aspect of the present invention relates to a chemiluminescence detection kit, comprising the microsphere composition according to the first aspect of the present invention or the acceptor reagent according to the second aspect of the present invention.
  • this kit When using this kit for detection, it has the advantages of both high sensitivity and wide detection range.
  • a fourth aspect of the present invention relates to a chemiluminescence analysis method, which comprises using the microsphere composition according to the first aspect of the present invention or the acceptor reagent according to the second aspect of the present invention or the third aspect of the present invention.
  • the kit detects whether the target molecule to be detected is present in the sample to be tested and / or the concentration of the target molecule to be tested in the sample to be tested. This method has the advantages of both high sensitivity and wide detection range.
  • a fifth aspect of the present invention provides a chemiluminescence analyzer using the microsphere composition according to the first aspect of the present invention or the acceptor reagent according to the second aspect of the present invention or the third aspect of the present invention.
  • the kit and / or the method according to the fourth aspect of the present invention detects whether the target molecule is present in the sample to be tested and / or the concentration of the target molecule in the sample to be tested is present.
  • the emulsion after the reaction is completed is cooled to room temperature and filtered with a suitable filter cloth.
  • the obtained emulsion was washed with deionized water by centrifugal sedimentation until the conductivity of the supernatant of the initial centrifugation was close to that of deionized water, and then diluted with water and stored in the form of an emulsion.
  • step 3 Slowly add the complex solution in step 1 to the three-necked flask in step 2, and stop the stirring after reacting at 70 ° C for 2 hours, and naturally cool.
  • Receptor microspheres having particle diameters of 50 nm, 80 nm, 110 nm, 140 nm, 170 nm, 250 nm, 300 nm, 350 nm, and 400 nm were prepared by the same method.
  • Example 2 Determination of sensitivity and upper limit of detection of microsphere composition
  • the sensitivity point is defined as when the signal of concentration 2C0 is higher than the signal of twice the concentration C0, that is, RLU (2C0)> 2RLU (C0), the corresponding detection reagent sensitivity is C0.
  • the upper limit of detection is defined as the corresponding concentration calculated by substituting the detection signal with a concentration of 1000 ng / ml into the curve of the concentration and signal.
  • cTnI antigen Dilute cTnI antigen to 5 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 100 pg / ml, 1000 pg / ml, 5000 pg / ml, 10000 pg / ml, 50000 pg / ml
  • cTnI monoclonal antibody 1 coated with different particle diameters 50nm, 80nm, 110nm, 140nm, 170nm, 200nm, 250nm, 300nm, 350nm, 400nm
  • Example 1 Dilute cTnI antigen to 5 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50
  • the recipient microspheres were diluted to 100 ug / ml, and then the same concentration of biotinylated cTnI mAb 2 (diluted to 2 ug / ml) and universal solution (donor microsphere solution) were used to detect the above-mentioned series of cTnI antigens.
  • the detection sensitivity and The upper detection limit is shown in Table 1.
  • the spheres were then tested with the same biotin-labeled PCT mAb 2 (diluted to 2ug / ml) and universal solution (donor microsphere solution) for the above-mentioned concentration series of PCT antigens.
  • the detection sensitivity and upper limit of detection are shown in Table 1.
  • Detection result of cTnI project The upper limit of detection of 50nm and 80nm receptor microspheres is very high, but the sensitivity is poor, while the 300nm receptor microsphere has the best sensitivity, but the lower detection limit.
  • the 50nm and 80nm acceptor microspheres were mixed with the 300nm acceptor microspheres to form a microsphere composition, and the sensitivity and upper detection limit of the corresponding microsphere composition were detected. The results are shown in Table 2.
  • a microsphere composition formed by combining a small particle size acceptor microsphere and a large particle size acceptor microsphere has both high sensitivity and a high detection limit (wide detection range), showing a large
  • the advantages of particle size acceptor microspheres and small size acceptor microspheres, compared with a single particle size acceptor microsphere, are that the performance of a composition containing two or more particle sizes is greatly improved.
  • the aldehyde group content of the latex microspheres was determined by conductometric titration to be 280 nmol / mg.
  • step 2 2) Prepare a 100 ml three-necked flask, add 10 ml of 95% ethanol, 10 ml of water, and 10 ml of the aldehyde-based polystyrene latex microspheres obtained in step 1.1, magnetically stir, and warm the water bath to 70 ° C;
  • step 1) Slowly add the complex solution in step 1) to the three-necked flask in step 2), stop stirring after reacting at 70 ° C for 2 hours, and naturally cool;
  • the paired antibody I was dialyzed to 50 mM CB buffer with a pH value of 10, and the concentration was measured to be 1 mg / ml.
  • the preparation method is the same as the preparation process of the acceptor microspheres with an average particle diameter of about 250nm in the above (1).
  • the Nicomp distribution is unimodal (as shown in Figure 6).
  • the preparation method is the same as the preparation process of the acceptor microspheres with an average particle diameter of about 250nm in the above (1).
  • the sensitivity point is defined as when the signal of concentration Cx is higher than the signal of double concentration C0, that is, RLU (Cx)> 2RLU (C0), then the corresponding detection reagent sensitivity is Cx.
  • the upper limit of detection is defined as the upper limit of the range determined using the methods in the documents of the National Committee for Clinical Laboratory Standards (NCCLS) Evaluation Protocol (EP) Series 6.

