CN113125702A

CN113125702A - Homogeneous phase chemiluminescence detection kit and application thereof

Info

Publication number: CN113125702A
Application number: CN201911414985.1A
Authority: CN
Inventors: 康蔡俊; 李临
Original assignee: Beyond Diagnostics Shanghai Co ltd; Chemclin Diagnostics Corp
Current assignee: Beyond Diagnostics Shanghai Co ltd; Chemclin Diagnostics Corp
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2021-07-16

Abstract

The invention relates to a homogeneous phase chemiluminescence detection kit and application thereof. The kit comprises a donor reagent and an acceptor reagent, wherein the donor reagent contains a donor particle, the acceptor reagent contains an acceptor particle, the donor particle comprises a first carrier, the first carrier is filled with a sensitizing agent, the surface of the first carrier is directly or indirectly connected with one member of a specific binding pair, and the sugar content in each mg of the donor particle is not higher than 25 ug; the acceptor particle comprises a second carrier, the inside of the second carrier is filled with a luminescent composition, the surface of the second carrier is coated with a coating layer, the surface of the coating layer is connected with a reporter molecule, the reporter molecule can be specifically combined with a target molecule to be detected, and the sugar content in the donor particle per milligram is not lower than 40 ug. The POCT detection method using the kit has the characteristics of high sensitivity, high precision, wide range, rapidness, portability and the like.

Description

Homogeneous phase chemiluminescence detection kit and application thereof

Technical Field

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

Background

Homogeneous chemiluminescence analysis refers to a method for performing chemiluminescence detection without the need to separate the complex formed after binding and the remaining free reactants.

The existing homogeneous phase chemiluminescence analysis has the following defects:

A. the instrument system is large in size and large in occupied area, and meanwhile, due to the fact that the testing flux is large, the reagent card takes 100 tests as a whole unit and has requirements on the sample scale of a laboratory;

B. instrument systems and reagents are expensive, maintenance cost is high, and the method is not suitable for basic medical institutions;

C. the instrument has large volume and cannot be carried about to enter a diagnosis and treatment site;

D. the chemiluminescence system mainly adopts serum and plasma as samples, and generally can not adopt whole blood, thereby limiting the application range of the chemiluminescence system.

Meanwhile, in recent years, a point-of-care testing (POCT) technology for clinical testing (bedside detection) near a patient, which is abbreviated as POCT technology, is emerging, wherein the POCT technology mainly adopts fluorescent quantitative chromatography or colloidal gold, and mainly adopts a rapid diagnosis technology for immunoassay by a membrane chromatography method through fluorescent microspheres or colloidal gold wrapped by fluorescent materials. However, since these two techniques mainly perform release detection on NC membranes, the CV of the membrane itself is 5% or more, so that the POCT detection CV by a solid-phase membrane method is generally 10% or more, the detection precision is poor, and it is extremely difficult to quantify items requiring high sensitivity, such as cTnI. In addition, a novel technology such as a microfluidic chip type POCT detection technology is adopted, so that the method has the advantages of high reaction speed, small sample demand and the like, but the problem of low detection sensitivity is caused due to insufficient reaction.

Therefore, it is highly desirable to provide a homogeneous chemiluminescent POCT assay kit and method with high sensitivity, high precision, and wide range, and which is fast and portable.

Disclosure of Invention

The invention aims to solve the technical problem of providing a homogeneous phase chemiluminescence POCT detection kit and a method, wherein the POCT detection method using the kit has the characteristics of high sensitivity, high precision, wide range, high speed, portability and the like of a chemiluminescence analysis technology.

To this end, in a first aspect, the present invention provides a homogeneous chemiluminescent assay kit comprising a donor reagent comprising a donor particle capable of generating reactive oxygen species upon excitation, and an acceptor reagent comprising a second buffer solution and an acceptor particle capable of reacting with received reactive oxygen species to generate a chemiluminescent signal,

the donor particles comprise a first carrier, the first carrier being filled internally with a sensitizer, the first carrier having a surface to which one of the specific binding pair members is directly or indirectly attached, and having a sugar content per mg of the donor particles of no more than 25 ug;

the acceptor particle comprises a second carrier, the inside of the second carrier is filled with a luminescent composition, the surface of the second carrier is coated with a coating layer, the surface of the coating layer is connected with a reporter molecule, the reporter molecule can be specifically combined with a target molecule to be detected, and the sugar content in the donor particle per milligram is not lower than 40 ug.

In some embodiments of the invention, the surface of the first support is directly bound to one of the members of the specific binding pair.

In other embodiments of the invention, the surface of the first support is not coated or linked with a polysaccharide substance that is directly bound to one of the members of the specific binding pair.

In some embodiments of the invention, the surface of the first support bears a bonding functionality for directly bonding one of the specific binding pair members to the surface of the first support.

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

In some embodiments of the invention, the specific binding pair member is selected from a pair of substances capable of specifically binding to each other, consisting of an antibody, an antibody fragment, a ligand, an oligonucleotide binding protein, a lectin, a hapten, an antigen, an immunoglobulin binding protein, avidin, or biotin.

In other embodiments of the invention, the specific binding pair member is avidin-biotin, said avidin being selected from the group consisting of ovalbumin, streptavidin, vitellin, neutravidin and an avidin-like, preferably neutravidin and/or streptavidin.

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

In other embodiments of the invention, the recipient particle has a particle size distribution variation coefficient C.V value of 5% or more in the recipient agent.

In some preferred embodiments of the invention, the acceptor particles have a particle size distribution variation coefficient C.V value of 8% or more in the acceptor reagent; preferably, the acceptor particle has a variation coefficient C.V value of 10% or more in the particle size distribution of the acceptor reagent.

In other preferred embodiments of the present invention, the recipient particle has a particle size distribution coefficient of variation C.V value of 40% or less in the recipient agent; still more preferably, the recipient particle has a particle size distribution variation coefficient C.V value of 20% or less in the recipient agent.

In some embodiments of the invention, the acceptor particles exhibit a particle size distribution in the acceptor agent that is polydisperse.

In other embodiments of the present invention, the value of the variation coefficient of particle size distribution C.V is calculated by a Gaussian distribution.

In some embodiments of the invention, the acceptor particle exhibits a Gaussian distribution curve in the acceptor agent that exhibits two or more peaks using a Gaussian distribution analysis.

In other embodiments of the present invention, the receptive agent comprises at least two distributions of average particle size receptive particles.

In some embodiments of the invention, the coating in the coating layer is selected from a polysaccharide, a high molecular polymer or a biomacromolecule, preferably a polysaccharide;

further preferably, the surface of the second carrier is coated with at least two continuous polysaccharide coatings having one of the members of the specific binding pair attached to its surface.

In other embodiments of the present invention, each polysaccharide layer in the continuous polysaccharide coating is spontaneously associated with a previous polysaccharide layer.

