CN116500020A - Chemiluminescence analysis method and application thereof - Google Patents
Chemiluminescence analysis method and application thereof Download PDFInfo
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- CN116500020A CN116500020A CN202310478945.3A CN202310478945A CN116500020A CN 116500020 A CN116500020 A CN 116500020A CN 202310478945 A CN202310478945 A CN 202310478945A CN 116500020 A CN116500020 A CN 116500020A
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Classifications
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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
-
- 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
Abstract
The invention relates to a chemiluminescent analysis method in the technical field of chemiluminescent analysis, which is used for analyzing and judging whether a sample to be detected contains a target molecule to be detected and/or the concentration of the target molecule to be detected by detecting the intensity of chemiluminescent signals generated by the reaction of at least two receptor microspheres with different particle diameters in the sample to be detected and active oxygen. The method of the invention has ultrahigh sensitivity and wide detection range. In addition, the reaction speed of the luminescent microspheres with small particle sizes is high, so that the detection time can be shortened and the reaction speed can be improved when the immunoassay method is adopted for detection.
Description
The application is a divisional application of Chinese patent application with the application number of 201910744763X, the application date of 2019, 8 and 13, and the invention name of 'a chemiluminescence analysis method and application'.
Technical Field
The invention belongs to the technical field of chemiluminescence analysis, and particularly relates to a chemiluminescence analysis method and application thereof.
Background
Chemiluminescent analysis is a method of detection using light waves emitted by chemiluminescent substances. Chemiluminescent substances are used as labels in nucleic acid detection and immunodetection. For example, a molecule of a specific binding pair may be bound to a luminescent substance by a variety of routes to form a luminescent microsphere composition. The microsphere composition can react with the analyte (the other molecule in the specific binding pair) in the sample, partition into a solid phase and a liquid phase, and the partition ratio is related to the amount of the analyte. The corresponding concentration of the detection object in the sample can be obtained by measuring the luminescence amount in the solid phase or the liquid phase.
With the progress of the detection industry, the requirement for a hypersensitive reagent is more and more, the sensitivity requirement is extremely high, the linear range requirement is extremely wide, and the detection condition is difficult to meet by the existing chemiluminescence analysis method.
Therefore, there is a need to develop a chemiluminescent assay that meets both sensitivity and linear range requirements.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a chemiluminescence analysis method which has ultrahigh sensitivity and wide detection range aiming at the defects of the prior art. In addition, the method can shorten the detection time.
Therefore, the invention provides a chemiluminescence analysis method, which is used for analyzing and judging whether a sample to be detected contains a target molecule to be detected and/or the concentration of the target molecule to be detected by detecting the intensity of a chemiluminescence signal generated by the reaction of at least two receptor microspheres with different particle diameters in the sample to be detected and active oxygen.
In some embodiments of the invention, the method analyzes and judges whether the sample to be tested contains the target molecule to be tested and/or the concentration of the target molecule to be tested by detecting the intensity of chemiluminescent signals generated by the reaction of two receptor microspheres with different particle sizes and active oxygen in the sample to be tested.
In some embodiments of the invention, the difference in particle size of the two different particle size receptor microspheres is no less than 100nm; preferably not less than 150nm; more preferably not less than 200nm.
In other embodiments of the invention, the ratio of particle sizes of the two different particle sizes of the receptor microspheres is selected from 1 (1.1-10); preferably selected from 1 (2-8); more preferably from 1 (3-6).
In some embodiments of the invention, the receptor microsphere is used at a concentration of 1ug/mL to 1000ug/mL; preferably 10ug/mL-500ug/mL; more preferably from 10ug/mL to 250ug/mL.
In some embodiments of the invention, the sample to be tested further comprises donor microspheres, which are capable of generating active oxygen in an excited state.
In other embodiments of the invention, the method is a homogeneous chemiluminescent assay.
In some preferred embodiments of the invention, the method comprises the steps of:
s1, mixing a sample to be tested with a reagent a containing at least two acceptor microspheres with different particle sizes, and then mixing the sample to be tested with a reagent b containing donor microspheres to obtain a sample to be tested;
s2, contacting the sample to be detected obtained in the step S1 by using energy or active chemical substances, and exciting a donor to generate active oxygen;
S3, analyzing and judging whether the sample to be detected contains the target molecule to be detected and/or the concentration of the target molecule to be detected by detecting the intensity of a chemiluminescent signal generated by the reaction of at least two receptor microspheres with different particle diameters in the sample to be detected and active oxygen.
In some embodiments of the invention, the sample to be tested is diluted with a diluent and then reacted.
In some embodiments of the invention, the method analyzes and judges whether the sample to be tested contains the target molecule to be tested and/or the concentration of the target molecule to be tested by detecting the intensity of chemiluminescent signals generated by the reaction of the three receptor microspheres with different particle sizes and active oxygen in the sample to be tested.
In some embodiments of the invention, the chemiluminescent signal has a detection wavelength of 520 to 620nm.
In other embodiments of the present invention, the laser irradiation is performed using 600 to 700nm of red excitation light.
In some embodiments of the invention, the receptor microsphere comprises a luminescent composition and a matrix, the luminescent composition being filled in the matrix and/or coated on the surface of the matrix.
In some preferred embodiments of the invention, the luminescent composition is capable of reacting with reactive oxygen species to produce a detectable chemiluminescent signal comprising a chemiluminescent compound and a metal chelate.
In some embodiments of the invention, the chemiluminescent compound is selected from the group consisting of olefinic compounds, preferably selected from the group consisting of dimethylthiophene, bis-butanedione compounds, dioxins, enol ethers, enamines, 9-alkylene xanthenes, 9-alkylene-N-9, 10-dihydroacridines, aryletherenes, arylimidazoles, and lucigenin, and their derivatives, more preferably selected from the group consisting of dimethylthiophene and its derivatives.
In other embodiments of the invention, the metal of the metal chelate is a rare earth metal or a group VIII metal, preferably selected from europium, terbium, dysprosium, samarium, osmium and ruthenium, more preferably selected from europium.
In some preferred embodiments of the invention, the metal chelate comprises a chelating agent selected from the group consisting of: MTTA, NHA, BHHT, BHHCT, DPP, TTA, NPPTA, NTA, TOPO, TPPO, BFTA 2, 2-dimethyl-4-perfluorobutanoyl-3-butanone (fod), 2' -bipyridine (bpy), bipyridylcarboxylic acid, azacrown ethers, aza-cryptands and trioctylphosphine oxide, and derivatives thereof.
In some embodiments of the invention, the luminescent compound is a derivative of dimethylthiophene and the metal chelate is a europium chelate.
In some embodiments of the invention, the substrate is selected from the group consisting of a tape, a sheet, a rod, a tube, a well, a microtiter plate, a bead, a particle, and a microsphere; preferably beads and microspheres.
In other embodiments of the invention, the matrix is a magnetic or non-magnetic particle.
In some embodiments of the invention, the matrix material of the receptor microspheres of different particle sizes is the same or different.
In some embodiments of the invention, the matrix material is selected from natural, synthetic or modified naturally occurring polymers, including but not limited to: agarose, cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polystyrene, polyethylene, polypropylene, poly (4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate, nylon, polyethylene butyrate or polyacrylate.
In some preferred embodiments of the invention, the matrix is an aldehyde-based latex microsphere; preferably, the polystyrene latex microspheres are aldehyde-modified.
In some embodiments of the invention, the surface of the substrate has directly attached thereto a biologically active substance capable of specifically binding to the target molecule to be detected.