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Abstract

一种化学发光分析技术领域的用于化学发光分析的微球组合物及其应用。微球组合物包括至少两种不同粒径的受体微球，受体微球能够与活性氧反应产生可检测的化学发光信号。微球组合物既有超高的灵敏度，又有很宽的检测量程。另外，由于小粒径受体微球的反应速度快，因此采用受体微球组合物进行检测时还能缩短检测时间，反应速度得到提升。

Description

一种用于化学发光分析的微球组合物及其应用
相关申请的交叉引用
本申请要求享有于2018年8月13日提交的名称为“一种用于均相化学发光检测的受体试剂及其应用”的中国专利申请CN201810915144.8的优先权，该申请的全部内容通过引用并入本文中。
技术领域
本发明属于化学发光分析技术领域，具体涉及一种用于化学发光分析的微球组合物及其应用。
背景技术
化学发光分析法是一种利用化学发光物质发射的光波进行检测的方法。化学发光物质作为标记应用于核酸检测与免疫检测中。例如，可通过多种途径将特异结合对中的某一分子与发光物质结合形成发光微球组合物。该微球组合物可与样品中被检测物(特异结合对中的另一分子)反应，分配于固相与液相中，且分配比例与检测物的量相关。通过测定固相或液相中发光量，即可得出样品中检测物的相应浓度。
目前随着检测行业的进步，对超敏试剂的需求越来越多，不但对灵敏度要求极高，而且线性范围又要求非常宽，现有的微球组合物很难满足上述检测条件。
因此，亟需开发一种既能满足灵敏度要求、又能满足线性范围要求的用于化学发光分析的微球组合物。
发明内容
本发明所要解决的技术问题是针对现有技术的不足，提供一种用于化学发光分析的微球组合物，将该微球组合物用于化学发光分析检测时，既有超高的灵敏度，又有很宽的检测量程。另外，还能够缩短检测时间。
为此，本发明第一方面提供了一种用于化学发光分析的微球组合物，其包括至少两种不同粒径的受体微球，所述受体微球能够与活性氧反应产生可检测的化学发光信号。
在本发明的一些实施方式中，所述受体微球包括发光组合物和基质两个部分，所述发光组合物填充于基质中和/或包被于基质表面。
在本发明的一些具体实施方式中，所述发光组合物能够与活性氧反应产生可检测的化学发光信号，其包含化学发光化合物和金属螯合物。
在本发明的另一些具体实施方式中，所述化学发光化合物选自烯烃化合物，优选选自二甲基噻吩、双丁二酮化合物、二氧杂环己烯、烯醇醚、烯胺、9-亚烷基苍耳烷、9-亚烷基-N-9,10二氢化吖啶、芳基乙醚烯、芳基咪唑和光泽精以及它们的衍生物，更优选选自二甲基噻吩及其衍生物。
在本发明的一些具体实施方式中，所述金属螯合物的金属是稀土金属或VIII族金属，优选选自铕、铽、镝、钐、锇和钌，更优选选自铕。
在本发明的另一些具体实施方式中，所述金属螯合物包含选自下列的螯合剂：MTTA、NHA、BHHT、BHHCT、DPP、TTA、NPPTA、NTA、TOPO、TPPO、BFTA、2,2-二甲基-4-全氟丁酰-3-丁酮(fod)、2,2’-联吡啶(bpy)、联吡啶基羧酸、氮杂冠醚、氮杂穴状配体和三辛基氧化膦以及它们的衍生物。
在本发明的一些具体实施方式中，所述发光化合物是二甲基噻吩的衍生物，所述金属螯合物是铕螯合物。
在本发明的一些实施方式中，所述基质选自带、片、棒、管、孔、微滴定板、珠、粒子和微球；优选为珠和微球。
在本发明的另一些实施方式中，所述基质是磁性或非磁性粒子。
在本发明的一些实施方式中，所述基质材料选自天然的、合成或改性的天然存在的聚合物，其包括但不限于：琼脂糖、纤维素、硝化纤维素、醋酸纤维素、聚氯乙烯、聚苯乙烯、聚乙烯、聚丙烯、聚(4-甲基丁烯)、聚丙烯酰胺、聚甲基丙烯酸酯、聚对苯二甲酸乙二醇酯、尼龙、聚丁酸乙烯或聚丙烯酸酯。
在本发明的另一些实施方式中，所述基质为醛基化乳胶微球；优选为醛基化聚苯乙烯乳胶微球。
在本发明的一些实施方式中，所述基质的表面直接连接有生物活性物质，所述生物活性物质能够与待测目标分子特异性结合。
在本发明的另一些实施方式中，所述基质的表面包被包覆层，所述包覆层的表面连接有生物活性物质，所述生物活性物质能够与待测目标分子特异性结合。
在本发明的一些实施方式中，所述包覆层中的包覆物选自多糖、高分子聚合物或生物大分子，优选为多糖。
在本发明的另一些实施方式中，所述基质的表面包被至少两个连续多糖层的涂层，其中第一多糖层与第二多糖层自发关联。
在本发明的一些实施方式中，所述连续多糖层中的每一层自发地与前一多糖层中的每一层相关联。
在本发明的另一些实施方式中，所述多糖具有侧基官能团，所述连续多糖层的所述官能团与所述前一多糖层的所述官能团所带电荷相反。
在本发明的一些实施方式中，所述多糖具有侧基官能团，并且所述多糖的所述连续层通过所述连续层的所述官能团与所述前一层的所述官能团之间的反应与所述前一多糖层共价连接。
在本发明的另一些实施方式中，所述连续多糖层的所述官能团在胺官能团和胺反应性官能团之间交替。
在本发明的一些实施方式中，所述胺反应性官能团是醛基或羧基。
在本发明的另一些实施方式中，所述第一多糖层自发地与所述载体相关联。
在本发明的一些实施方式中，所述涂层的最外一层多糖层具有至少一个侧基官能团。
在本发明的另一些实施方式中，所述涂层的最外一层多糖层的侧基官能团选自醛基、羧基、巯基、氨基、羟基和马来胺基中的至少一种；优选选自醛基和/或羧基。
在本发明的一些实施方式中，所述涂层的最外一层多糖层的侧基官能团直接地或间接地与生物活性物质连接。
在本发明的另一些实施方式中，所述多糖选自含有三个或更多个未修饰或修饰的单糖单元的碳水化合物；优选选自葡聚糖、淀粉、糖原、菊粉、果聚糖、甘露聚糖、琼脂糖、半乳聚糖、羧基葡聚糖和氨基葡聚糖；更优选选自葡聚糖、淀粉、糖原和聚核糖。
在本发明的一些实施方式中，所述不同粒径的受体微球的基质粒径相同。
在本发明的一些实施方式中，所述不同粒径的受体微球的基质粒径不同。
在本发明的一些优选的实施方式中，所述活性氧为单线态氧。
在本发明的一些实施方式中，所述微球组合物包括两种不同粒径的受体微球。
在本发明的一些具体实施方式中，所述两种不同粒径的受体微球的粒径的差值不低于100nm；优选不低于150nm；更优选不低于200nm。
在本发明的另一些具体实施方式中，所述两种不同粒径的受体微球的粒径比选自1:(1.1-10)；优选选自1:(2-8)；更优选选自1:(3-6)。
在本发明的一些实施方式中，所述微球组合物的使用浓度为1ug/mL-1000ug/mL；优选为10ug/mL-500ug/mL，更优选为10ug/mL-250ug/mL。
在本发明的一些实施方式中，所述微球组合物包括至少三种不同粒径的受体微球。
本发明第二方面提供了一种受体试剂，其包括如本发明第一方面所述的微球组合物。
本发明第三方面提供了一种化学发光检测试剂盒，其包括如本发明第一方面所述的微球组合物或本发明第二方面所述的受体试剂。
本发明第四方面提供了一种化学发光分析方法，其包括利用如本发明第一方面所述的微球组合物或本发明第二方面所述的受体试剂或本发明第三方面所述的试剂盒检测待测样本中待测目标分子是否存在和/或待测目标分子在待测样本中的浓度。