In some embodiments of the invention, the polysaccharide has pendant functional groups, the pendant functional groups of any one of the successive polysaccharide layers being oppositely charged from the pendant functional groups of the previous polysaccharide layer.

In other embodiments of the present invention, the polysaccharide has pendant functional groups, and any one polysaccharide layer in the continuous polysaccharide coating is covalently linked to a preceding polysaccharide layer by a chemical bonding reaction between the pendant functional groups and the pendant functional groups of the preceding polysaccharide layer.

In some embodiments of the invention, the pendant functional groups of the continuous polysaccharide coating alternate between amine functional groups and amine-reactive functional groups.

In other embodiments of the present invention, the amine-reactive functional group is an aldehyde group or a carboxyl group.

In some embodiments of the invention, the outermost polysaccharide layer of the continuous polysaccharide coating has at least one pendant functional group.

In other embodiments of the present invention, the pendant functional group is selected from at least one of aldehyde, carboxyl, thiol, amino, hydroxyl, and maleic groups; preferably selected from aldehyde groups and/or carboxyl groups.

In some embodiments of the invention, the continuous polysaccharide coating pendant functional groups are chemically bonded directly or indirectly to one of the members of the specific binding pair.

In other embodiments of the invention, the polysaccharide is selected from the group consisting of carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from the group consisting of dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably selected from dextran, starch, glycogen and polyribose.

In some embodiments of the invention, the sugar content is detected by the anthrone method;

preferably, the saccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units, preferably from dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably selected from dextran, starch, glycogen and polyribose;

further preferably, the molecular weight distribution Mw of the glucan is 1000-1000000 KDa, preferably 10000-800000 KDa, more preferably 30000-700000 KDa.

In a second aspect, the invention provides a homogeneous chemiluminescent POCT test kit comprising a kit according to the first aspect of the invention, wherein the POCT is a point-of-care test or a clinical test performed in the vicinity of a patient.

In some embodiments of the present invention, the kit comprises a reagent strip, wherein a plurality of holes for containing reagents are disposed on the reagent strip, and the holes at least comprise:

a first reagent well site for holding said donor reagent;

a second reagent well site for holding said acceptor reagent.

In a third aspect, the invention provides a homogeneous chemiluminescence POCT detection method, which uses the kit according to the second aspect of the invention to detect a target molecule to be detected in a sample to be detected.

The fourth aspect of the present invention provides a homogeneous chemiluminescence POCT detection apparatus, which uses the kit according to the second aspect or the method according to the third aspect of the present invention to detect a target molecule to be detected in a sample to be detected.

In some embodiments of the invention, the apparatus comprises:

an incubation module for controlling the temperature of the reagent strip according to the second aspect of the invention;

the optical excitation and detection module is arranged on one side of the incubation module and is used for emitting excitation light to the reagent strip to enable the reagent strip to generate a light-excited chemiluminescence reaction; and detecting the chemiluminescent signal produced by the reagent.

The fifth aspect of the present invention provides a method for performing homogeneous chemiluminescence analysis by using the POCT detection apparatus according to the fourth aspect of the present invention, comprising the steps of:

s1, contacting the sample to be detected with the receptor reagent and the donor reagent, and generating a mixture to be detected after reaction;

s2, exciting the mixture to be detected to perform chemiluminescence by using exciting light with the wavelength of 600-700 nm, and detecting the signal intensity of the chemiluminescence; the detection wavelength of the chemiluminescence is 520-620 nm;

and S3, judging whether the sample to be detected contains the target molecules to be detected and/or the concentration of the target molecules to be detected in the sample to be detected according to the analysis of the chemiluminescence signal intensity.

The invention has the beneficial effects that: the kit comprises a donor reagent of a specific donor particle and/or an acceptor reagent of a specific acceptor particle, the efficiency of generating active oxygen by the donor particle is high, the active oxygen is more easily transferred to the acceptor particle in a homogeneous system and is not easily interfered by other substances, the stability of the donor particle is high, and the donor particle can stably exist in the donor reagent and is not easily inactivated; meanwhile, the sugar content in each milligram of the donor particles is not higher than 25ug, the sugar content in each milligram of the donor particles is not lower than 40ug, and the variation coefficient C.V value of the particle size distribution of the acceptor particles is not less than 5%, so that the phase chemiluminescence POCT detection performed by using the kit disclosed by the invention has the characteristics of high sensitivity, high precision and wide range of a chemiluminescence analysis technology, and the POCT detection technology is rapid and portable.

Drawings

The invention will be further explained with reference to the drawings.

Fig. 1 is a Gaussian distribution plot of donor particles prepared in example 1.

Fig. 2 is a Gaussian distribution plot of donor particles prepared in example 2.

FIG. 3 is a Gaussian distribution plot of acceptor particles prepared in example 3 with an average particle size around 250 nm.

FIG. 4 is a standard curve for sugar content determination in example 4.

FIG. 5 is a schematic view showing the structure of a reagent strip in example 5.

FIG. 6 is a graph of correlation coefficients for CRP assays at different concentrations in serum and whole blood in example 9.

Detailed Description

In order that the invention may be readily understood, a detailed description of the invention is provided below. However, before the invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. The practice of the invention is not limited to the following examples, and any variations and/or modifications made thereto are intended to fall within the scope of the invention.

Where a range of values is provided, it is understood that each intervening value, to the extent that there is no stated or intervening value in that stated range, to the extent that there is no such intervening value, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where a specified range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

Term (I)

The term "active oxygen" as used herein refers to a general term for a substance which is composed of oxygen, contains oxygen, and is active in nature, and is mainly an excited oxygen molecule, including superoxide anion (O) which is an electron reduction product of oxygen₂(-) and the two-electron reduction product hydrogen peroxide (H)₂O₂) The three-electron reduction product hydroxyl radical (. OH) and nitric oxide and singlet oxygen (1O)₂) And the like.

The term "donor particles" as used herein refers to particles containing a sensitizer capable of generating a reactive intermediate, such as a reactive oxygen species, upon activation by energy or a reactive compound, to react with the acceptor particles. The donor particles may be light activated (e.g., dyes and aromatic compounds) or chemically activated (e.g., enzymes, metal salts, etc.). In some embodiments of the invention, the donor particles are polymeric microspheres filled with photosensitizers, which may be known in the art, preferably relatively light stable and not reactive with singlet oxygen, non-limiting examples of which include compounds such as methylene blue, rose bengal, porphyrins, phthalocyanines, and chlorophylls, as disclosed in, for example, U.S. patent No. 5709994, which is incorporated herein by reference in its entirety, and derivatives of these compounds having 1-50 atom substituents that are used to render these compounds more lipophilic or more hydrophilic and/or as linkers to specific binding partner members. Examples of other photosensitizers known to those skilled in the art may also be used in the present invention, such as those described in US patent No. US6406913, which is incorporated herein by reference.