In other embodiments of the invention, the surface of the substrate is coated with a coating, and the surface of the coating is connected with a bioactive substance, wherein the bioactive substance can be specifically combined with a target molecule to be detected.
In some embodiments of the invention, the coating in the coating layer is selected from polysaccharides, high molecular polymers or biological macromolecules, preferably polysaccharides.
In other embodiments of the invention, the surface of the substrate is coated with a coating of at least two successive polysaccharide layers, wherein the first polysaccharide layer is spontaneously associated with the second polysaccharide layer.
In some embodiments of the invention, each of the continuous polysaccharide layers is spontaneously associated with each of the previous polysaccharide layers.
In other embodiments of the invention, the polysaccharide has pendant functional groups, the functional groups of the continuous polysaccharide layer being oppositely charged to the functional groups of the preceding polysaccharide layer.
In some embodiments of the invention, the polysaccharide has pendant functional groups and the continuous layer of the polysaccharide is covalently linked to the previous polysaccharide layer by a reaction between the functional groups of the continuous layer and the functional groups of the previous layer.
In other embodiments of the invention, the functional groups of the continuous polysaccharide layer alternate between amine functional groups and amine reactive functional groups.
In some embodiments of the invention, the amine-reactive functional group is an aldehyde group or a carboxyl group.
In other embodiments of the invention, the first polysaccharide layer is spontaneously associated with the carrier.
In some embodiments of the invention, the outermost polysaccharide layer of the coating has at least one pendant functional group.
In other embodiments of the present invention, the pendant functional groups of the outermost polysaccharide layer of the coating are selected from at least one of aldehyde groups, carboxyl groups, mercapto groups, amino groups, hydroxyl groups, and maleamine groups; preferably selected from aldehyde groups and/or carboxyl groups.
In some embodiments of the invention, the pendant functional groups of the outermost polysaccharide layer of the coating are directly or indirectly attached to the biologically active substance.
In other embodiments of the invention, the polysaccharide is selected from the group consisting of carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from the group consisting of dextran, starch, glycogen, inulin, levan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably selected from the group consisting of dextran, starch, glycogen and polyribose.
In some embodiments of the invention, the matrix particle size of the different particle size receptor microspheres is the same.
In other embodiments of the invention, the different particle sizes of the matrix of the receptor microspheres differ.
In some embodiments of the invention, the active oxygen is singlet oxygen.
In a second aspect the present invention provides a chemiluminescent analyzer for detecting the presence and/or concentration of a test target molecule in a test sample by a method according to the first aspect of the present invention.
In some embodiments of the invention, the chemiluminescent analyzer comprises at least the following:
the incubation module is used for providing a proper temperature environment for the chemiluminescent reaction of the sample to be tested and at least two receptor microspheres with different particle diameters after being mixed;
the detection module is used for detecting a chemiluminescent signal generated by the reaction of the receptor microsphere and the active oxygen;
and the processor is used for judging whether the target molecule to be detected exists in the sample to be detected or not and/or the concentration of the target molecule to be detected in the sample to be detected according to the condition of the chemiluminescent signal detected by the detection module.
In a third aspect the invention provides the use of a method according to the first aspect of the invention and/or a chemiluminescent analyzer according to the second aspect of the invention for detecting markers of myocardial damage including cTnI and/or markers of inflammation including procalcitonin.
The beneficial effects of the invention are as follows: according to the chemiluminescence analysis method, at least two receptor microspheres (small-particle-size receptor microspheres and large-particle-size receptor microspheres) are added into a sample to be detected, and the small-particle-size receptor microspheres can widen the detection range and the large-particle-size receptor microspheres can improve the detection sensitivity, so that the detection performance of the method is greatly improved compared with the prior art, and the method has ultrahigh sensitivity and wide detection range. In addition, the reaction speed of the small-particle-size receptor microspheres is high, so that the detection time can be shortened and the reaction speed can be improved when the method is used for detection.
Drawings
The invention will be further described with reference to the accompanying drawings.
FIG. 1 is a graph showing the Gaussian distribution of the aldehyde-based polystyrene latex microspheres prepared in example 3.
FIG. 2 is a graph showing the Gaussian distribution of the aldehyde-based polystyrene latex microspheres impregnated with the luminescent composition prepared in example 3.
FIG. 3 is a Gaussian distribution diagram of dextran-coated aldehyde-based polystyrene latex microspheres embedded with a luminescent composition prepared in example 3.
FIG. 4 is a Gaussian distribution diagram of the receptor microsphere having an average particle diameter of about 250nm prepared in example 3.
FIG. 5 is a Gaussian distribution diagram of the receptor microsphere with a particle diameter of about 110nm prepared in example 3.
FIG. 6 is a Nicomp profile of the receptor microsphere having a particle diameter of about 110nm prepared in example 3.
FIG. 7 is a Gaussian distribution diagram of the receptor microsphere having a particle diameter of about 350nm prepared in example 3.
FIG. 8 is a Nicomp profile of the receptor microsphere having a particle diameter of about 350nm prepared in example 3.
FIG. 9 is a Gaussian distribution of the particle size distribution of the mixed receptor microspheres of example 4.
FIG. 10 is a Nicomp profile of the particle size distribution of the mixed receptor microspheres of example 4.
Detailed Description
In order that the invention may be readily understood, the invention will be described in detail. Before the present 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, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless defined otherwise, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. 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.
I terminology
The term "active oxygen" as used herein refers to a substance which is composed of oxygen in the body or in the natural environment, contains oxygen and is active in nature, and is mainly an excited oxygen molecule, including an electron reduction product of oxygen, superoxide anion (O 2 Hydrogen peroxide (H), a two-electron reduction product 2 O 2 ) Hydroxyl radical (OH) of three-electron reduction product, nitric oxide and singlet oxygen (1O) 2 ) Etc.
In the present invention, the term "receptor microsphere" refers to a nanoparticle capable of reacting with active oxygen to produce a detectable chemiluminescent signal, which may also be referred to as an oxygen-receiving microsphere or a luminescent microsphere. Preferably, the acceptor microsphere may be a polymer particle filled with a light emitting composition including a chemiluminescent compound capable of reacting with active oxygen formed by filling a functional group in a matrix. In some embodiments of the invention, the chemiluminescent compound undergoes a chemical reaction with reactive oxygen species to form an unstable metastable intermediate that may decompose with or subsequent to luminescence. Typical examples of such substances include, but are not limited to: enol ethers, enamines, 9-alkylidene xanthan gums, 9-alkylidene-N-alkyl acridines, aryl ether olefins, bisoxyethylene, dimethylthiophene, aryl imidazoles or gloss concentrates.
In the present invention, the "chemiluminescent compound", a compound known as a label, may undergo a chemical reaction to cause luminescence, such as by being converted to another compound formed in an electronically excited state. The excited state may be a singlet state or a triplet excited state. The excited state may relax to the ground state to emit light directly or by transferring excitation energy to an emission energy acceptor, thereby restoring itself to the ground state. In this process, the energy acceptor microsphere will be transitioned to an excited state to emit light.
The chemiluminescent compound may be bound to a specific binding partner member that is capable of directly or indirectly binding to a test molecule or test component whose concentration is affected by the presence of the test molecule. By "capable of binding directly or indirectly" is meant that the specified entity is capable of specifically binding to the entity (directly), or that the specified entity is capable of specifically binding to a specific binding pair member, or a complex having two or more specific binding partners capable of binding to other entities (indirectly).