本发明第五方面提供了一种化学发光分析仪，其利用如本发明第一方面所述的微球组合物或本发明第二方面所述的受体试剂或本发明第三方面所述的试剂盒和/或本发明第四方面所述的方法检测待测样本中待测目标分子是否存在和/或待测目标分子在待测样本中的浓度。
在本发明的一些优选的实施方式中，所述的化学发光分析仪至少包括如下部分：
孵育模块，其用于为待测样本与微球组合物发生化学发光反应提供合适的温度环境，所述微球组合物包含至少两种不同粒径的受体微球；
检测模块，其用于检测受体微球与活性氧反应产生的化学发光信号；
处理器，其根据检测模块检测到的化学发光信号的情况判断待测样本中是否存在待测目标分子和/或待测目标分子在待测样本中的浓度。
本发明的有益效果为：本发明所述用于化学发光分析的微球组合物，同时包括至少两种不同粒径的受体微球。由于小粒径的受体微球能拓宽检测量程，大粒径的受体微球能提高检测灵敏度，因此本发明所述微球组合物的性能较现有技术得到大幅提升，既有超高的灵敏度，又有很宽的量程。另外，由于小粒径受体微球的反应速度快，因此采用本发明所述微球组合物进行检测时还能缩短检测时间，反应速度得到提升。
附图说明
下面将结合附图对本发明作进一步说明。
图1为实施例3中制备的醛基聚苯乙烯乳胶微球的Gaussian分布曲线图。
图2为实施例3中制备的填埋有发光组合物的醛基聚苯乙烯乳胶微球的Gaussian分布曲线图。
图3为实施例3制备的包被葡聚糖的填埋有发光组合物的醛基聚苯乙烯乳胶微球的Gaussian分布图
图4为实施例3制备的平均粒径在250nm左右的受体微球的Gaussian分布图。
图5为实施例3制备的粒径在110nm左右的受体微球的Gaussian分布图。
图6为实施例3制备的粒径在110nm左右的受体微球的Nicomp分布图。
图7为实施例3制备的粒径在350nm左右的的受体微球的Gaussian分布图。
图8为实施例3制备的粒径在350nm左右的的受体微球的Nicomp分布图。
图9为实施例4中混合受体微球粒径分布的Gaussian分布图。
图10为实施例4中混合受体微球粒径分布的Nicomp分布图。
具体实施方式
为使本发明容易理解，下面将详细说明本发明。但在详细描述本发明前，应当理解本发明不限于描述的具体实施方式。还应当理解，本文中使用的术语仅为了描述具体实施方式，而并不表示限制性的。
在提供了数值范围的情况下，应当理解所述范围的上限和下限和所述规定范围中的任何其他规定或居间数值之间的每个居间数值均涵盖在本发明内。这些较小范围的上限和下限可以独立包括在较小的范围中，并且也涵盖在本发明内，服从规定范围中任何明确排除的限度。在规定的范围包含一个或两个限度的情况下，排除那些包括的限度之任一或两者的范围也包含在本发明中。
除非另有定义，本文使用的所有术语与本发明所属领域的普通技术人员的通常理解具有相同的意义。虽然与本文中描述的方法和材料类似或等同的任何方法和材料也可在本发明的实施或测试中使用，但是现在描述了优选的方法和材料。
Ⅰ.术语
本发明所述用语“活性氧”是指机体内或者自然环境中由氧组成，含氧并且性质活泼的物质的总称，主要为一种激发态的氧分子，包括氧的一电子还原产物超氧阴离子(O 2·-)、二电子还原产物过氧化氢(H 2O 2)、三电子还原产物羟基自由基(·OH)以及一氧化氮和单线态氧(1O 2)等。
本发明中，所述用语“受体微球”是指能够与活性氧反应产生可检测的化学发光信号的纳米微球，其也可以称为受氧微球或发光微球。优选地，所述受体微球可以是通过功能基团填充于基质中形成填充有发光组合物的高分子微粒，所述发光组合物包含有能够与活性氧发生反应的化学发光化合物。在本发明的一些具体实施例中，所述化学发光化合物，其经历与活性氧的化学反应以形成不稳定的亚稳态中间体，所述亚稳态中间体可以分解，同时或随后发光。这些物质的典型例子包括但不限于：烯醇醚、烯胺、9-烷叉黄原胶、9-烷叉-N-烷基吖啶满、芳基乙醚烯、双环氧乙烯、二甲基噻吩、芳基咪唑或光泽精。
本发明中，所述“化学发光化合物”即一种被称作为标记物的化合物，可进行化学反应以便引起发光，比如通过被转化为在电子激发态下形成的另一种化合物。激发态可以是单线态或是三重激发态。激发态可弛豫到基态直接发光，或者是通过将激发能量传递到发射能量受体，从而自身恢复到基态。在此过程中，能量受体微球将被跃迁为激发态而发光。
化学发光化合物可以结合到特异性结合伴侣成员上，该特异性结合伴侣成员能够直接或间接地与待测目标分子或测试成分结合，该测试成分的浓度受存在的待测目标分子的影响。用于“能够直接或间接地结合”是指指定的实体物能够特 异地结合到实体物上(直接地)，或者指定的实体物能够特异性地结合至特异性结合配对成员、或具有两个或更多能够结合其他实体物的特异性结合伴侣的复合物上(间接地)。
本发明所述“特异性结合配对成员”选自(1)小分子和对于所述小分子的结合配偶体，以及(2)大分子和对于所述大分子的结合配偶体
在本发明中，所述活性氧可以由“供体微球”提供，所述供体微球是能够在激发状态下生成活性氧的纳米微球。优选地，所述供体微球可以是通过功能基团被包被在基体上形成填充有感光化合物的高分子微粒，在光激发下能够产生单线态氧，此时感光微球也可以称为供氧微球或感光微球。所述供体微球表面可以有亲水性的醛基葡聚糖，内部填充有光敏剂。所述光敏剂可以是本领域已知的光敏剂，优选相对光稳定且不与单线态氧有效反应的化合物，其非限定性的例子包括亚甲基蓝、玫瑰红、卟啉、和酞菁等化合物，以及这些化合物的具有1-50个原子取代基的衍生物，所述取代基用于使得这些化合物更具有亲脂性或更具有亲水性、和/或作为连接至特异性结合配对成员的连接基团。所述供体微球表面还可以填充其他敏化剂，其非限定性的例子是某些化合物，它们催化过氧化氢转化为单线态氧和水。其他一些供体的例子包括：1,4-二羧基乙基-1,4-萘内过氧化物、9,10-二苯基蒽-9,10-内过氧化物等，加热这些化合物或者这些化合物直接吸收光会释放活性氧，例如单线态氧。
所述敏化剂是一种化合物，当被激发或被诱导反应时可导致另一种化合物或物质发生化学反应。敏化剂包括光敏剂，其能够被光照射所诱导，从而形成活化的激发态。敏化剂也包括能发生化学反应，从而产生亚稳态活性氧物质诸如单线态氧的化合物。
光敏剂一般通过照射含有上述反应物的介质来活化化学发光化合物。介质必须用具有一定的波长、且能量足以将光敏剂转化至激发态、进而使得它能够将分子氧活化为单线态氧的光照射。能够激发分子氧的光敏剂的激发态一般位于三重态，其比处于基态的光敏剂的能量高大约20Kcal/mol、通常至少23Kcal/mol。虽然可以使用较短的波长，如230-950nm，但是优选地，使用波长大约是450-950nm的光来照射介质。可以以任何传统方式，诸如照相、肉眼直观、光度计等，来测定产生的光，以便确定其与介质中分析物含量相关的量。光敏剂优选是相对非极性的，以确保可溶解到亲脂性成员中。