The term "acceptor particle" as used herein refers to a particle that contains a compound that reacts with reactive oxygen species to produce a detectable signal. The donor particles are induced by energy or an active compound to activate and release reactive oxygen species in a high energy state that are captured by the acceptor particles in close proximity, thereby transferring energy to activate the acceptor particles. In some embodiments of the present invention, the acceptor particle comprises a luminescent composition and a carrier, wherein the luminescent composition is filled in the carrier and/or coated on the surface of the carrier.

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

In the present invention, the "chemiluminescent compound", i.e., a compound referred to as a label, may undergo a chemical reaction to cause luminescence, such as by being converted to another compound formed in an electronically excited state. The excited state may be a singlet state or a triplet excited state. The excited state may relax to the ground state to emit light directly, or may return to the ground state itself by transferring excitation energy to an emission energy acceptor. In this process, the energy acceptor particle will be transitioned to an excited state to emit light.

A "specific binding pair member" as used herein refers to a pair of substances that are capable of specifically binding to each other.

The "variation coefficient C.V value of particle size distribution" described in the present invention refers to the variation coefficient of particle size in Gaussian distribution in the detection result of the nanometer particle size analyzer. The coefficient of variation is calculated as: C.V value (standard deviation SD/Mean) x 100%.

The term "Nicomp distribution" as used herein refers to an algorithmic distribution in the US PSS nanometer particle sizer, NICOMP. Compared with a Gaussian single-peak algorithm, the Nicomp multi-peak algorithm has unique advantages in the analysis of multi-component liquid dispersion systems with nonuniform particle size distribution and the stability analysis of colloidal systems.

The term "test sample" as used herein refers to a mixture containing or suspected of containing a target molecule to be tested. The test sample that can be used in the present invention includes body fluids such as blood (which may be anticoagulated blood commonly seen in collected blood samples), plasma, serum, urine, semen, saliva, cell cultures, tissue extracts, and the like. Other types of samples to be tested include solvents, seawater, industrial water samples, food samples, environmental samples such as soil or water, plant material, eukaryotic cells, bacteria, plasmids, viruses, fungi, and cells from prokaryotes. The sample to be tested can be diluted with a diluent as required before use. For example, to avoid the HOOK effect, the sample to be tested may be diluted with a diluent before the on-line detection and then detected on the detection instrument.

The term "target molecule to be detected" as used herein refers to a substance in a sample to be detected during detection. One or more substances having a specific binding affinity for the target molecule to be detected will be used for the detection of the target molecule. The target molecule to be detected may be a protein, a peptide, an antibody or a hapten which allows it to bind to an antibody. The target molecule to be detected may be a nucleic acid or oligonucleotide that binds to a complementary nucleic acid or oligonucleotide. The target molecule to be detected may be any other substance that can form a member of a specific binding pair. Other examples of typical target molecules to be detected include: drugs such as steroids, hormones, proteins, glycoproteins, mucins, nucleoproteins, phosphoproteins, drugs of abuse, vitamins, antibacterial agents, antifungal agents, antiviral agents, purines, antitumor agents, amphetamines, heteroazoids, nucleic acids, and prostaglandins, and metabolites of any of these drugs; pesticides and metabolites thereof; and a receptor. Analytes also include cells, viruses, bacteria, and fungi.

The term "antibody" as used herein is used in the broadest sense and includes antibodies of any isotype, antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, bispecific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. In any case desired, the antibody may be further conjugated to other moieties, such as a member of a specific binding pair member, e.g., biotin or avidin (a member of a biotin-avidin specific binding pair member), and the like.

The term "antigen" as used herein refers to a substance that stimulates the body to produce an immune response and that binds to the immune response product antibodies and sensitized lymphocytes in vitro and in vivo to produce an immune effect.

The term "binding" as used herein refers to direct association between two molecules due to interactions such as covalent, electrostatic, hydrophobic, ionic and/or hydrogen bonding, including but not limited to interactions such as salt and water bridges. The term "specific binding" as used herein refers to the mutual discrimination and selective binding reaction between two substances, and is the conformation correspondence between the corresponding reactants in terms of the three-dimensional structure. Under the technical idea disclosed by the invention, the detection method of the specific binding reaction comprises but is not limited to the following steps: double antibody sandwich, competition, neutralization competition, indirect or capture.

Detailed description of the preferred embodiments

The present invention will be described in more detail with reference to examples.

After researching the sugar content and the uniformity of the particle size in the donor particles and the acceptor particles, the inventor of the application finds that the sugar content in the donor particles is not higher than 25ug per mg, the sugar content in the acceptor particles is not lower than 40ug per mg, and the sensitivity of light-activated chemiluminescence detection can be ensured and the detection range can be widened by adopting microspheres with the uniformity of the particle size (such as the variation coefficient of the microsphere particle size distribution is more than 5%).

Accordingly, the invention in a first aspect relates to a homogeneous chemiluminescent assay kit comprising: comprising a donor reagent comprising donor particles capable of generating reactive oxygen species upon excitation, and an acceptor reagent comprising a second buffer solution and acceptor particles capable of reacting with the reactive oxygen species received to generate a chemiluminescent signal,

In some embodiments of the present invention, the bonding functional group content on the surface of the first support is 100 to 500nmol/mg, preferably 200 to 400 nmol/mg.

In some embodiments of the invention, the donor particles have a coefficient of variation of size distribution C.V value ≧ 5% in the donor agent.

In other embodiments of the present invention, the donor particles have a size distribution coefficient of variation C.V value of 8% or more in the donor reagent; preferably, the donor particles have a variation coefficient C.V value of 10% or more in the particle size distribution of the donor particles in the reagent.

In some embodiments of the invention, the donor particles have a coefficient of variation of particle size distribution C.V value ≦ 40% in the donor agent; still more preferably, the donor particles have a coefficient of variation of particle size distribution C.V value ≦ 20% in the donor agent.

It should be noted that the C.V value of the donor particle size distribution variation coefficient refers to C.V value of the donor particle size distribution variation coefficient after it is coated with the desired material.

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

In other embodiments of the present invention, the donor particles exhibit a polydispersity in their size distribution in the donor agent.

In some embodiments of the invention, the value of the variation coefficient C.V of the particle size distribution is calculated by Gaussian distribution.

In other embodiments of the invention, the acceptor particle exhibits two or more peaks in the acceptor agent's Gaussian distribution curve using a Gaussian distribution analysis.

In some embodiments of the invention, the concentration of the donor particles in the donor agent is from 10 μ g/ml to 1mg/ml, preferably from 20 μ g/ml to 500 μ g/ml, more preferably from 50 μ g/ml to 200 μ g/ml.

In other embodiments of the present invention, the donor reagent further comprises a buffer solution having a pH of 7.0 to 9.0, wherein the donor particles are suspended in the buffer solution.