The "specific binding pair member" of the 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
In the present invention, the active oxygen may be provided by "donor microspheres", which are nano-microspheres capable of generating active oxygen in an excited state. Preferably, the donor microsphere may be polymer particles filled with a photosensitive compound formed by coating a functional group on a substrate, and the photosensitive microsphere may be also called an oxygen supply microsphere or a photosensitive microsphere when the donor microsphere is capable of generating singlet oxygen under light excitation. The surface of the donor microsphere can be provided with hydrophilic aldehyde dextran, and the inside of the donor microsphere is filled with a photosensitizer. The photosensitizers may be photosensitizers known in the art, preferably compounds that are relatively light stable and do not effectively react with singlet oxygen, non-limiting examples of which include methylene blue, rose bengal, porphyrin, and phthalocyanine compounds, and derivatives of these compounds having 1-50 atom substituents that are used to render these compounds more lipophilic or hydrophilic, and/or as linking groups to specific binding pair members. The donor microspheres may also be filled with other sensitizers, non-limiting examples of which are certain compounds that catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Examples of other donors include: 1, 4-dicarboxyethyl-1, 4-naphthalene endoperoxide, 9, 10-diphenylanthracene-9, 10-endoperoxide, and the like, and heating these compounds or direct absorption of light by these compounds may release active oxygen, such as singlet oxygen.
The "matrix" according to the invention, which may be of any size, may be organic or inorganic, may be expandable or non-expandable, may be porous or non-porous, has any density, but preferably has a density close to that of water, is preferably floatable in water, and is composed of transparent, partially transparent or opaque material. The matrix may or may not be charged and when charged is preferably negatively charged. The matrix may be a solid (e.g., polymers, metals, glass, organic and inorganic substances such as minerals, salts, and diatoms), oil droplets (e.g., hydrocarbons, fluorocarbons, siliceous fluids), vesicles (e.g., synthetic such as phospholipids, or natural such as cells, and organelles). The matrix may be latex particles or other particles containing organic or inorganic polymers, lipid bilayers such as liposomes, phospholipid vesicles, oil droplets, silica particles, metal sols, cells and microcrystalline dyes. The matrix is generally multifunctional or capable of binding to a donor or acceptor by specific or non-specific covalent or non-covalent interactions. Many functional groups are available or incorporated. Typical functional groups include carboxylic acid, acetaldehyde, amino, cyano, vinyl, hydroxyl, mercapto, and the like. One non-limiting example of a matrix suitable for use in the present invention is an aldehyde-based polystyrene latex microsphere.
The photosensitizer and/or chemiluminescent compound may be selected to be dissolved in, or non-covalently bound to, the surface of the particle. In this case, the compounds are preferably hydrophobic to reduce their ability to dissociate from the particles, thereby allowing both compounds to bind to the same particles.
The term "particle size" as used herein refers to the average particle size of the luminescent microspheres as determined by conventional particle sizers. The "receptor microsphere" comprises at least a matrix, a luminescent composition and bioactive molecules, and preferably also comprises a coating layer; the luminescent composition may be filled in the matrix and/or coated on the surface of the matrix. When the receptor microsphere does not include a coating, the bioactive substance is directly attached to the surface of the matrix. When the receptor microsphere comprises a coating layer, the coating layer is coated on the surface of a substrate, and the outermost layer of the coating layer is connected with a bioactive substance.
It is noted that the term "average particle size of the receptor microsphere" as used herein refers to the average particle size of the receptor microsphere after being attached and/or coated with the corresponding substance. The particle sizes of the matrixes in the receptor microspheres with different particle sizes can be the same or different, so long as the particle sizes of the receptor microspheres finally formed are different.
The term "test sample" as used herein refers to a mixture that is tested for the presence or suspected presence of a test target molecule. Samples to be tested that may be used in the present disclosure include body fluids such as blood (which may be anticoagulated blood as is commonly found in collected blood samples), plasma, serum, urine, semen, saliva, cell cultures, tissue extracts, and the like. Other types of samples to be tested include solvents, seawater, industrial water samples, food samples, environmental samples such as soil or water, plant material, eukaryotic cells, bacteria, plasmids, viruses, fungi, and cells from prokaryotes. The sample to be measured can be diluted with a diluent as required before use. For example, in order to avoid the HOOK effect, the sample to be tested may be diluted with a diluent before on-machine testing and then tested on a testing instrument.
The term "target molecule to be detected" as used herein refers to a substance in a sample to be detected during detection. One or more substances having a specific binding affinity for the target molecule to be detected may be used to detect the target molecule. The target molecule to be tested may be a protein, peptide, antibody or hapten which can be conjugated to an antibody. The target molecule to be detected may be a nucleic acid or oligonucleotide that binds to a complementary nucleic acid or oligonucleotide. The target molecule to be tested may be any other substance that can form a specific binding pair member. Examples of other typical target molecules to be measured include: drugs such as steroids, hormones, proteins, glycoproteins, mucins, nucleoproteins, phosphoproteins, drugs of abuse, vitamins, antibacterial agents, antifungal agents, antiviral agents, purines, antitumor agents, amphetamines, heteronitrogen compounds, nucleic acids and prostaglandins, and metabolites of any of these drugs; pesticides and metabolites thereof; and a recipient. Analytes also include cells, viruses, bacteria, and fungi.
The term "sample to be measured" refers to a mixed liquid to be measured containing multiple components such as the sample to be measured, the acceptor microsphere, the donor microsphere and the like before on-machine detection and analysis.
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. In any desired case, the antibody may be further conjugated to other moieties, such as specific binding pair members, e.g., biotin or streptavidin (one of the biotin-streptavidin specific binding pair members), and the like.
The term "antigen" as used herein refers to a substance that stimulates the body to produce an immune response and binds to antibodies and sensitized lymphocytes, which are the products of the immune response, in vivo and in vitro, resulting in an immune effect.
The term "binding" as used herein refers to the 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 recognition and selective binding reaction between two substances, and from a steric perspective, corresponds to the conformational correspondence between the corresponding reactants. Under the technical ideas disclosed in the present invention, the detection method of the specific binding reaction includes, but is not limited to: a diabody sandwich method, a competition method, a neutralization competition method, an indirect method or a capture method.
The term "homogeneous" as used herein is defined as "homogeneous" and refers to the fact that the detection is accomplished without the need to separate the bound antigen-antibody complex from the remaining free antigen or antibody.
The term "C.V value of the particle size distribution coefficient of variation" as used herein refers to the coefficient of variation of the particle size in the Gaussian distribution in the result of the detection by the nanoparticle analyzer. The calculation formula of the variation coefficient is as follows: C.V values = (standard deviation SD/Mean) x 100%.
The term "Nicomp distribution" as used herein refers to an algorithmic distribution in the united states PSS nanoparticle sizer Nicomp. The Nicomp multimodal algorithm has unique advantages over the Gaussian unimodal algorithm for the analysis of multicomponent, non-uniform particle size distribution liquid dispersions and stability analysis of colloidal systems.
Embodiment II
The present invention will be described in more detail below.
The first aspect of the present invention relates to a chemiluminescent analysis method for determining whether a sample to be tested contains a target molecule to be tested and/or the concentration of the target molecule to be tested by detecting the intensity of chemiluminescent signals generated by the reaction of at least two acceptor microspheres with different particle diameters in the sample to be tested with active oxygen.
In some embodiments of the invention, the method analyzes and judges whether the sample to be tested contains the target molecule to be tested and/or the concentration of the target molecule to be tested by detecting the intensity of chemiluminescent signals generated by the reaction of two receptor microspheres with different particle sizes and active oxygen in the sample to be tested.