本发明所述的“基质”其可以是任何尺寸的，其可以是有机的或是无机的，其可以是可膨胀或不可膨胀的，其可以是多孔的或非多孔的，其具有任何密度，但优选具有和水接近的密度，优选能漂浮于水中，且由透明、部分透明或不透明的材料构成。所述基质可以有或没有电荷，当带有电荷时，优选是负电荷。所述基质可以是固体(如聚合物、金属、玻璃、有机和无机物诸如矿物、盐和硅藻)、小油滴(如碳氢化合物、碳氟化合物、硅质流体)、囊泡(如合成的诸如磷脂、或天然的诸如细胞、及细胞器官)。基质可以是乳胶颗粒或是含有有机或无机聚合物的其他颗粒、脂双层如脂质体、磷脂囊泡、小油滴、硅颗粒、金属溶胶、细胞和微晶染料。基质通常具有多功能性，或者能够通过特异或非特异的共价或非共价相互作用而结合到供体或受体上。有许多官能团是可用的或者将其合并进来。典型的官能团包括羧酸、乙醛、氨基、氰基、乙烯基、羟基、巯基等。适用于本发明的基质的一个非限制性的例子是醛基聚苯乙烯乳胶微球。
可以选择光敏剂和/或化学发光化合物溶解在、或非共价地结合到颗粒的表面。在这种情况下，这些化合物优选是疏水性的，以降低它们从颗粒解离下来的能力，从而使两种化合物都能和相同的颗粒结合。
本发明所述用语“粒径”是指受体微球的平均粒径，它是用常规粒径仪测定的。本发明所述“受体微球”至少包括基质、发光组合物以及生物活性分子，优选还包括包覆层；所述发光组合物可以填充于基质中和/或包被于基质表面。当所述受体微球不包括包覆层时，所述生物活性物质直接连接于基质表面。当所述受体微球包括包覆层时，所述包覆层包被于基质表面，且所述包覆层的最外层连接生物活性活性物质。
值得注意的是，本发明所述“受体微球的平均粒径”指的是连接和/或包覆上相应的物质后的受体微球的平均粒径。不同粒径的受体微球中的基质的粒径可以相同也可以不同，只要最终形成的受体微球的粒径不同即可，本发明最优选的技术方案是所述不同粒径的受体微球中的基质的粒径也不相同。
本发明所述用语“待测样本”是指检测待测的含有或疑似含有待测目标分子的一种混合物。可以被用在本发明公开的待测样本包括体液，如血液(可以是在收集的血液样品中通常看到的抗凝血)、血浆、血清、尿、精液、唾液、细胞培养物、组织提取物等。其他类型的待测样本包括溶剂、海水、工业水样、食品样品、环境样本诸如土或水、植物材料、真核细胞、细菌、质粒、病毒、真菌、及 来自于原核的细胞。待测样本可以在使用前根据需要利用稀释液进行稀释。例如，为了避免HOOK效应，可以在上机检测前使用稀释液对待测样本进行稀释后再在检测仪器上进行检测。
本发明所述用语“待测目标分子”是指检测时待检测样本中的物质。与待测目标分子具有特异性结合亲合力的一种或多种物质会被用于检测该目标分子。待测目标分子可以是蛋白、肽、抗体或可以使其与抗体结合的半抗原。待测目标分子可以是与互补核酸或寡聚核苷酸结合的核酸或寡聚核苷酸。待测目标分子可以是可形成特异性结合配对成员的任何其他物质。其他典型的待测目标分子的例子包括：药物，诸如类固醇、激素、蛋白、糖蛋白、粘蛋白、核蛋白、磷蛋白、滥用的药物、维生素、抗细菌药、抗真菌药、抗病毒药、嘌呤、抗肿瘤试剂、安非他命、杂氮化合物、核酸和前列腺素，以及任何这些药物的代谢物；杀虫剂及其代谢物；以及受体。分析物也包括细胞、病毒、细菌和真菌。
本发明所述用语“抗体”以最广含义使用，包括任何同种型的抗体，保留对抗原的特异性结合的抗体片段，包括但不限于Fab、Fv、scFv、和Fd片段、嵌合抗体、人源化抗体、单链抗体、双特异性抗体、和包含抗体的抗原结合部分和非抗体蛋白的融合蛋白。在任何需要的情况下，抗体可以进一步与其它部分，诸如特异性结合配对成员，例如生物素或链霉亲和素(生物素-链霉亲和素特异性结合配对成员中的一员)等缀合。
本发明所述用语“抗原”是指能够刺激机体产生免疫应答，并能与免疫应答产物抗体和致敏淋巴细胞在体内外结合，发生免疫效应的物质。
本发明所述用语“结合”指由于例如共价、静电、疏水、离子和/或氢键等相互作用，包括但不限于如盐桥和水桥等相互作用引起的两个分子间的直接联合。
本发明所述用语“特异性结合”，是指两种物质之间的相互辨别和选择性结合反应，从立体结构角度上说就是相应的反应物之间构象的对应性。在本发明公开的技术思想下，特异性结合反应的检测方法包括但不限于：双抗体夹心法、竞争法、中和竞争法、间接法或捕获法。
本发明所述“粒径分布变异系数C.V值”是指在纳米粒度仪的检测结果中，粒径在Gaussian分布中的变异系数。变异系数的计算公式为：C.V值＝(标准偏差SD/平均值Mean)×100％。
本发明所述用语“Nicomp分布”是指美国PSS纳米粒度仪NICOMP中的一种算法分布。相对于Gaussian单峰算法，Nicomp多峰算法对于多组分、粒径分布不均匀液态分散体系的分析以及胶体体系的稳定性分析具有独特优势。
Ⅱ.具体实施方案
下面将更详细地说明本发明。
本发明通过控制微球组合物中受体微球的粒径，进而控制每个受体微球表面生物活性物质(如，抗体/抗原)的量(小粒径微球比表面积大，单位质量微球表面报告分子的量多，大粒径微球比表面积小，单位质量微球表面报告分子的量少)，从而提高检测的灵敏度和拓宽检测量程。另外小粒径受体微球由于直径较小，使得供体微球产生的单线氧的活化效率提高，从而也能提高受体微球微球的发光效率。
本发明第一方面涉及一种用于化学发光分析的微球组合物，其包括至少两种不同粒径的受体微球，所述受体微球能够与活性氧反应产生可检测的化学发光信号。
在本发明的一些实施方式中，所述受体微球包括发光组合物和基质两个部分，所述发光组合物填充于基质中和/或包被于基质表面。
在本发明的一些具体实施方式中，所述发光组合物能够与活性氧反应产生可检测的化学发光信号，其包含化学发光化合物和金属螯合物。
在本发明的另一些具体实施方式中，所述化学发光化合物选自烯烃化合物，烯烃化合物是能够与活性氧(如单线态氧)反应的化合物。合适的富含电子的烯化合物的例子在美国专利号5,709,994中列出，相关的内容通过引用并入本文。在本发明的一些优选的实施方式中，所述烯烃化合物选自二甲基噻吩、双丁二酮化合物、二氧杂环己烯、烯醇醚、烯胺、9-亚烷基苍耳烷、9-亚烷基-N-9,10二氢化吖啶、芳基乙醚烯、芳基咪唑和光泽精以及它们的衍生物，更优选选自二甲基噻吩及其衍生物。
除了烯烃化合物外，所述化学发光化合物还包括金属和一个或多个螯合剂的复合物(金属螯合物)。在本发明的一些具体实施方式中，所述金属螯合物的金属是稀土金属或VIII族金属，优选选自铕、铽、镝、钐、锇和钌，更优选选自铕。
在本发明的另一些具体实施方式中，所述金属螯合物包含选自下列的螯合剂：4’-(10-甲基-9-蒽基)-2,2’:6’2”-联三吡啶-6,6”-二甲胺]四乙酸(MTTA)、2-(1’,1’,2’,2’,3’,3’-七氟-4’,6’-己二酮-6’-基)-萘(NHA)、4,4’-二(2”,3”,3”-七氟-4”,6”-己二酮-6”-基)-邻-三联苯(BHHT)、4,4’-二(1”,1”,1”,2”,2”,3”,3”-七氟-4”,6”-己二酮-6”-基)-氯代磺基-邻-三联苯(BHHCT)、4,7-联苯-1,10-菲咯啉(DPP)、1,1,1-三氟丙酮(TTA)、3-萘酰-1,1,1-三氟丙酮(NPPTA)、萘基三氟丁二酮(NTA)、三辛基氧化膦(TOPO)、三苯基氧化膦(TPPO)、3-苯甲酰-1,1,1-三氟丙酮(BFTA)、2,2-二甲基-4-全氟丁酰-3-丁酮(fod)、2,2’-联吡啶(bpy)、联吡啶基羧酸、氮杂冠醚、氮杂穴状配体和三辛基氧化膦以及它们的衍生物。