In some embodiments of the invention, the buffer solution contains a polysaccharide selected from carbohydrates containing three or more unmodified or modified monosaccharide units, preferably selected from dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxydextran, and aminodextran; more preferably selected from dextran, starch, glycogen and polyribose.

In further embodiments of the present invention, the dextran has a molecular weight distribution Mw selected from 10000 to 1000000kDa, preferably from 100000 to 800000kDa, more preferably from 300000 to 700000 kDa.

In some embodiments of the invention, the content of dextran in the buffer solution is 0.01 to 1 wt%, preferably 0.05 to 0.5 wt%.

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

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

In some embodiments of the invention, the material of the first support and/or the second support is selected from natural, synthetic or modified naturally occurring polymers; preferably a synthetic polymer.

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

In some embodiments of the invention, the first support and/or the second support are polystyrene latex microspheres.

In some embodiments of the invention, the sensitizer is a photoactivated photosensitizer and/or a chemically activated initiator, preferably a photoactivated photosensitizer; further preferably, the sensitizer is selected from methylene blue, rose bengal, porphyrins, phthalocyanines and chlorophylls.

In some 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 present invention, the chemiluminescent compound is selected from the group consisting of olefinic compounds, preferably from the group consisting of dimethylthiophene, dibutyldione compounds, dioxins, enol ethers, enamines, 9-alkylidenexanthanes, 9-alkylene-N-9, 10 dihydroacridines, arylethyletherenes, arylimidazoles, and lucigenins and derivatives thereof, more preferably from the group consisting of dimethylthiophene and derivatives thereof.

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 europium.

In some embodiments of the invention, the metal chelate comprises a chelating agent selected from the group consisting of: 4 ' - (10-methyl-9-anthracenyl) -2,2 ': 6 ' 2 "-bipyridine-6, 6" -dimethylamine ] tetraacetic acid (MTTA), 2- (1 ', 1 ', 2 ', 2 ', 3 ', 3 ' -heptafluoro-4 ', 6 ' -hexanedion-6 ' -yl) -Naphthalene (NHA), 4 ' -bis (2 ', 3 ', 3 "-heptafluoro-4 ', 6" -hexanedion-6 "-yl) -o-terphenyl (BHHT), 4 ' -bis (1 ', 2 ', 3 ', 3" -heptafluoro-4 ', 6 "-hexanedion-6" -yl) -chlorosulphonyl-o-terphenyl (BHHCT), 4, 7-biphenyl-1, 10-phenanthroline (DPP), 1,1, 1-trifluoroacetone (TTA), 3-naphthoyl-1, 1, 1-trifluoroacetone (NPPTA), Naphthyltrifluorobutanedione (NTA), trioctylphosphine oxide (TOPO), triphenylphosphine oxide (TPPO), 3-benzoyl-1, 1, 1-trifluoroacetone (BFTA), 2-dimethyl-4-perfluorobutanoyl-3-butanone (fod), 2' -bipyridine (bpy), bipyridylcarboxylic acid, azacrown ether, azacryptand trioctylphosphine oxide and derivatives thereof.

The homogeneous chemiluminescent POCT test kit according to the second aspect of the present invention comprises the kit according to the first aspect of the present invention, wherein the POCT is a point-of-care test or a clinical test performed in the vicinity of a patient.

a first reagent well site for holding said donor reagent;

a second reagent well site for holding said acceptor reagent.

The homogeneous chemiluminescence POCT detection method in the third aspect of the invention uses the kit in the second aspect of the invention to detect a target molecule to be detected in a sample to be detected.

The homogeneous chemiluminescence POCT detection apparatus according to the fourth aspect of the present invention detects a target molecule to be detected in a sample to be detected by using the kit according to the second aspect of the present invention or the method according to the third aspect of the present invention.

In some embodiments of the invention, the apparatus comprises:

The fifth aspect of the present invention relates to a method for performing homogeneous chemiluminescence analysis by using the POCT detection apparatus according to the fourth aspect of the present invention, comprising the steps of:

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

In some embodiments of the present invention, the test sample is selected from materials suspected of containing the target molecule to be tested, which include but are not limited to: blood, serum, plasma, sputum, lymph, semen, vaginal mucus, feces, urine, or spinal fluid.

Examples

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

Example 1: preparation of Donor reagent

Preparation of aldehyde group polystyrene latex microsphere

a) A100 ml three-necked flask was prepared, 40mmol of styrene, 5mmol of acrolein and 10ml of water were added thereto, and after stirring for 10min, N was introduced thereinto₂ 30min。

b) 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 a), and continuously introducing N₂ 30min。

c) The reaction system was warmed to 70 ℃ and reacted for 15 hours.

d) The emulsion after completion of the reaction was cooled to room temperature and filtered through a suitable filter cloth. And washing the obtained emulsion by using deionized water through centrifugal sedimentation for a plurality of times until the conductivity of the supernatant at the beginning of centrifugation is close to that of the deionized water, then diluting the supernatant with water, and storing the diluted supernatant in an emulsion form.

e) The average particle size of the latex microspheres in a gaussian distribution measured by a nanometer particle size analyzer was 201.3nm, and the coefficient of variation (C.V.) -8.0%.

Filling of (II) sensitizers

a) A25 ml round bottom flask was prepared, 0.11g of copper phthalocyanine and 10ml of N, N-dimethylformamide were added, magnetic stirring was carried out, and the temperature in the water bath was raised to 75 ℃ to obtain a photosensitizer solution.

b) Preparing a 100ml three-neck flask, adding 10ml 95% ethanol, 10ml water and 10ml aldehyde polystyrene latex microspheres obtained in the step (I) with the concentration of 10%, magnetically stirring, and heating in a water bath to 70 ℃.

c) Slowly dropwise adding the solution obtained in the step a) into the three-neck flask obtained in the step b), reacting at 70 ℃ for 2 hours, stopping stirring, and naturally cooling to obtain an emulsion.

d) The emulsion was centrifuged for 1 hour at 30000G, the supernatant discarded after centrifugation and resuspended in 50% ethanol. After repeated centrifugation washing three times, the suspension was resuspended in 50mM CB buffer at pH 10 to a final concentration of 20 mg/ml.