In some embodiments of the invention, the difference in particle size of the two different particle size receptor microspheres is no less than 100nm; preferably not less than 150nm; more preferably not less than 200nm. In some embodiments of the invention, the difference in particle size of the two different particle size receptor microspheres is no less than 100nm, 130nm, 150nm, 170nm, 190nm, 200nm, 220nm, 240nm, or 250nm.
In other embodiments of the invention, the ratio of particle sizes of the two different particle sizes of the receptor microspheres is selected from 1 (1.1-10); preferably selected from 1 (2-8); more preferably from 1 (3-6). In some embodiments of the invention, the particle size ratio of the two different particle size receptor microspheres is selected from 1:1.5, 1:2, 1:2.7, 1:3, 1:3.2, 1:3.75, 1:4, 1:5, or 1:6.
In some preferred embodiments of the invention, one of the receptor microspheres has a particle size selected from 50nm to 300nm and the other receptor microsphere has a particle size selected from 200nm to 400nm. For example, one of the receptor microspheres has a particle size selected from 50nm, 80nm, 110nm, 140nm, 170nm, 200nm or 300nm, and the other receptor microsphere has a particle size selected from 200nm, 250nm, 300nm, 350nm or 400nm.
In a further preferred embodiment of the present invention, wherein the particle size of one of the receptor microspheres is selected from 50nm to 200nm and the particle size of the other receptor microsphere is selected from 200nm to 350nm.
In a still further embodiment of the present invention, wherein the particle size of one of the receptor microspheres is selected from 80nm to 150nm and the particle size of the other receptor microsphere is selected from 220nm to 350nm. In the present invention, the size of the receptor microsphere particle should be such that a uniform and stable latex solution is produced, and the particle size of the receptor microsphere that can generally meet this requirement should be in the nanometer range. Therefore, the upper limit of the particle size of the large-size receptor microspheres is preferably about 300nm, which enables the formation of a stable latex solution. Meanwhile, the coating and cleaning of the receptor microspheres can be performed under the existing technical conditions so as to meet the production of reagents.
The method controls the particle size of the receptor microspheres in the used microsphere composition, so as to control the amount of bioactive substances (such as antibodies/antigens) on the surface of each receptor microsphere (the specific surface area of the microspheres with small particle size is large, the amount of the surface reporter molecules of the microspheres with large particle size is large, the specific surface area of the microspheres with large particle size is small, and the amount of the surface reporter molecules of the microspheres with large particle size is small), thereby improving the detection sensitivity and widening the detection range. In addition, the small-particle-size acceptor microsphere has smaller diameter, so that the activation efficiency of single-wire oxygen generated by the donor microsphere is improved, and the luminous efficiency of the acceptor microsphere can also be improved.
In some embodiments of the invention, the receptor microsphere is used at a concentration of 1ug/mL to 1000ug/mL; preferably 10ug/mL-500ug/mL, more preferably 50ug/mL-250ug/mL. In the invention, the use concentration of the receptor microsphere is determined by the concentration of different target molecules to be detected in blood and the characteristics of the target molecules to be detected.
In some embodiments of the invention, the sample to be tested further comprises donor microspheres, which are capable of generating active oxygen in an excited state.
In other embodiments of the invention, the method is a homogeneous chemiluminescent assay.
In some preferred embodiments of the invention, the method comprises the steps of:
s1, mixing a sample to be tested with a reagent a containing at least two acceptor microspheres with different particle sizes, and then mixing the sample to be tested with a reagent b containing donor microspheres to obtain a sample to be tested;
s2, contacting the sample to be detected obtained in the step S1 by using energy or active chemical substances, and exciting a donor to generate active oxygen;
s3, analyzing and judging whether the sample to be detected contains the target molecule to be detected and/or the concentration of the target molecule to be detected by detecting the intensity of a chemiluminescent signal generated by the reaction of at least two receptor microspheres with different particle diameters in the sample to be detected and active oxygen.
In some embodiments of the invention, the receptor microsphere may have a particle size distribution coefficient of variation C.V in agent a of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35% or 40%, etc.
It is noted that the C.V value of the particle size distribution variation coefficient of the receptor microsphere according to the present invention refers to C.V value of the particle size distribution variation coefficient of the receptor microsphere after being coated with the desired substance.
In some preferred embodiments of the present invention, in step S1, the sample to be tested is mixed with the first reagent before being mixed with the reagent a comprising at least two receptor microspheres of different particle sizes. It should be noted that, the first reagent according to the present invention, which is not specific to a certain type of reagent, is added to ensure the smooth or optimal performance of some detection methods based on specific reactions, and includes, but is not limited to: biotinylated antigens or antibodies.
In some preferred embodiments of the invention, the detection method of the specific reaction is a sandwich method. For example, immune complex patterns are: donor microsphere-streptavidin-biotin-antibody 1-antigen-antibody 2-acceptor microsphere, where the first reagent is a biotinylated antigen or antibody; the donor microsphere is coupled with the donor microsphere of streptavidin, and the acceptor microsphere is coupled with the antigen or antibody.
In order to further improve the accuracy of the final detection result and the stability of the sample to be tested, in some preferred embodiments of the present invention, in step S1, the sample to be tested is diluted with a diluent, then mixed with the first reagent, and then mixed with the donor microsphere reagent.
In some embodiments of the invention, the method analyzes and judges whether the sample to be tested contains the target molecule to be tested and/or the concentration of the target molecule to be tested by detecting the intensity of chemiluminescent signals generated by the reaction of the three receptor microspheres with different particle sizes and active oxygen in the sample to be tested.
In some embodiments of the invention, the chemiluminescent signal has a detection wavelength of 520 to 620nm, preferably 610 to 620nm, more preferably 615nm.
In other embodiments of the present invention, the laser irradiation is performed using 600 to 700nm of red excitation light; preferably, the red excitation light with the wavelength of 640-680nm is used for laser irradiation; more preferably, the laser irradiation is performed with 660nm of red excitation light.
In some embodiments of the invention, the receptor microsphere comprises a luminescent composition and a matrix, the luminescent composition being filled in the matrix and/or coated on the surface of the matrix.
In some preferred embodiments of the invention, the luminescent composition is capable of reacting with reactive oxygen species to produce a detectable chemiluminescent signal comprising a chemiluminescent compound and a metal chelate.
In some embodiments of the invention, the chemiluminescent compound is selected from the group consisting of olefin compounds, which are compounds that are capable of reacting with reactive oxygen species (e.g., singlet oxygen). Examples of suitable electron-rich alkylene compounds are listed in U.S. patent No. 5,709,994, the relevant content of which is incorporated herein by reference. In some preferred embodiments of the present invention, the olefinic compound is selected from the group consisting of dimethylthiophene, a bisbutanedione compound, a dioxine, an enol ether, an enamine, 9-alkylene xanthane, 9-alkylene-N-9, 10-dihydroacridine, an aryletherene, an arylimidazole, and a gloss essence, and their derivatives, more preferably from the group consisting of dimethylthiophene and its derivatives.
In addition to olefin compounds, the chemiluminescent compounds also include complexes of a metal and one or more chelating agents (metal chelates). In some embodiments of the invention, the metal of the metal chelate is a rare earth metal or a group VIII metal, preferably selected from europium, terbium, dysprosium, samarium, osmium and ruthenium, more preferably selected from europium.