在本发明的一些具体实施方式中，所述发光化合物是二甲基噻吩的衍生物，所述金属螯合物是铕螯合物。
在本发明的一些实施方式中，所述基质选自带、片、棒、管、孔、微滴定板、珠、粒子和微球；优选为珠和微球。
在本发明的另一些实施方式中，所述基质是磁性或非磁性粒子。
在本发明的一些实施方式中，所述不同粒径的受体微球的基质的材质相同或不同。
在本发明的另一些实施方式中，所述基质材料选自天然的、合成或改性的天然存在的聚合物，其包括但不限于：琼脂糖、纤维素、硝化纤维素、醋酸纤维素、聚氯乙烯、聚苯乙烯、聚乙烯、聚丙烯、聚(4-甲基丁烯)、聚丙烯酰胺、聚甲基丙烯酸酯、聚对苯二甲酸乙二醇酯、尼龙、聚丁酸乙烯或聚丙烯酸酯。
在本发明的另一些实施方式中，所述基质为醛基化乳胶微球；优选为醛基化聚苯乙烯乳胶微球。
在本发明的一些实施方式中，所述基质的表面直接连接有生物活性物质，所述生物活性物质能够与待测目标分子特异性结合。
在本发明的另一些实施方式中，所述基质的表面包被包覆层，所述包覆层的表面连接有生物活性物质，所述生物活性物质能够与待测目标分子特异性结合；举例说明但非限定地，所述的生物活性物质-待测目标分子结合配偶体的离子包括抗原-抗体、激素-激素受体、核酸双链体、IgG-蛋白质A、多核苷酸对例如DNA-DNA、DNA-RNA等。
在本发明的一些优选的实施方式中，所述生物活性物质为抗原和/或抗体；所述抗原是指具有免疫原性的物质；所述抗体是指机体产生的能识别特定外来物的免疫球蛋白。
在本发明的一些实施方式中，所述包覆层中的包覆物选自多糖、高分子聚合物或生物大分子，优选为多糖。
在本发明的另一些实施方式中，所述基质的表面包被至少两个连续多糖层的涂层，其中第一多糖层与第二多糖层自发关联。
在本发明的一些实施方式中，所述连续多糖层中的每一层自发地与前一多糖层中的每一层相关联。
在本发明的另一些实施方式中，所述多糖具有侧基官能团，所述连续多糖层的所述官能团与所述前一多糖层的所述官能团所带电荷相反。
在本发明的一些实施方式中，所述多糖具有侧基官能团，并且所述多糖的所述连续层通过所述连续层的所述官能团与所述前一层的所述官能团之间的反应与所述前一多糖层共价连接。
在本发明的另一些实施方式中，所述连续多糖层的所述官能团在胺官能团和胺反应性官能团之间交替。
在本发明的一些实施方式中，所述胺反应性官能团是醛基或羧基。
在本发明的另一些实施方式中，所述第一多糖层自发地与所述载体相关联。
在本发明的一些实施方式中，所述涂层的最外一层多糖层具有至少一个侧基官能团。
在本发明的另一些实施方式中，所述涂层的最外一层多糖层的侧基官能团选自醛基、羧基、巯基、氨基、羟基和马来胺基中的至少一种；优选选自醛基和/或羧基。
在本发明的一些实施方式中，所述涂层的最外一层多糖层的侧基官能团直接地或间接地与生物活性物质连接。
在本发明的另一些实施方式中，所述多糖选自含有三个或更多个未修饰或修饰的单糖单元的碳水化合物；优选选自葡聚糖、淀粉、糖原、菊粉、果聚糖、甘露聚糖、琼脂糖、半乳聚糖、羧基葡聚糖和氨基葡聚糖；更优选选自葡聚糖、淀粉、糖原和聚核糖。
在本发明的一些实施方式中，所述不同粒径的受体微球的基质粒径相同。
在本发明的另一些实施方式中，所述不同粒径的受体微球的基质粒径不同。
在本发明的一些实施方式中，所述化学发光信号的检测波长为450-680nm；优选为520-620nm，更优选为610-620nm，最优选为615nm。相应地，用于激发的激发光的波长范围为640-680nm，优选所述激发光的波长为660nm。
在本发明的一些优选的实施方式中，所述活性氧为单线态氧。
在本发明的一些实施方式中，所述微球组合物包括两种不同粒径的受体微球。
在本发明的一些具体实施方式中，所述两种不同粒径的受体微球的粒径的差值不低于100nm；优选不低于150nm；更优选不低于200nm。在本发明的一些具体实施例中，所述两种不同粒径的受体微球的粒径的差值不低于100nm、130nm、150nm、170nm、190nm、200nm、220nm、240nm或250nm。
在本发明的另一些具体实施方式中，所述两种不同粒径的受体微球的粒径比选自1:(1.1-10)；优选选自1:(2-8)；更优选选自1:(3-6)。在本发明的一些具体实施例中，所述两种不同粒径的受体微球的的粒径比选自1:1.5、1:2、1:2.7、1:3、1:3.2、1:3.75、1:4、1:5或1:6。
在本发明的一些优选的具体实施方式中，其中一种受体微球的粒径选自50nm-300nm，而另一种受体微球的粒径选自200nm-400nm。例如，其中一种受体微球的粒径选自50nm、80nm、110nm、140nm、170nm、200nm或300nm，而另一种受体微球的粒径选自200nm、250nm、300nm、350nm或400nm。在本发明的一些更优选的具体实施方式中，其中一种受体微球的粒径选自50nm-200nm，而另一种受体微球的粒径选自200nm-350nm。
在本发明的一些最优选的具体实施方式中，其中一种受体微球的粒径选自80nm-150nm，而另一种受体微球的粒径选自220nm-350nm。本发明中，所述受体微球粒径的大小应能产生均匀稳定的乳胶溶液，一般能达到这个要求的受体微球的粒径应该在纳米范围内。因此大粒径受体微球的粒径上限是能生成稳定的乳胶溶液，一般选择300nm左右为宜。同时，受体微球的包被和清洗要能够在现有的技术条件下进行从而满足试剂的生产。
在本发明的一些实施方式中，所述微球组合物的使用浓度为1ug/mL-1000ug/mL；优选为10ug/mL-500ug/mL，更优选为50ug/mL-250ug/mL。 本发明中，所述微球组合物的使用浓度由不同待测目标分子在血液中的浓度及待测目标分子的特性所决定。
在本发明的一些实施方式中，所述微球组合物包括至少三种不同粒径的受体微球。
在本发明的一些实施方式中，所述不同粒径的受体微球的组成及化学结构，各自可以相同，也可以各不同。例如，不同粒径的受体微球各自的发光组合物和/或基质可以相同，亦可以不同，只要满足能够与活性氧反应产生可检测的化学发光信号即可。
本发明第二方面涉及一种受体试剂，其包括如本发明第一方面所述的微球组合物。
在本发明的一些具体实施方式中，所述微球组合物在受体试剂中的粒径分布变异系数C.V值可以为5％、6％、7％、8％、9％、10％、11％、12％、13％、14％、15％、16％、17％、18％、19％、20％、25％、30％、35％或40％等。
值得注意的是，本发明所述的微球组合物粒径分布变异系数C.V值指的是受体微球包被上所需的物质后的粒径分布变异系数C.V值。
本发明第三方面涉及一种化学发光检测试剂盒，其包括如本发明第一方面所述的微球组合物或本发明第二方面所述的受体试剂。采用该试剂盒进行检测时同时具有高灵敏度和宽检测量程的优点。