(III) preparation of Donor reagent by modifying avidin on the surface of microsphere

a) Treating microsphere suspension: and (3) sucking a certain amount of the microspheres prepared in the step (II) to centrifuge in a high-speed refrigerated centrifuge, removing the supernatant, adding a certain amount of MES buffer solution, performing ultrasonic treatment on an ultrasonic cell disruption instrument until the particles are resuspended, and adding the MES buffer solution to adjust the concentration of the microspheres to 100 mg/ml.

b) Preparing an avidin solution: a certain amount of streptavidin was weighed and dissolved in MES buffer to 8 mg/ml.

c) Mixing: mixing the treated microsphere suspension, 8mg/ml avidin and MES buffer solution in a volume ratio of 2:5:1, and quickly mixing to obtain a reaction solution.

d) Reaction: preparing 25mg/ml NaBH by MES buffer solution₃Adding CN solution according to the volume ratio of 1:25 to the reaction solution, and quickly and uniformly mixing. The reaction was rotated at 37 ℃ for 48 hours.

e) And (3) sealing: MES buffer solution is prepared into 75mg/ml Gly solution and 25mg/ml NaBH₃Adding CN solution into the solution according to the volume ratio of 2:1:10 of the reaction solution, mixing uniformly, and carrying out rotary reaction for 2 hours at 37 ℃. Then, 200mg/ml BSA solution (MES buffer) was added thereto at a volume ratio of 5:8, and the mixture was rapidly mixed and subjected to a rotary reaction at 37 ℃ for 16 hours.

f) Cleaning: adding MES buffer solution into the reacted solution, centrifuging by a high-speed refrigerated centrifuge, removing supernatant, adding fresh MES buffer solution, resuspending by an ultrasonic method, centrifuging again, washing for 3 times, finally suspending by using a small amount of donor particle buffer solution, measuring solid content, and adjusting the concentration to 150 mu g/ml by using the donor particle buffer solution to obtain the donor reagent containing donor particles.

g) The average diameter of the donor particles in the gaussian distribution was 227.7nm as measured by a nanometer particle sizer, and the coefficient of variation (C.V.) -6.5%, as shown in fig. 1.

Example 2: preparation of Donor reagent

The preparation of aldehyde-based polystyrene latex microspheres and the filling process of the sensitizer were the same as the preparation steps of (a) and (b) in example 1.

Preparation of (mono) aminodextran

a) A500 mL four-necked flask was placed in an oil bath pan, equipped with a condenser tube, and purged with nitrogen.

b) 10g of dextran with the average molecular weight distribution of 500000KDa, 100ml of deionized water, 2g of NaOH and 10g N- (2, 3-epoxypropyl) phthalimide are sequentially added, and the mixture is mechanically stirred.

c) After the oil bath is carried out for 2 hours at the temperature of 90 ℃, the heating is closed, and the stirring is maintained for natural cooling.

d) The reaction mixture separated out the main mixture in 2L of methanol, the solid was collected and dried.

e) A200 mL four-necked flask was placed in an oil bath pan, equipped with a condenser tube, and purged with nitrogen.

f) The dried solid, 100mL of deionized water, 1.8g of sodium acetate, and 5mL of 50% hydrazine hydrate were sequentially added, the pH was adjusted to 4, and the mixture was mechanically stirred.

g) After the oil bath is carried out for 1 hour at the temperature of 85 ℃, the heating is closed, and the stirring is maintained for natural cooling.

h) The pH of the reaction solution is adjusted to be neutral and then filtered, and the filtrate is collected.

i) The filtrate is put into a dialysis bag, and is dialyzed for 2 days at the temperature of 4 ℃ by deionized water, and the water is changed for 3 to 4 times every day.

j) After dialysis, the resulting solution was lyophilized to obtain 9.0g of an aminodextran solid.

Preparation of (di) aldehyde dextran

a) 10g of dextran with a mean molecular weight distribution of 500000kDa were weighed into a 250 beaker, and 100mL of 0.1M/pH 6.0 phosphate buffer was added and dissolved with stirring at room temperature.

b) 1.8g of sodium metaperiodate was weighed into a 50mL beaker, and 10mL of 0.1M/pH 6.0 phosphate buffer was added and dissolved with stirring at room temperature.

c) Slowly dropwise adding the sodium metaperiodate solution into the glucan solution, reacting until no bubbles are generated, and continuing stirring for 1 hour.

d) The reaction mixture is put into a dialysis bag, and is dialyzed for 2 days at the temperature of 4 ℃ by deionized water, and the water is changed for 3 to 4 times every day.

e) After dialysis, the mixture was freeze-dried to obtain 9.6g of aldehyde dextran solid.

(III) microsphere-coated dextran

a) 50mg of the aminodextran solid was placed in a 20mL round-bottom flask, and 5mL of 50mM/pH 10 carbonate buffer was added and dissolved with stirring at 30 ℃ in the dark.

b) 100mg of donor particles were added to the aminodextran solution and stirred for 2 hours.

c) 10mg of sodium borohydride was dissolved in 0.5mL of 50mM/pH 10 carbonate buffer solution, and the solution was added dropwise to the reaction solution, followed by overnight reaction at 30 ℃ in the absence of light.

d) After the reaction, the mixture 30000G was centrifuged, the supernatant was discarded, and 50mM/pH 10 carbonate buffer was added thereto for ultrasonic dispersion. After repeating the centrifugal washing three times, the volume was adjusted to 20mg/ml by using 50mM/pH 10 carbonate buffer.

e) 100mg of aldehyde dextran solid was placed in a 20mL round-bottom flask, 5mL of 50mM/pH 10 carbonate buffer was added, and the mixture was dissolved with stirring at 30 ℃ in the dark.

f) The particles are added into the aldehyde dextran solution and stirred for 2 hours.

g) 15mg of sodium borohydride was dissolved in 0.5mL of 50mM/pH 10 carbonate buffer solution, and the solution was added dropwise to the reaction solution and reacted overnight at 30 ℃ with exclusion of light.

h) After the reaction, the mixture 30000G was centrifuged, the supernatant was discarded, and 50mM/pH 10 carbonate buffer was added thereto for ultrasonic dispersion. After repeating the centrifugal washing three times, the volume was adjusted to 20mg/ml by using 50mM/pH 10 carbonate buffer.

i) The gaussian distribution average particle size of the microspheres was 235.6nm as measured by a nano-particle sizer, and the coefficient of variation (C.V.) -8.1%.

(IV) preparing donor reagent by modifying avidin on the surface of microsphere

h) Treating microsphere suspension: and (3) sucking a certain amount of microspheres prepared in the step (three) into a high-speed freezing centrifugal machine for centrifugation, removing a supernatant, adding a certain amount of MES buffer solution, performing ultrasonic treatment on an ultrasonic cell disruption instrument until the microspheres are resuspended, and adding the MES buffer solution to adjust the concentration of donor particles to 100 mg/ml.

i) Preparing an avidin solution: a certain amount of neutravidin was weighed and dissolved to 8mg/ml by adding MES buffer.

j) Mixing: mixing the treated microsphere suspension, 8mg/ml avidin and MES buffer solution in a volume ratio of 2:5:1, and quickly mixing to obtain a reaction solution.

k) Reaction: preparing 25mg/ml NaBH by MES buffer solution₃Adding CN solution according to the volume ratio of 1:25 to the reaction solution, and quickly and uniformly mixing. The reaction was rotated at 37 ℃ for 48 hours.

l) sealing: MES buffer solution is prepared into 75mg/ml Gly solution and 25mg/ml NaBH₃Adding CN solution into the solution according to the volume ratio of 2:1:10 of the reaction solution, mixing uniformly, and carrying out rotary reaction for 2 hours at 37 ℃. Then, 200mg/ml BSA solution (MES buffer) was added thereto at a volume ratio of 5:8, and the mixture was rapidly mixed and subjected to a rotary reaction at 37 ℃ for 16 hours.

m) cleaning: adding MES buffer solution into the reacted solution, centrifuging by a high-speed refrigerated centrifuge, removing supernatant, adding fresh MES buffer solution, resuspending by an ultrasonic method, centrifuging again, washing for 3 times, finally suspending by using a small amount of donor particle buffer solution, measuring solid content, and adjusting the concentration to 150 mu g/ml by using the donor particle buffer solution to obtain the donor reagent containing donor particles.

n) the average diameter of the donor particles in the gaussian distribution measured by a nano-particle sizer was 249.9nm, and the coefficient of variation (C.V.) -11.6%, as shown in fig. 2.