In some preferred embodiments of the present invention, the metal chelate comprises a chelating agent selected from the group consisting of: 4' - (10-methyl-9-anthracenyl) -2,2':6'2 "-Bifide-6, 6" -dimethylamine ] tetraacetic acid (MTTA), 2- (1 ',1',2',2',3',3' -heptafluoro-4 ',6' -hexanedione-6 ' -yl) -Naphthalene (NHA), 4' -bis (2 ",3",3 "-heptafluoro-4", 6 "-hexanedione-6" -yl) -o-terphenyl (BHHT), 4' -bis (1 ",1",1",2",2",3",3 "-heptafluoro-4", 6' -hexanedione-6 ' -yl) -chlorosulfonyl-o-terphenyl (BHHCT), 4, 7-biphenyl-1, 10-phenanthroline (DPP), 1-trifluoroacetone (TTA), 3-naphthaloyl-1, 1-trifluoroacetone (NPPTA), naphthalene Trifluorobutanedione (NTA), trioctylphosphine oxide (TOPO) triphenylphosphine oxide (TPPO), 3-benzoyl-1, 1-trifluoroacetone (BFTA), 2-dimethyl-4-perfluorobutyryl-3-butanone (fod), 2' -bipyridine (bpy), bipyridylcarboxylic acid, azacrown ethers, aza-cryptands and trioctylphosphine oxide, and derivatives thereof.
In some embodiments of the invention, the luminescent compound is a derivative of dimethylthiophene and the metal chelate is a europium chelate.
In some embodiments of the invention, the substrate is selected from the group consisting of a tape, a sheet, a rod, a tube, a well, a microtiter plate, a bead, a particle, and a microsphere; preferably beads and microspheres.
In other embodiments of the invention, the matrix is a magnetic or non-magnetic particle.
In some embodiments of the invention, the matrix material of the receptor microspheres of different particle sizes is the same or different.
In some embodiments of the invention, the matrix material is selected from natural, synthetic or modified naturally occurring polymers, including but not limited to: agarose, cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polystyrene, polyethylene, polypropylene, poly (4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate, nylon, polyethylene butyrate or polyacrylate.
In some preferred embodiments of the invention, the matrix is an aldehyde-based latex microsphere; preferably, the polystyrene latex microspheres are aldehyde-modified.
In some embodiments of the invention, the surface of the substrate has directly attached thereto a biologically active substance capable of specifically binding to the target molecule to be detected.
In other embodiments of the invention, the surface of the substrate is coated with a coating layer, and the surface of the coating layer is connected with a bioactive substance, wherein the bioactive substance can be specifically combined with a target molecule to be detected; by way of example and not limitation, the ions of the bioactive substance-test target molecule binding partner include antigen-antibodies, hormone-hormone receptors, nucleic acid duplex, igG-protein a, polynucleotide pairs such as DNA-DNA, DNA-RNA, and the like.
In some preferred embodiments of the invention, the biologically active substance is an antigen and/or an antibody; the antigen refers to a substance having immunogenicity; the antibody refers to an immunoglobulin produced by an organism and capable of recognizing a specific foreign object.
In some embodiments of the invention, the coating in the coating layer is selected from polysaccharides, high molecular polymers or biological macromolecules, preferably polysaccharides.
In other embodiments of the invention, the surface of the substrate is coated with a coating of at least two successive polysaccharide layers, wherein the first polysaccharide layer is spontaneously associated with the second polysaccharide layer.
In some embodiments of the invention, each of the continuous polysaccharide layers is spontaneously associated with each of the previous polysaccharide layers.
In other embodiments of the invention, the polysaccharide has pendant functional groups, the functional groups of the continuous polysaccharide layer being oppositely charged to the functional groups of the preceding polysaccharide layer.
In some embodiments of the invention, the polysaccharide has pendant functional groups and the continuous layer of the polysaccharide is covalently linked to the previous polysaccharide layer by a reaction between the functional groups of the continuous layer and the functional groups of the previous layer.
In other embodiments of the invention, the functional groups of the continuous polysaccharide layer alternate between amine functional groups and amine reactive functional groups.
In some embodiments of the invention, the amine-reactive functional group is an aldehyde group or a carboxyl group.
In other embodiments of the invention, the first polysaccharide layer is spontaneously associated with the carrier.
In some embodiments of the invention, the outermost polysaccharide layer of the coating has at least one pendant functional group.
In other embodiments of the present invention, the pendant functional groups of the outermost polysaccharide layer of the coating are selected from at least one of aldehyde groups, carboxyl groups, mercapto groups, amino groups, hydroxyl groups, and maleamine groups; preferably selected from aldehyde groups and/or carboxyl groups.
In some embodiments of the invention, the pendant functional groups of the outermost polysaccharide layer of the coating are directly or indirectly attached to the biologically active substance.
In other embodiments of the invention, the polysaccharide is selected from the group consisting of carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from the group consisting of dextran, starch, glycogen, inulin, levan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably selected from the group consisting of dextran, starch, glycogen and polyribose.
In some embodiments of the invention, the matrix particle size of the different particle size receptor microspheres is the same.
In other embodiments of the invention, the different particle sizes of the matrix of the receptor microspheres differ.
In some preferred embodiments of the invention, the active oxygen is singlet oxygen.
In the present invention, the composition and chemical structure of the receptor microspheres with different particle diameters may be the same or different. For example, the luminescent compositions and/or matrices of the receptor microspheres of different particle sizes may be the same or may be different, provided that they are capable of reacting with reactive oxygen species to produce a detectable chemiluminescent signal.
A second aspect of the invention relates to a chemiluminescent analyzer for detecting the presence and/or concentration of a test target molecule in a test sample by means of a method according to the first aspect of the invention.
In some embodiments of the invention, the chemiluminescent analyzer comprises at least the following:
the incubation module is used for providing a proper temperature environment for the chemiluminescent reaction of the sample to be tested and at least two receptor microspheres with different particle diameters after being mixed;
the detection module is used for detecting a chemiluminescent signal generated by the reaction of the receptor microsphere and the active oxygen;
And the processor is used for judging whether the target molecule to be detected exists in the sample to be detected or not and/or the concentration of the target molecule to be detected in the sample to be detected according to the condition of the chemiluminescent signal detected by the detection module.
A third aspect of the invention relates to the use of a method according to the first aspect of the invention and/or a chemiluminescent analyzer according to the second aspect of the invention for detecting markers of myocardial damage including cTnI and/or markers of inflammation including procalcitonin.
III. Detailed description of the invention
In order that the invention may be more readily understood, the invention will be further described in detail with reference to the following examples, which are given by way of illustration only and are not limiting in scope of application. The starting materials or components used in the present invention may be prepared by commercial or conventional methods unless specifically indicated.
Example 1: preparation of receptor microspheres with different particle sizes
(1) Preparation and characterization process of aldehyde polystyrene latex microsphere
1. A100 ml three-necked flask was prepared, 40mmol of styrene, 5mmol of acrolein and 10ml of water were added thereto, and after stirring for 10 minutes, N was introduced 2 30min。
2. 0.11g of ammonium persulfate and 0.2g of sodium chloride were weighed and dissolved in 40ml of water to prepare an aqueous solution. Adding the aqueous solution into the reaction system of the step 1, and continuing to introduce N 2 30min。
3. The reaction system was warmed to 70℃and reacted for 15 hours.
4. The emulsion after completion of the reaction was cooled to room temperature and filtered through a suitable filter cloth. The obtained emulsion is washed by centrifugal sedimentation with deionized water until the conductivity of the supernatant fluid at the beginning of centrifugation is close to that of the deionized water, and then diluted with water, and is stored in the form of emulsion.