本发明第四方面涉及一种化学发光分析方法，其包括利用如本发明第一方面所述的微球组合物或本发明第二方面所述的受体试剂或本发明第三方面所述的试剂盒检测待测样本中待测目标分子是否存在和/或待测目标分子在待测样本中的浓度。该方法同时具有高灵敏度和宽检测量程的优点。
本发明第五方面提供了一种化学发光分析仪，其利用如本发明第一方面所述的微球组合物或本发明第二方面所述的受体试剂或本发明第三方面所述的试剂盒和/或本发明第四方面所述的方法检测待测样本中待测目标分子是否存在和/或待测目标分子在待测样本中的浓度。
Ⅲ.具体实施例
为使本发明更加容易理解，下面将结合实施例来进一步详细说明本发明，这些实施例仅起说明性作用，并不局限于本发明的应用范围。本发明中所使用的原料或组分若无特殊说明均可以通过商业途径或常规方法制得。
实施例1：微球组合物的制备
(1)醛基聚苯乙烯乳胶微球的制备及表征过程
1.准备100ml的三口烧瓶，加入40mmol苯乙烯、5mmol丙烯醛、10ml水，搅拌10min后通N 2 30min。
2.称取0.11g过硫酸铵和0.2g氯化钠，溶于40ml水中配置成水溶液。将该水溶液加入到步骤1的反应体系中，继续通N 2 30min。
3.将反应体系升温至70℃，反应15小时。
4.将反应完成后的乳液冷却至室温，用合适的滤布过滤。得到的乳液用去离子水过次离心沉降清洗，直至离心初的上清液的电导率接近去离子水，然后用水稀释，以乳液形式保存。
5.由纳米粒度仪测得该乳胶微球粒径为190.4nm，CV＝5.1％；由电导滴定法测得该乳胶微球醛基含量为280nmol/mg。
(2)发光组合物的包被过程及表征过程
1.准备25ml的圆底烧瓶，加入0.1g二甲基噻吩衍生物和0.1g铕(Ⅲ)配合物(MTTA-EU 3+)，10ml 95％乙醇，磁力搅拌，水浴升温至70℃，获得配合物溶液。
2.准备100ml的三口烧瓶，加入10ml 95％乙醇、10ml水和10ml浓度为10％、粒径140nm的醛基聚苯乙烯乳胶微球，磁力搅拌，水浴升温至70℃。
3.将步骤1中的配合物溶液缓慢滴加至步骤2中的三口烧瓶中，70℃反应2小时后停止搅拌，自然冷却。
4.将上述乳液离心1小时，30000G，离心后弃去上清液，用50％乙醇重新悬浮。重复离心清洗三次后用PH值＝10的50mM CB缓冲液重新悬浮，使其终浓度为20mg/ml。
(3)抗体的偶联过程
1.将PCT抗体1透析至PH值＝10的50mM CB缓冲液，测得浓度为1mg/ml。
2.在2ml离心管中加入0.5ml带有醛基的聚苯乙烯乳胶微球，0.5ml步骤1获得的配对抗体Ⅰ，混匀后加入100μl 10mg/ml NaBH 4溶液(50mM CB缓冲液)，2-8℃反应4小时。
3.反应完毕后加入0.5ml 100mg/ml BSA溶液(50mM CB缓冲液)，2-8℃反应2小时。
4.反应完毕后将离心45min，30000G，离心后弃去上清液，用50mM MES缓冲液重新悬浮。重复离心清洗四次，获得偶联PCT抗体1的受体微球。
5.由纳米粒度仪测得该受体微球的粒径为210.4nm，CV＝5.1％。
用同样方法制得粒径为50nm、80nm、110nm、140nm、170nm、250nm、300nm、350nm、400nm的受体微球。
实施例2：微球组合物灵敏度和检测上限的测定
定义灵敏度点为当浓度2C0的信号高于两倍浓度C0的信号，即RLU(2C0)>2RLU(C0)，则对应的检测试剂灵敏度为C0。定义检测上限点为浓度1000ng/ml的检测信号代入浓度与信号的曲线反算出的对应浓度。
(1)将cTnI抗原稀释到5pg/ml、10pg/ml、20pg/ml、30pg/ml、40pg/ml、50pg/ml、100pg/ml、1000pg/ml、5000pg/ml、10000pg/ml、50000pg/ml、1000ng/ml的系列浓度，将采用与实施例1相同的方法制备不同粒径(50nm、80nm、110nm、140nm、170nm、200nm、250nm、300nm、350nm、400nm)包被cTnI单抗1的受体微球分别稀释到100ug/ml，然后与相同的生物素标记的cTnI单抗2(稀释到2ug/ml)和通用液(供体微球溶液)检测上述浓度系列cTnI抗原，检测灵敏度和检测上限如表1所示。
(2)将PCT抗原稀释到20pg/ml、30pg/ml、40pg/ml、60pg/ml、80pg/ml、160pg/ml、500pg/ml、1000pg/ml、5000pg/ml、20000pg/ml、100000pg/ml及2000ng/ml的系列浓度，将采用实施例1制备的不同粒径(50nm、80nm、110nm、140nm、170nm、200nm、250nm、300nm、350nm、400nm)包被PCT单抗1的受体微球，然后与相同的生物素标记的PCT单抗2(稀释到2ug/ml)和通用液(供 体微球溶液)检测上述浓度系列PCT抗原，检测灵敏度和检测上限如表1所示。
表1
Figure PCTCN2019100340-appb-000001
从表1可知，
(1)cTnI项目检测结果：在50nm、80nm的受体微球的检测上限很高，但是灵敏度较差，而300nm的受体微球有最佳的灵敏度，但是检测上限较低。将50nm和80nm的受体微球分别与300nm受体微球混合，形成微球组合物，检测相应的微球组合物的灵敏度和检测上限，结果如表2所示。
(2)PCT项目检测结果：在110nm的受体微球的检测上限很高，但是灵敏度较差，而300nm、350nm的受体微球有最佳的灵敏度，但是检测上限较低。将110nm受体微球分别与300nm、350nm受体微球混合，形成微球组合物，检测相应的微球组合物的灵敏度和检测上限，结果如表2所示。
表2
Figure PCTCN2019100340-appb-000002
从表2可知，通过将小粒径的受体微球和大粒径的受体微球组合后形成的微球组合物，同时具有高灵敏度和高检测上限(宽检测量程)，展现出大粒径受体微球和小粒径受体微球的优势，与单一粒径的受体微球相比含有两种以上粒径的微球组合物的性能得到极大的提高。
实施例3：微球组合物的制备
(一)平均粒径在250nm左右的偶联抗体的受体微球的制备
1.1醛基聚苯乙烯乳胶微球的制备及表征过程
1)准备100ml的三口烧瓶，加入40mmol苯乙烯、5mmol丙烯醛、10ml水，搅拌10min后通N 2 30min；
2)称取0.11g过硫酸铵和0.2g氯化钠，溶于40ml水中配置成水溶液。将该水溶液加入到步骤1的反应体系中，继续通N 2 30min；
3)将反应体系升温至70℃，反应15小时；
4)将反应完成后的乳液冷却至室温，用合适的滤布过滤。得到的乳液用去离子水过次离心沉降清洗，直至离心初的上清液的电导率接近去离子水，然后用水稀释，以乳液形式保存；
5)由纳米粒度仪测得此时乳胶微球粒径的Gaussian分布平均粒径为202.2nm，变异系数(C.V.)＝4.60％，Gaussian分布曲线如图1所示。由电导滴定法测得该乳胶微球醛基含量为280nmol/mg。
1.2发光组合物的填埋过程及表征