Example 3: preparation of receptor reagents

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 10min, N was introduced thereinto₂ 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 in the step 1, and continuously introducing N₂ 30min；

3) Heating the reaction system to 70 ℃ and reacting 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 with deionized water by secondary centrifugal sedimentation until the conductivity of the supernatant at the beginning of centrifugation is close to that of the deionized water, then diluting with water, and storing in an emulsion form;

5) the mean particle size of the latex microspheres in the Gaussian distribution was 202.2nm and the coefficient of variation (C.V.) -4.60% as measured by a nanometer particle sizer.

2. Process and characterization of embedding luminescent compositions within microspheres

1) A25 ml round-bottom flask was prepared, and 0.1g of a dimethylthiophene derivative and 0.1g of europium (III) complex (MTTA-EU) were added³⁺) 10ml of 95% ethanol, magnetically stirring, heating in a water bath to 70 ℃ to obtain a complex solution;

2) preparing a 100ml three-neck flask, adding 10ml of 95% ethanol, 10ml of water and 10ml of aldehyde polystyrene latex microspheres with the concentration of 10% obtained in the step 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), reacting for 2 hours at 70 ℃, stopping stirring, and naturally cooling;

4) and centrifuging the emulsion for 1 hour at 30000G, and removing supernatant after centrifugation to obtain the aldehyde polystyrene microspheres embedded with the luminescent composition.

5) The average particle size of Gaussian distribution of the particle size of the microspheres measured by a nanometer particle size analyzer was 204.9nm, and the coefficient of variation (C.V.) -5.00%.

3. Process and characterization for coating polysaccharide coating on microsphere surface

1) Taking 50mg of aminodextran solid, putting the aminodextran solid in a 20mL round-bottom flask, adding 5mL of 50mM/pH 10 carbonate buffer solution, and stirring and dissolving the aminodextran solid at 30 ℃ in the dark;

2) adding 100mg of aldehyde polystyrene microspheres which are prepared in the step 2 and are filled with the luminescent composition into the aminodextran solution, and stirring for 2 hours;

3) dissolving 10mg of sodium borohydride in 0.5mL of 50mM/pH 10 carbonate buffer solution, dropwise adding the solution into the reaction solution, and reacting overnight at 30 ℃ in a dark place;

4) after the reaction, the mixture 30000G was centrifuged, the supernatant was discarded, and 50mM/pH 10 carbonate buffer was added thereto for ultrasonic dispersion. After repeated centrifugal washing for three times, the solution is fixed by 50mM/pH 10 carbonate buffer solution to a final concentration of 20 mg/ml;

5) adding 100mg aldehyde dextran solid into a 20mL round-bottom flask, adding 5mL 50mM/pH 10 carbonate buffer, and stirring and dissolving at 30 ℃ in the dark;

6) adding the microspheres into an aldehyde dextran solution and stirring for 2 hours;

7) dissolving 15mg of sodium borohydride in 0.5mL of 50mM/pH 10 carbonate buffer solution, dropwise adding the solution into the reaction solution, and reacting overnight at 30 ℃ in a dark place;

8) after the reaction, the mixture 30000G was centrifuged, the supernatant was discarded, and 50mM/pH 10 carbonate buffer was added thereto for ultrasonic dispersion. After repeating the centrifugal washing three times, the volume was adjusted to 20mg/ml by using 50mM/pH 10 carbonate buffer.

9) The average particle size of Gaussian distribution of the particle size of the microspheres measured by a nanometer particle size analyzer was 241.6nm, and the coefficient of variation (C.V.) was 12.90%.

Conjugation procedure for PCT antibody

1) The paired PCT antibody was dialyzed into 50mM CB buffer at PH 10 to a measured concentration of 1 mg/ml.

2) Adding 0.5ml of microspheres obtained in the step 3 and 0.5ml of paired antibody I obtained in the step 1) into a 2ml centrifuge tube, uniformly mixing, and adding 100 mu l of 10mg/ml NaBH₄The solution (50mM CB buffer) was reacted at 2-8 ℃ for 4 hours.

3) After completion of the reaction, 0.5ml of 100mg/ml BSA solution (50mM CB buffer) was added thereto, and the reaction was carried out at 2 to 8 ℃ for 2 hours.

4) After completion of the reaction, the reaction mixture was centrifuged at 30000G for 45min, and the supernatant was discarded after centrifugation and resuspended in 50mM MES buffer. The centrifugal washing was repeated four times, and diluted with a buffer solution to a final concentration of 50. mu.g/ml to obtain an antibody I-conjugated receptor reagent.

5) The mean particle size of the Gaussian distribution of the acceptor particle size at this time was 253.5nm as measured by a nanometer particle sizer, and the coefficient of variation (C.V value) was 9.60% (as shown in fig. 3).

Example 4: detection of sugar content of microspheres by anthrone method

a) Pretreatment of microsphere samples:

the donor reagent A containing 1mg of donor microspheres a in example 1, the donor reagent B containing 1mg of donor microspheres B in example 2 and the acceptor reagent containing 1mg of acceptor microspheres in example 3 were respectively taken, centrifuged at 20000g for 40min, the supernatant was removed, ultrasonically dispersed with purified water, and repeatedly centrifuged and dispersed three times, and then the volume was adjusted to 1mg/mL with purified water.

b) Preparing a glucose standard solution:

the 1mg/mL glucose stock solution was prepared as a standard solution at 0mg/mL, 0.025mg/mL, 0.05mg/mL, 0.075mg/mL, 0.10mg/mL, 0.15mg/mL with purified water.

c) Preparing an anthrone solution: the solution is prepared into 2mg/mL by 80 percent sulfuric acid solution (the solution is stable within 24 hours at room temperature, and the solution is prepared as before use).

d) 0.1mL of glucose standard solution with each concentration and a sample to be detected are respectively added into a centrifuge tube, and 1mL of anthrone test solution is respectively added into each tube.

e) Incubate at 85 ℃ for 30 min.

f) And centrifuging the sample reaction tube at 15000g for 40min, and sucking clear liquid from the bottom of the tube by a pipette tip to measure the absorbance so as to avoid sucking suspended matters out of the upper part.

g) The temperature was returned to room temperature and the absorbance at 620nm was measured (the measurement was preferably carried out within 2 h).

h) And (3) performing linear regression by taking the concentration of the standard solution as an X value and the absorbance as a Y value, and detecting the sugar concentration of the sample to be detected by using the detection result and the obtained standard curve as shown in table 1 and figure 4 respectively.