5. The latex microsphere particle size was 190.4nm, cv=5.1% as measured by a nanoparticle sizer; the aldehyde group content of the latex microsphere is 280nmol/mg measured by a conductivity titration method.
(2) Coating process and characterization process of luminescent composition
1. A25 ml round-bottomed flask was prepared and charged with 0.1g of a dimethylthiophene derivative and 0.1g of europium (III) complex (MTTA-EU) 3+ ) 10ml of 95% ethanol was magnetically stirred and heated to 70℃in a water bath to obtain a complex solution.
2. A100 ml three-necked flask was prepared, 10ml of 95% ethanol, 10ml of water and 10ml of aldehyde-based polystyrene latex microspheres with a concentration of 10% and a particle size of 140nm were added, and the mixture was magnetically stirred and heated to 70℃in a water bath.
3. Slowly dripping the complex solution in the step 1 into the three-neck flask in the step 2, stopping stirring after reacting for 2 hours at 70 ℃, and naturally cooling.
4. The emulsion was centrifuged for 1 hour, 30000G, after which the supernatant was discarded and resuspended in 50% ethanol. After repeating the centrifugation washing three times, the mixture was resuspended in 50mM CB buffer at pH=10 to a final concentration of 20mg/ml.
(3) Coupling procedure of antibodies
1. PCT antibody 1 was dialyzed to 50mM CB buffer at ph=10 to give a concentration of 1mg/ml.
2. Adding 0.5ml of receptor microspheres with different particle sizes and aldehyde groups into a 2ml centrifuge tube, adding 0.5ml of the paired antibody I obtained in the step 1, uniformly mixing, and adding 100 μl of 10mg/ml NaBH 4 The solution (50 mM CB buffer) was reacted at 2-8℃for 4 hours.
3. After completion of the reaction, 0.5ml of 100mg/ml BSA solution (50 mM CB buffer) was added, and the reaction was carried out at 2-8℃for 2 hours.
4. After the reaction, the mixture was centrifuged for 45min at 30000G, the supernatant was discarded after centrifugation, and resuspended in 50mM MES buffer. The centrifugation washing was repeated four times and diluted to a final concentration of 100. Mu.g/ml to obtain a receptor microsphere coupled to PCT antibody 1.
5. The particle size of the receptor microsphere was measured by a nanoparticle sizer to be 210.4nm, cv=5.1%.
The receptor microspheres with aldehyde groups and the particle diameters of 50nm, 80nm, 110nm, 170nm, 250nm, 300nm, 350nm and 400nm are prepared by the same method.
Example 2: chemiluminescent assay method sensitivity and detection upper limit determination using receptor microspheres of different particle sizes
The sensitivity point is defined as when the signal at concentration 2C0 is higher than the signal at double concentration C0, i.e., RLU (2C 0) >2RLU (C0), then the corresponding detection reagent sensitivity is C0. The upper limit point of detection is defined as the corresponding concentration of the detection signal with the concentration of 1000ng/ml calculated by substituting the curve of the concentration and the signal.
(1) The cTnI antigen was diluted to a series of concentrations of 5pg/ml, 10pg/ml, 20pg/ml, 30pg/ml, 40pg/ml, 50pg/ml, 100pg/ml, 1000pg/ml, 5000pg/ml, 10000pg/ml, 50000pg/ml, 1000ng/ml, and the receptor microspheres coated with cTnI mab 1 having different particle diameters (50 nm, 80nm, 110nm, 140nm, 170nm, 200nm, 250nm, 300nm, 350nm, 400 nm) were prepared by the same method as in example 1, respectively diluted to 100ug/ml, and then the above concentration series cTnI antigen was detected with the same biotin-labeled cTnI mab 2 (diluted to 2 ug/ml) and universal solution (donor microsphere solution), and the detection sensitivity and upper detection limit are shown in table 1.
(2) PCT antigen was diluted to a series of concentrations of 20pg/ml, 30pg/ml, 40pg/ml, 60pg/ml, 80pg/ml, 160pg/ml, 500pg/ml, 1000pg/ml, 5000pg/ml, 20000pg/ml, 100000pg/ml and 2000ng/ml, and PCT monoclonal antibody 1 receptor microspheres were coated with different particle sizes (50 nm, 80nm, 110nm, 140nm, 170nm, 200nm, 250nm, 300nm, 350nm, 400 nm) prepared in example 1, and then tested with the same biotin-labeled PCT monoclonal antibody 2 (diluted to 2 ug/ml) and universal solution (donor microsphere solution), the test sensitivities and upper test limits were as shown in Table 1.
TABLE 1
As can be seen from the table 1,
(1) cTnI project detection results: the upper detection limit of the receptor microsphere at 50nm and 80nm is high, but the sensitivity is poor, while the receptor microsphere at 300nm has the best sensitivity, but the upper detection limit is low. The 50nm and 80nm receptor microspheres were mixed with 300nm receptor microspheres, respectively, to form a composition of receptor microspheres with different particle diameters, and the sensitivity and upper limit of detection of the chemiluminescent analysis method using the corresponding composition were measured, and the results are shown in Table 2.
(2) PCT project detection results: the upper detection limit of the receptor microsphere at 110nm is high, but the sensitivity is poor, while the receptor microsphere at 300nm and 350nm has the best sensitivity, but the upper detection limit is low. 110nm receptor microspheres are respectively mixed with 300nm receptor microspheres and 350nm receptor microspheres to form a composition of the receptor microspheres with different particle diameters, and the sensitivity and the upper detection limit of a chemiluminescent analysis method of the corresponding composition are adopted for detection, and the results are shown in Table 2.
TABLE 2
As can be seen from table 2, the chemiluminescent analysis method employing the composition formed by combining small-sized receptor microspheres and large-sized receptor microspheres has both high sensitivity and high upper detection limit (wide detection range), exhibits the advantages of large-sized receptor microspheres and small-sized receptor microspheres, and greatly improves the performance of a microsphere composition containing two or more particle sizes as compared with a single-sized receptor microsphere.
Example 3: preparation of receptor microspheres with different particle sizes (one) preparation of conjugated antibody receptor microspheres with average particle size of about 250nm
1.1 preparation and characterization Process of aldehyde polystyrene latex microspheres
1) A100 ml three-necked flask was prepared, 40mmol of styrene, 5mmol of acrolein and 10ml of water were added thereto, and after stirring for 10 minutes, N was introduced 2 30min；
2) 0.11g of ammonium persulfate and 0.2g of sodium chloride were weighed and dissolved in 40ml of water to prepare an aqueous solution. Adding the aqueous solution into the reaction system of the step 1, and continuing to introduce N 2 30min；
3) Heating the reaction system to 70 ℃ for reaction for 15 hours;
4) The emulsion after completion of the reaction was cooled to room temperature and filtered through a suitable filter cloth. Washing the obtained emulsion by centrifugal sedimentation with deionized water until the conductivity of the supernatant fluid at the beginning of centrifugation is close to that of the deionized water, diluting with water, and preserving in an emulsion form;
5) The average particle diameter of the Gaussian distribution of the latex microsphere particle diameter at this time was 202.2nm as measured by a nanoparticle analyzer, and the coefficient of variation (c.v.) was=4.60%, and the Gaussian distribution curve is shown in fig. 1. The aldehyde group content of the latex microsphere is 280nmol/mg measured by a conductivity titration method.