1)准备25ml的圆底烧瓶，加入0.1g二甲基噻吩衍生物和0.1g铕(Ⅲ)配合物(MTTA-EU 3+)，10ml 95％乙醇，磁力搅拌，水浴升温至70℃，获得配合物溶液；
2)准备100ml的三口烧瓶，加入10ml 95％乙醇、10ml水和10ml浓度为10％、步骤1.1中获得的醛基聚苯乙烯乳胶微球，磁力搅拌，水浴升温至70℃；
3)将步骤1)中的配合物溶液缓慢滴加至步骤2)中的三口烧瓶中，70℃反应2小时后停止搅拌，自然冷却；
4)将上述乳液离心1小时，30000G，离心后弃去上清液，得到填埋有发光组合物的醛基聚苯乙烯微球。
5)由纳米粒度仪测得此时微球粒径的Gaussian分布平均粒径为204.9nm，变异系数(C.V.)＝5.00％(如图2所示)。
1.3受体微球的表面包被葡聚糖
1)取50mg氨基葡聚糖固体于20mL圆底烧瓶中，加入5mL 50mM/pH＝10碳酸盐缓冲液，30℃避光搅拌溶解；
2)取100mg已制备好的填埋有发光组合物的醛基聚苯乙烯微球，加入到氨基葡聚糖溶液中搅拌2小时；
3)将10mg硼氢化钠溶于0.5mL 50mM/pH＝10碳酸盐缓冲液后滴加到上述反应液中，30℃避光反应过夜；
4)将反应后的混合液30000G离心后弃去上清液，加入50mM/pH＝10碳酸盐缓冲液超声分散。重复离心清洗三次后用50mM/pH＝10碳酸盐缓冲液定容，使其终浓度为20mg/ml；
5)取100mg醛基葡聚糖固体于20mL圆底烧瓶中，加入5mL 50mM/pH＝10碳酸盐缓冲液，30℃避光搅拌溶解；
6)将上述微球加入到醛基葡聚糖溶液中搅拌2小时；
7)将15mg硼氢化钠溶于0.5mL 50mM/pH＝10碳酸盐缓冲液后滴加到上述反应液中，30℃避光反应过夜；
8)将反应后的混合液30000G离心后弃去上清液，加入50mM/pH＝10碳酸盐缓冲液超声分散。重复离心清洗三次后用50mM/pH＝10碳酸盐缓冲液定容，使其终浓度为20mg/ml。
9)由纳米粒度仪测得此时微球粒径的Gaussian分布平均粒径为241.6nm，变异系数(C.V.)＝12.90％(如图3所示)。
1.4抗体的偶联过程
1)将配对抗体Ⅰ透析至PH值＝10的50mM CB缓冲液，测得浓度为1mg/ml。
2)在2ml离心管中加入0.5ml(3)中获得的受体微球以及0.5ml步骤1)获得的配对抗体Ⅰ，混匀后加入100μl 10mg/ml NaBH 4溶液(50mM CB缓冲液)，2-8℃反应4小时。
3)反应完毕后加入0.5ml 100mg/ml BSA溶液(50mM CB缓冲液)，2-8℃ 反应2小时。
4)反应完毕后将离心45min，30000G，离心后弃去上清液，用50mM MES缓冲液重新悬浮。重复离心清洗四次，并稀释至终浓度为100μg/ml，获得偶联抗体Ⅰ的受体微球溶液。
5)由纳米粒度仪测得此时微球粒径的Gaussian分布平均粒径值为253.5nm，变异系数(C.V值)＝9.60％(如图4所示)。
(二)平均粒径在110nm左右的偶联抗体的受体微球的制备
制备方法同上述(一)中平均粒径为250nm左右的受体微球的制备过程，由纳米粒度仪测得该受体微球粒径的Gaussian分布(如图5所示)平均粒径值为107.1nm，变异系数(C.V.)＝7.6％。Nicomp分布为单峰(如图6所示)。
(三)平均粒径在350nm左右的偶联PCT抗体的受体微球的制备
制备方法同上述(一)中平均粒径为250nm左右的受体微球的制备过程，由纳米粒度仪测得该受体微球粒径的Gaussian分布(如图7所示)平均粒径值为347.5nm，变异系数(C.V.)＝3.9％，Nicomp分布为单峰(如图8所示)。
实施例4：试剂盒灵敏度和检测上限的测定
定义灵敏度点为当浓度Cx的信号高于两倍浓度C0的信号，即RLU(Cx)>2RLU(C0)，则对应的检测试剂灵敏度为Cx。定义检测上限点为使用美国临床实验室标准化委员会(NCCLS)评价方案(EP)系列6的文件中的方法确定的范围上限。
(1)将PCT抗原稀释到20pg/ml、30pg/ml、40pg/ml、50pg/ml、60pg/ml、80pg/ml、160pg/ml、500pg/ml、1000pg/ml、5000pg/ml、20000pg/ml、50000pg/ml、100000pg/ml及200000pg/ml的系列浓度，将采用实施例3中制备的分别包含不同平均粒径(110nm、250nm和350nm)偶联PCT抗体Ⅰ的受体微球的受体试剂(浓度为100ug/ml)，然后与相同的生物素标记的PCT单抗2(稀释到2ug/ml)和通用液(含供体微球的试剂)检测上述浓度系列PCT抗原，利用博阳生物科技(上海)有限公司开发的光激化学发光分析系统检测灵敏度和检测上限如表3所示。
表3
Figure PCTCN2019100340-appb-000003
从表3可知，110nm平均粒径的受体微球的检测上限较高，但是灵敏度较差；而在350nm平均粒径的受体微球有最佳的灵敏度，但是检测上限较低。
(2)将平均粒径110nm偶联PCT抗体Ⅰ的受体微球溶液与平均粒径350nm偶联PCT抗体Ⅰ的受体微球溶液混合后，得到新的受体试剂。新的受体试剂中，受体微球粒径的测定结果如下：
Gaussian分布平均粒径317.7nm，粒径分布变异系数(C.V值)＝37.2％(如图9所示)；
Nicomp分布为双峰：#1：平均粒径103.1nm变异系数(C.V值)＝11.8％；#2：平均粒径328.8nm，粒径分布变异系数(C.V值)＝13.0％(如图10所示)。
将上述新的受体试剂与生物素标记的PCT单抗2(稀释到2ug/ml)和通用液(含供体微球的试剂)检测上述浓度系列PCT抗原，利用博阳生物科技(上海)有限公司开发的光激化学发光分析系统检测灵敏度和检测上限如表4所示。
表4
Figure PCTCN2019100340-appb-000004
从表4可知，适当增加受体微球粒径的不均一性，所述试剂盒的检测性能得到明显提升。
应当注意的是，以上所述的实施例仅用于解释本发明，并不构成对本发明的任何限制。通过参照典型实施例对本发明进行了描述，但应当理解为其中所用的词语为描述性和解释性词汇，而不是限定性词汇。可以按规定在本发明权利要求 的范围内对本发明作出修改，以及在不背离本发明的范围和精神内对本发明进行修订。尽管其中描述的本发明涉及特定的方法、材料和实施例，但是并不意味着本发明限于其中公开的特定例，相反，本发明可扩展至其他所有具有相同功能的方法和应用。

Claims (38)

  1. 一种用于化学发光分析的微球组合物，其包括至少两种不同粒径的受体微球，所述受体微球能够与活性氧反应产生可检测的化学发光信号。
  2. 根据权利要求1所述的微球组合物，其特征在于，所述受体微球包括发光组合物和基质两个部分，所述发光组合物填充于基质中和/或包被于基质表面。
  3. 根据权利要求2所述的微球组合物，其特征在于，所述发光组合物能够与活性氧反应产生可检测的化学发光信号，其包含化学发光化合物和金属螯合物。