TABLE 1

Serial number	Concentration mg/mL	Absorbance A	Absorbance B	Absorbance mean value
					1	0	0.0008	0.0008	0.0008
2	0.025	0.0880	0.0916	0.0898
					3	0.05	0.1547	0.1611	0.1579
4	0.075	0.2375	0.2471	0.2423
					5	0.1	0.3190	0.332	0.3255
6	0.15	0.4855	0.5053	0.4954

And (3) detection results:

sugar content of donor microsphere a in example 1: 11.3. mu.g/mg

Sugar content of donor microsphere b in example 2: 40.8. mu.g/mg

Sugar content of acceptor microspheres in example 3: 60.4. mu.g/mg

Example 5: preparation reagent strip and kit

The prepared reagent strip 11 is shown in fig. 5, and is provided with a plurality of reagent containing holes 111, wherein the holes comprise:

a first reagent well site for holding the donor reagent prepared in example 1 or example 2, wherein the concentration of the donor particles is 150 ug/ml.

A second reagent well site for holding the receptor reagent prepared in example 3, wherein the concentration of the receptor particles is 50 ug/ml;

a sample hole site for containing a sample to be tested; and

and detecting the hole position by using the signal.

Alternatively, the cross-section of the hole site 111 may be circular, elliptical, or rectangular. Preferably, the plurality of wells differ from each other in cross-section to distinguish different reagents contained therein.

Optionally, a third reagent well and/or a fourth reagent well for carrying diluent and/or additional reagent may also be provided in the reagent strip 11. When the wells for holding the diluent and the additional reagent are provided, the order of adding the diluent and the additional reagent is as follows. And adding the sample to be detected into the third reagent hole site carrying the diluent, and mixing the sample to be detected with the diluent, thereby performing dilution operation. And after the dilution is finished, adding a certain volume of the diluted sample to be detected into a fifth reagent hole site carrying the additional reagent, and mixing the sample with the additional reagent. And then, continuously taking a certain volume of mixed liquid and adding the mixed liquid into the hole position carrying the first reagent. After a certain time of reaction, continuously taking a certain volume of mixed liquid, adding the mixed liquid into a hole carrying a second reagent, and carrying out subsequent processes.

In this embodiment, the end faces of the holes 111 are coated with a film to close the openings. The coating film can be a disposable sealing film or a repeatedly used sealing film.

In this embodiment, a barcode region 112 is provided on the side of the reagent strip. The barcode region 112 contains information on the reagent strip. For example, the barcode region 112 is provided with a barcode, which may be a one-dimensional code or a two-dimensional code.

The donor reagent prepared in example 1 and/or example 2, the acceptor reagent prepared in example 3, and the reagent strip prepared above are assembled to obtain the homogeneous chemiluminescent POCT assay kit described herein.

Example 6: homogeneous phase chemiluminescence POCT detection device

The principle of the homogeneous chemiluminescence POCT detection device in the embodiment is as follows: the target molecules to be detected in the sample to be detected react with the donor particles and the acceptor particles to form immune complexes, the donor particles and the acceptor particles are drawn close by the interaction, and under the irradiation of laser (the wavelength is 680nm), the sensitizer in the donor particles converts oxygen in the surrounding environment into more active monomer oxygen. The monomer oxygen diffuses to the acceptor particle and reacts with the chemiluminescence agent in the acceptor particle to further activate the luminescent group on the acceptor particle to enable the luminescent group to emit light with the wavelength of 520-620 nm. The half-life of the monomeric oxygen is 4 [ mu ] Sec, and the diffusion distance in the solution is about 200 nm. If there is no interaction between the biomolecules and singlet oxygen cannot diffuse to the receptor particle, no light signal is generated. Therefore, the concentration of the target molecule to be detected in the sample to be detected can be calculated by measuring the intensity of light emitted by the mixture. Wherein the donor particle comprises a first support having a sensitizer filled therein and having one of the members of the specific binding pair attached to a surface thereof; the receptor particles comprise a second carrier, the interior of the second carrier is filled with a luminescent composition, the surface of the second carrier is coated with a coating layer, the surface of the coating layer is connected with a reporter molecule, and the reporter molecule can be specifically combined with a target molecule to be detected.

A preferred structure of the photo-excitation chemiluminescence immunoassay analyzer in this embodiment includes the following modules:

the reagent sample adding module is used for adding a sample to be detected, an acceptor reagent and/or a donor reagent; wherein the donor reagent comprises donor particles, and the variation coefficient C.V value of the particle size distribution of the donor particles in the donor reagent is more than or equal to 5%; the variation coefficient C.V value of the particle size distribution of the receptor particles in the receptor reagent is more than or equal to 5%;

an incubation module for controlling the temperature of the reagent strip; the incubation module can adopt a metal bath, a water bath or an oil bath and the like;

Example 7: test results and analysis on computer (test substance: PCT antigen)

(1) The donor reagents of example 1 and example 2 were simultaneously loaded with the acceptor reagent of example 3 using the assay device of example 5 to detect PCT antigens, and the results are shown in table 2. The PCT quantitative assay detection kit (photo-activated chemiluminescence method) used in this example was composed of a reagent 1(R1 ') containing acceptor particles coated with a primary anti-PCT antibody, a reagent 2(R2 ') containing a biotin-labeled secondary anti-PCT antibody, and additionally included a general-purpose liquid (R3 ') containing donor particles. Wherein R1' is the receptor reagent prepared using the receptor particles (particle size distribution variation coefficient C.V value: 9.6%) in example 3; r3' is the donor reagent prepared using the donor particles of examples 1 and 2.

TABLE 2

As can be seen from the results in table 2, the sensitivity and the upper limit of detection of the kit provided in the present application are excellent. And the sensitivity and upper limit of detection of the kit comprising the donor reagent in example 1 are superior to those of the kit comprising the donor reagent in example 2. It can be seen that the performance of the donor particles using the non-polysaccharide coated particles is more excellent.