1.2 landfill Process and characterization of luminescent compositions
1) A25 ml round-bottomed flask was prepared and charged with 0.1g of a dimethylthiophene derivative and 0.1g of europium (III) complex (MTTA-EU) 3+ ) 10ml of 95% ethanol is magnetically stirred, and the temperature of the water bath is raised to 70 ℃ to obtain a complex solution;
2) Preparing a 100ml three-neck flask, adding 10ml 95% ethanol, 10ml water and 10ml aldehyde polystyrene latex microspheres with concentration of 10% obtained in the step 1.1, magnetically stirring, and heating to 70 ℃ in a water bath;
3) Slowly dripping the complex solution in the step 1) into the three-neck flask in the step 2), stopping stirring after reacting for 2 hours at 70 ℃, and naturally cooling;
4) Centrifuging the emulsion for 1 hour, 30000G, and discarding supernatant after centrifuging to obtain aldehyde polystyrene microsphere filled with luminous composition.
5) The average particle diameter of the Gaussian distribution of the particle diameters of the microspheres at this time was 204.9nm as measured by a nanoparticle analyzer, and the coefficient of variation (c.v.) was=5.00% (as shown in fig. 2).
1.3 surface coating of receptor microspheres with dextran
1) 50mg of aminodextran solid was taken in a 20mL round bottom flask, 5mL of 50 mm/ph=10 carbonate buffer was added, and the solution was stirred at 30 ℃ in the absence of light;
2) Taking 100mg of prepared aldehyde polystyrene microspheres filled with the luminous composition, adding the prepared aldehyde polystyrene microspheres into an aminodextran solution, and stirring for 2 hours;
3) 10mg of sodium borohydride was dissolved in 0.5ml of 50 mM/pH=10 carbonate buffer, and then added dropwise to the above reaction solution, followed by reaction at 30℃overnight in the absence of light;
4) After the reaction mixture was centrifuged at 30000G, the supernatant was discarded, and 50 mM/ph=10 carbonate buffer was added for ultrasonic dispersion. After repeating the centrifugal washing three times, the volume is fixed by 50 mM/pH=10 carbonate buffer solution to make the final concentration of the solution be 20mg/ml;
5) 100mg of aldehyde dextran solid was taken in a 20mL round bottom flask, 5mL of 50 mm/ph=10 carbonate buffer was added, and stirred at 30 ℃ in the absence of light for dissolution;
6) Adding the microsphere into an aldehyde dextran solution, and stirring for 2 hours;
7) 15mg of sodium borohydride was dissolved in 0.5ml of 50 mM/pH=10 carbonate buffer, and then added dropwise to the above reaction solution, followed by reaction at 30℃overnight in the absence of light;
8) After the reaction mixture was centrifuged at 30000G, the supernatant was discarded, and 50 mM/ph=10 carbonate buffer was added for ultrasonic dispersion. After repeating the centrifugation washing three times, the final concentration was set to 20mg/ml by constant volume with 50 mM/pH=10 carbonate buffer.
9) The average particle diameter of the Gaussian distribution of the particle diameters of the microspheres at this time was 241.6nm as measured by a nanoparticle analyzer, and the coefficient of variation (c.v.) was=12.90% (as shown in fig. 3).
1.4 coupling procedure of antibodies
1) The paired antibody I was dialyzed to 50mM CB buffer at pH=10 to give a concentration of 1mg/ml.
2) Adding 0.5ml of the receptor microsphere obtained in step (3) and 0.5ml of the paired antibody I obtained in step 1) into a 2ml centrifuge tube, uniformly mixing, and adding 100. Mu.l of 10mg/ml NaBH 4 The solution (50 mM CB buffer) was reacted at 2-8℃for 4 hours.
3) After completion of the reaction, 0.5ml of 100mg/ml BSA solution (50 mM CB buffer) was added, and the reaction was carried out at 2-8℃for 2 hours.
4) After the reaction, the mixture was centrifuged for 45min at 30000G, the supernatant was discarded after centrifugation, and resuspended in 50mM MES buffer. The centrifugation washing was repeated four times and diluted to a final concentration of 100. Mu.g/ml to obtain a receptor microsphere solution of the conjugated antibody I.
5) The average particle diameter of the Gaussian distribution of the particle diameters of the microspheres at this time was 253.5nm as measured by a nanoparticle analyzer, and the coefficient of variation (C.V value) =9.60% (as shown in fig. 4).
Preparation of conjugated antibody receptor microspheres having an average particle size of about 110nm
The preparation method is the same as the preparation process of the receptor microsphere with the average particle diameter of about 250nm in the first step, and the average particle diameter value of the Gaussian distribution (shown in fig. 5) of the particle diameter of the receptor microsphere is 107.1nm as measured by a nano-particle sizer, and the coefficient of variation (c.v.) is=7.6%. Nicomp distribution was unimodal (as shown in fig. 6).
(III) preparation of PCT antibody-coupled receptor microspheres with average particle size of about 350nm
The preparation method is the same as the preparation process of the acceptor microsphere with the average particle diameter of about 250nm in the first step, the average particle diameter value of the Gaussian distribution (shown in fig. 7) of the particle diameter of the acceptor microsphere is 347.5nm, the variation coefficient (C.V.) is=3.9%, and the Nicomp distribution is unimodal (shown in fig. 8).
Example 4: chemiluminescent assay method sensitivity and detection upper limit determination using receptor microspheres of different particle sizes
The sensitivity point is defined as when the signal of concentration Cx is higher than the signal of double concentration C0, i.e. RLU (Cx) >2RLU (C0), the corresponding detection reagent sensitivity is Cx. The upper limit of detection point was defined as the upper limit of the range determined using the method in the U.S. clinical laboratory standardization committee (NCCLS) Evaluation Protocol (EP) series 6 file.
(1) PCT antigen was diluted to a series of concentrations of 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 and 200000pg/ml, and then the receptor reagents (100 ug/ml concentration) comprising the receptor microspheres of PCT antibody I coupled with different average particle diameters (110 nm, 250nm and 350 nm) prepared in example 3 were used, and then the same biotin-labeled PCT monoclonal antibody 2 (diluted to 2 ug/ml) and universal solution (donor microsphere-containing reagent) were detected for the above-mentioned series of PCT antigens, and the detection sensitivity and upper limit of the detection using the photo-excitation chemiluminescence analysis system developed by Boyang biotechnology (Shanghai) limited were shown in Table 3.
TABLE 3 Table 3
As can be seen from Table 3, the upper limit of detection of the receptor microspheres with an average particle diameter of 110nm is high, but the sensitivity is poor; while the receptor microsphere with the average particle diameter of 350nm has the best sensitivity, the upper detection limit is lower.
(2) And mixing the acceptor microsphere solution with the average particle size of 110nm and the acceptor microsphere solution with the average particle size of 350nm and the acceptor microsphere solution with the average particle size of the PCT antibody is obtained. The results of the measurement of the particle size of the receptor microspheres in the novel receptor reagent are as follows:
gaussian distribution average particle diameter 317.7nm, particle diameter distribution coefficient of variation (C.V value) =37.2% (as shown in fig. 9);
the Nicomp distribution was bimodal: #1: average particle diameter 103.1nm coefficient of variation (C.V value) =11.8%; #2: average particle diameter 328.8nm, particle diameter distribution coefficient of variation (C.V value) =13.0% (as shown in fig. 10).
The novel acceptor reagent was subjected to detection of the above-described concentration series of PCT antigens with biotin-labeled PCT mab 2 (diluted to 2 ug/ml) and universal solution (reagent containing donor microspheres), and the detection sensitivity and upper limit of detection using a photoexcitation chemiluminescent assay system developed by bosch biotechnology (shanghai) are shown in table 4.