  4. 根据权利要求3所述的微球组合物，其特征在于，所述化学发光化合物选自烯烃化合物，优选选自二甲基噻吩、双丁二酮化合物、二氧杂环己烯、烯醇醚、烯胺、9-亚烷基苍耳烷、9-亚烷基-N-9,10二氢化吖啶、芳基乙醚烯、芳基咪唑和光泽精以及它们的衍生物，更优选选自二甲基噻吩及其衍生物。
  5. 根据权利要求3或4所述的微球组合物，其特征在于，所述金属螯合物的金属是稀土金属或VIII族金属，优选选自铕、铽、镝、钐、锇和钌，更优选选自铕。
  6. 根据权利要求3-5中任意一项所述的微球组合物，其特征在于，所述金属螯合物包含选自下列的螯合剂：MTTA、NHA、BHHT、BHHCT、DPP、TTA、NPPTA、NTA、TOPO、TPPO、BFTA、2,2-二甲基-4-全氟丁酰-3-丁酮(fod)、2,2’-联吡啶(bpy)、联吡啶基羧酸、氮杂冠醚、氮杂穴状配体和三辛基氧化膦以及它们的衍生物。
  7. 根据权利要求3-6中任意一项所述的微球组合物，其特征在于，所述发光化合物是二甲基噻吩的衍生物，所述金属螯合物是铕螯合物。
  8. 根据权利要求1-7中任意一项所述的微球组合物，其特征在于，所述基质选自带、片、棒、管、孔、微滴定板、珠、粒子和微球；优选为珠和微球。
  9. 根据权利要求1-8中任意一项所述的微球组合物，其特征在于，所述基质是磁性或非磁性粒子。
  10. 根据权利要求1-9中任意一项所述的微球组合物，其特征在于，所述不同粒径的受体微球的基质的材质相同或不同。
  11. 根据权利要求1-10中任意一项所述的微球组合物，其特征在于，所述基质材料选自天然的、合成或改性的天然存在的聚合物，其选自琼脂糖、纤维素、硝化纤维素、醋酸纤维素、聚氯乙烯、聚苯乙烯、聚乙烯、聚丙烯、聚(4-甲基 丁烯)、聚丙烯酰胺、聚甲基丙烯酸酯、聚对苯二甲酸乙二醇酯、尼龙、聚丁酸乙烯或聚丙烯酸酯；优选地，所述基质为聚苯乙烯乳胶微球，进一步优选为羧基和/或醛基化聚苯乙烯乳胶微球。
  12. 根据权利要求1-11中任意一项所述的微球组合物，其特征在于，所述基质的表面直接连接有生物活性物质，所述生物活性物质能够与待测目标分子特异性结合。
  13. 根据权利要求1-11中任意一项所述的微球组合物，其特征在于，所述基质的表面包被包覆层，所述包覆层的表面连接有生物活性物质，所述生物活性物质能够与待测目标分子特异性结合。
  14. 根据权利要求13所述的微球组合物，其特征在于，所述包覆层中的包覆物选自多糖、高分子聚合物或生物大分子，优选为多糖。
  15. 根据权利要求14所述的微球组合物，其特征在于，所述基质的表面包被至少两个连续多糖层的涂层，其中第一多糖层与第二多糖层自发关联。
  16. 根据权利要求15所述的微球组合物，其特征在于，所述连续多糖层中的每一层自发地与前一多糖层中的每一层相关联。
  17. 根据权利要求14-16中任意一项所述的微球组合物，其特征在于，所述多糖具有侧基官能团，所述连续多糖层的所述官能团与所述前一多糖层的所述官能团所带电荷相反。
  18. 根据权利要求14-16中任意一项所述的微球组合物，其特征在于，所述多糖具有侧基官能团，并且所述多糖的所述连续层通过所述连续层的所述官能团与所述前一层的所述官能团之间的反应与所述前一多糖层共价连接。
  19. 根据权利要求18所述的微球组合物，其特征在于，所述连续多糖层的所述官能团在胺官能团和胺反应性官能团之间交替。
  20. 根据权利要求19所述的微球组合物，其特征在于，所述胺反应性官能团是醛基或羧基。
  21. 根据权利要求15-20中任意一项所述的微球组合物，其特征在于，所述第一多糖层自发地与所述载体相关联。
  22. 根据权利要求15-21中任意一项所述的微球组合物，其特征在于，所述涂层的最外一层多糖层具有至少一个侧基官能团。
  23. 根据权利要求15-22中任意一项所述的微球组合物，其特征在于，所述涂层的最外一层多糖层的侧基官能团选自醛基、羧基、巯基、氨基、羟基和马来胺基中的至少一种；优选选自醛基和/或羧基。
  24. 根据权利要求22或23所述的微球组合物，其特征在于，所述涂层的最外一层多糖层的侧基官能团直接地或间接地与生物活性物质连接。
  25. 根据权利要求14-24中任意一项所述的微球组合物，其特征在于，所述多糖选自含有三个或更多个未修饰或修饰的单糖单元的碳水化合物；优选选自葡聚糖、淀粉、糖原、菊粉、果聚糖、甘露聚糖、琼脂糖、半乳聚糖、羧基葡聚糖和氨基葡聚糖；更优选选自葡聚糖、淀粉、糖原和聚核糖。
  26. 根据权利要求1-25中任意一项所述的微球组合物，其特征在于是，所述不同粒径的受体微球的基质粒径相同。
  27. 根据权利要求1-25中任意一项所述的微球组合物，其特征在于是，所述不同粒径的受体微球的基质粒径不同。
  28. 根据权利要求1-27中任意一项所述的微球组合物，其特征在于，所述活性氧为单线态氧。
  29. 根据权利要求1-28中任意一项所述的微球组合物，其特征在于，所述微球组合物包括两种不同粒径的受体微球。
  30. 根据权利要求29所述的微球组合物，其特征在于，所述两种不同粒径的受体微球的粒径的差值不低于50nm；优选不低于100nm；更优选不低于150nm。
  31. 根据权利要求29或30所述的微球组合物，其特征在于，所述两种不同粒径的受体微球的粒径比选自1:(1.1-10)；优选选自1:(2-8)；更优选选自1:(3-6)。
  32. 根据权利要求1-31中任意一项所述的微球组合物，其特征在于，所述微球组合物的使用浓度为1ug/mL-1000ug/mL；优选为10ug/mL-500ug/mL，更优选为10ug/mL-250ug/mL。
  33. 根据权利要求1-28中任意一项所述的微球组合物，其特征在于，所述微球组合物包括至少三种不同粒径的受体微球。
  34. 一种受体试剂，其包括如权利要求1-33中任意一项所述的微球组合物。
  35. 一种化学发光检测试剂盒，其包括如权利要求1-33中任意一项所述的微球组合物或权利要求34所述的受体试剂。
  36. 一种化学发光分析方法，其包括利用如权利要求1-33中任意一项所述的微球组合物或权利要求34所述的受体试剂或权利要求35所述的试剂盒检测待测样本中待测目标分子是否存在和/或待测目标分子在待测样本中的浓度。
  37. 一种化学发光分析仪，其利用如权利要求1-33中任意一项所述的微球组合物或权利要求34所述的受体试剂或权利要求35所述的试剂盒和/或权利要求36所述的方法检测待测样本中待测目标分子是否存在和/或待测目标分子在待测样本中的浓度。
  38. 根据权利要求37所述的化学发光分析仪，其特征在于，所述化学发光分析仪至少包括如下部分：
    孵育模块，其用于为待测样本与微球组合物发生化学发光反应提供合适的温度环境，所述微球组合物包含至少两种不同粒径的受体微球；
    检测模块，其用于检测受体微球与活性氧反应产生的化学发光信号；
    处理器，其根据检测模块检测到的化学发光信号的情况判断待测样本中是否存在待测目标分子和/或待测目标分子在待测样本中的浓度。
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