TABLE 4 Table 4
As can be seen from Table 4, the detection performance of the method is significantly improved by appropriately increasing the non-uniformity of the particle size of the receptor microspheres.
It should be noted that the above-described embodiments are only for explaining the present invention and do not constitute any limitation of the present invention. The invention has been described with reference to exemplary embodiments, but it is understood that the words which have been used are words of description and illustration, rather than words of limitation. Modifications may be made to the invention as defined in the appended claims, and the invention may be modified without departing from the scope and spirit of the invention. Although the invention is described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, as the invention extends to all other means and applications which perform the same function.
Claims (10)
1. A chemiluminescent analysis method is provided, which is used for analyzing and judging whether a sample to be tested contains a target molecule to be tested and/or the concentration of the target molecule to be tested by detecting the intensity of chemiluminescent signals generated by the reaction of at least two receptor microspheres with different particle diameters in the sample to be tested and active oxygen.
2. The method of claim 1, wherein the difference in particle size of the two different particle size receptor microspheres is no less than 100nm; preferably not less than 150nm; more preferably not less than 200nm; and/or
The particle size ratio of the two receptor microspheres with different particle sizes is selected from 1 (1.1-10); preferably selected from 1 (2-8); more preferably selected from 1 (3-6); and/or
The receptor microsphere is used at a concentration of 1ug/mL-1000ug/mL; preferably 10ug/mL-500ug/mL, more preferably 10ug/mL-250ug/mL.
3. The method according to claim 1 or 2, wherein the method is a homogeneous chemiluminescent assay method,
the method comprises the following steps:
s1, mixing a sample to be tested with a reagent a containing at least two acceptor microspheres with different particle sizes, and then mixing the sample to be tested with a reagent b containing donor microspheres to obtain a sample to be tested;
s2, contacting the sample to be detected obtained in the step S1 by using energy or active chemical substances, and exciting a donor to generate active oxygen;
S3, analyzing and judging whether the sample to be detected contains the target molecule to be detected and/or the concentration of the target molecule to be detected by detecting the intensity of a chemiluminescent signal generated by the reaction of at least two receptor microspheres with different particle diameters in the sample to be detected and active oxygen.
4. A method according to any one of claims 1 to 3, wherein the chemiluminescent signal has a detection wavelength of 520 to 620nm; and/or
And (3) adopting 600-700 nm red excitation light to perform laser irradiation.
5. The method of any one of claims 1-4, wherein the receptor microsphere comprises a light-emitting composition and a matrix, the light-emitting composition being filled in the matrix and/or coated on the surface of the matrix;
preferably, the luminescent composition is capable of reacting with active oxygen to produce a detectable chemiluminescent signal comprising a chemiluminescent compound and a metal chelate;
preferably, the chemiluminescent compound is selected from the group consisting of olefinic compounds, preferably selected from the group consisting of dimethylthiophene, bisbutanedione compounds, dioxines, enol ethers, enamines, 9-alkylene xanthenes, 9-alkylene-N-9, 10-dihydroacridine, aryletherenes, arylimidazoles and gloss concentrates and their derivatives, more preferably selected from the group consisting of dimethylthiophene and its derivatives;
Preferably, the metal of the metal chelate is a rare earth metal or a group VIII metal, preferably selected from europium, terbium, dysprosium, samarium, osmium and ruthenium, more preferably selected from europium;
more preferably, the metal chelate comprises a chelating agent selected from the group consisting of: MTTA, NHA, BHHT, BHHCT, DPP, TTA, NPPTA, NTA, TOPO, TPPO, BFTA 2, 2-dimethyl-4-perfluorobutanoyl-3-butanone (fod), 2' -bipyridine (bpy), bipyridylcarboxylic acid, azacrown ether, aza-cryptand and trioctylphosphine oxide, and derivatives thereof;
further preferably, the luminescent compound is a derivative of dimethylthiophene and the metal chelate is a europium chelate.
6. The method of claim 5, wherein the matrix material is selected from natural, synthetic, or modified naturally occurring polymers including, but not limited to: agarose, cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polystyrene, polyethylene, polypropylene, poly (4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate, nylon, polyethylene butyrate or polyacrylate; preferably, the matrix is polystyrene latex microspheres; further preferred are carboxyl and/or aldehyde polystyrene latex microspheres; and/or
The surface of the matrix is directly connected with a bioactive substance, and the bioactive substance can be specifically combined with a target molecule to be detected; and/or
The surface of the substrate is coated with a coating layer, and the surface of the coating layer is connected with a bioactive substance which can be specifically combined with a target molecule to be detected;
preferably, the coating in the coating layer is selected from polysaccharides, high molecular polymers or biological macromolecules, preferably polysaccharides;
preferably, the surface of the substrate is coated with a coating of at least two successive polysaccharide layers, wherein each of the successive polysaccharide layers is spontaneously associated with each of the preceding polysaccharide layers.
7. The method of claim 6, wherein the polysaccharide has pendant functional groups, the functional groups of the continuous polysaccharide layer being oppositely charged to the functional groups of the preceding polysaccharide layer; and/or
The polysaccharide has pendant functional groups and the continuous layer of the polysaccharide is covalently linked to the previous polysaccharide layer by a reaction between the functional groups of the continuous layer and the functional groups of the previous layer;
preferably, the functional groups of the continuous polysaccharide layer alternate between amine functional groups and amine reactive functional groups; preferably the amine-reactive functional group is an aldehyde group or a carboxyl group;
Preferably, the polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from the group consisting of dextran, starch, glycogen, inulin, levan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably selected from the group consisting of dextran, starch, glycogen and polyribose.
8. The method of claim 6 or 7, wherein the outermost polysaccharide layer of the coating has at least one pendant functional group;
preferably, the pendant functional groups are selected from at least one of aldehyde, carboxyl, sulfhydryl, amino, hydroxyl, and maleamine groups; preferably selected from aldehyde groups and/or carboxyl groups.
9. A chemiluminescent analyzer for detecting the presence and/or concentration of a test target molecule in a test sample by the method of any one of claims 1-8;
preferably, the chemiluminescent analyzer comprises at least the following parts:
the incubation module is used for providing a proper temperature environment for the chemiluminescent reaction of the sample to be tested and at least two receptor microspheres with different particle diameters after being mixed;
the detection module is used for detecting a chemiluminescent signal generated by the reaction of the receptor microsphere and the active oxygen;
And the processor is used for judging whether the target molecule to be detected exists in the sample to be detected or not and/or the concentration of the target molecule to be detected in the sample to be detected according to the condition of the chemiluminescent signal detected by the detection module.
10. Use of a method according to any one of claims 1-8 and/or a chemiluminescent analyzer according to claim 9 for detecting markers of myocardial damage including cTnI and/or markers of inflammation including procalcitonin.
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| CN111912839B (en) * | 2020-08-13 | 2022-10-28 | 广东药科大学 | Catalytic light-emitting reactor, and butanone peroxide detection device and detection method |
| CN114152742A (en) * | 2021-11-30 | 2022-03-08 | 深圳市易瑞生物技术股份有限公司 | Kit for light-activated chemiluminescence immunoassay containing magnetic luminescent microspheres and application thereof |
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| US20090075397A1 (en) * | 2007-09-13 | 2009-03-19 | Academia Sinica | Method for characterizing sugar-binding interactions of biomolecules |
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