Example 8:

a series of kits (shown in the following table) containing donor and acceptor microspheres with different sugar contents were prepared according to the methods given in examples 1-3, and then cross-assembled into a kit as shown in example 5, and the signal level of each kit for the same batch of samples was detected on a phase-chemiluminescence POCT detection device as given in example 6. the detection flow of the POCT device is as follows: after respectively adding 25 muL of a sample to be detected, 25 muL of a biotin-labeled antibody, 175 muL of a donor reagent and 25 muL of an acceptor reagent into a sample hole site, an additional reagent hole site, a first reagent hole site and a second reagent hole site of the reagent strip, placing the reagent strip into a POCT analyzer developed by Boyang biotechnology (Shanghai) Limited, adding a sample with a corresponding volume to be detected into the additional reagent hole site by a sample adding mechanism, vibrating and incubating at 37 ℃ for 10 minutes; adding the incubated liquid in the additional reagent hole site into the first reagent hole site, vibrating, and incubating at 37 ℃ for 10 minutes to form mixed liquid; the mixed liquid is continuously added into the second reagent hole site, vibrated, incubated at 37 ℃ for 10 minutes to form a mixture to be detected, and then moved to the signal detection hole site. The laser irradiation signal emitted by the exciter in the optical excitation and detection module is used to detect the mixed liquid to be detected in the hole site, and the generated chemiluminescence signal is detected, and the result is shown in table 3.

TABLE 3

Example 9: detection of clinical samples of CRP at different concentrations

On the test device in example 6, 50 μ L of clinical samples (containing serum and whole blood) with different concentrations of CRP were added to the test strip, the mean value was taken in parallel channels of each well, 50 μ L of biotinylated anti-CRP antibody, 50 μ L of acceptor reagent containing coupled CRP acceptor particles (sugar content per mg acceptor particles is 59.3ug, average particle size of acceptor particles in Gaussian distribution curve is 253.6nm, and coefficient of variation of particle size distribution is C.V value is 10.9%), reaction was carried out at 37 ℃ for 7.5min, 50 μ L of donor reagent (sugar content per mg donor particles is 9.5ug/mg microparticles, average particle size of donor particles in Gaussian distribution curve is 226.7nm, and coefficient of variation of particle size distribution is C.V value is 8.1%) was further added, and photo-excitation test was carried out at 37 ℃ for 5min, and the test results are shown in table 4 and fig. 6. As can be seen from table 4 and fig. 6, the correlation coefficient using serum and whole blood reached 0.9988. The experimental result shows that the donor particle greatly reduces the nonspecific adsorption in the sample, has very good correlation between the measurement results aiming at the serum and the whole blood, greatly enhances the adaptability of the donor reagent to the clinical sample, and can be directly used for the detection of the clinical whole blood sample.

TABLE 4

It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.

Claims

1. A homogeneous chemiluminescent assay kit comprising a donor reagent comprising a donor particle capable of generating reactive oxygen species upon excitation and an acceptor reagent comprising a second buffer solution and an acceptor particle capable of reacting with the reactive oxygen species received to generate a chemiluminescent signal,

2. The kit of claim 1, wherein the surface of the first support has directly bound thereto one of the members of the specific binding pair.

3. The kit of claim 2, wherein the surface of the first carrier is free of coating or has attached thereto a polysaccharide substance that directly binds to one of the members of the specific binding pair.

4. A kit according to claim 3, wherein the surface of the first support carries a bonding functionality for directly bonding one of the members of the specific binding pair to the surface of the first support.

5. The kit according to claim 4, wherein the bonding functional group is selected from the group consisting of an amine group, an amide group, a hydroxyl group, an aldehyde group, a carboxyl group, a maleimide group and a thiol group; preferably selected from aldehyde groups and/or carboxyl groups.

6. The kit of any one of claims 1 to 5, wherein the specific binding pair member is selected from a pair of substances capable of specifically binding to each other, consisting of an antibody, an antibody fragment, a ligand, an oligonucleotide binding protein, a lectin, a hapten, an antigen, an immunoglobulin binding protein, avidin or biotin.

7. Kit according to claim 6, characterized in that the specific binding pair member is avidin-biotin, said avidin being selected from the group consisting of ovalbumin, streptavidin, vitellin, neutravidin and avidin-like, preferably neutravidin and/or streptavidin.

8. The kit according to claim 6 or 7, wherein the avidin is chemically bonded to the surface of the first carrier by reacting an amino group with an aldehyde group on the surface of the first carrier to form a Schiff base.

9. The kit according to any one of claims 1 to 8, wherein the acceptor particle has a particle size distribution variation coefficient C.V value of 5% or more in the acceptor reagent.

10. The kit of claim 9, wherein the acceptor particle has a particle size distribution variation coefficient C.V value of 8% or more in the acceptor reagent; preferably, the acceptor particle has a variation coefficient C.V value of 10% or more in the particle size distribution of the acceptor reagent.

11. The kit of claim 9 or 10, wherein the recipient particle has a particle size distribution coefficient of variation C.V value of 40% or less in the recipient reagent; still more preferably, the recipient particle has a particle size distribution variation coefficient C.V value of 20% or less in the recipient agent.

12. The kit of any one of claims 9 to 11, wherein the acceptor particles have a particle size distribution that exhibits polydispersity in the acceptor reagent.

13. The kit according to any one of claims 9 to 12, wherein the value of the variation coefficient C.V of the particle size distribution is calculated by a Gaussian distribution.

14. The kit according to any one of claims 9 to 13, wherein the receptor particle exhibits two or more peaks in a Gaussian distribution curve in the receptor reagent by a Gaussian distribution analysis method.

15. The kit of any one of claims 9 to 14, wherein the receptor reagent comprises at least two receptor particles having an average particle size distribution.

16. The kit according to any one of claims 1 to 15, wherein the coating in the coating layer is selected from polysaccharides, high molecular polymers or biological macromolecules, preferably polysaccharides;

17. The kit of claim 16, wherein each polysaccharide layer in the continuous polysaccharide coating spontaneously associates with a previous polysaccharide layer.

18. The kit of claim 16 or 17, wherein the polysaccharide has pendant functional groups, the pendant functional groups of any one of the successive polysaccharide layers being oppositely charged from the pendant functional groups of the previous polysaccharide layer.

19. The kit of claim 16 or 17, wherein the polysaccharide has pendant functional groups and any polysaccharide layer in the continuous polysaccharide coating is covalently linked to a preceding polysaccharide layer by a chemical bonding reaction between the pendant functional groups and the pendant functional groups of the preceding polysaccharide layer.

20. The kit of claim 18 or 19, wherein the pendant functional groups of the continuous polysaccharide coating alternate between amine functional groups and amine reactive functional groups.

21. The kit of claim 20, wherein the amine-reactive functional group is an aldehyde group or a carboxyl group.

22. The kit of any one of claims 16 to 21, wherein the outermost polysaccharide layer of the continuous polysaccharide coating has at least one pendant functional group.

23. The kit according to claim 18 to 22, wherein the pendant functional group is at least one selected from the group consisting of aldehyde group, carboxyl group, mercapto group, amino group, hydroxyl group and maleic amine group; preferably selected from aldehyde groups and/or carboxyl groups.

24. The kit of claim 22 or 23, wherein the continuous polysaccharide coating pendant functional groups are chemically bonded directly or indirectly to one of the members of the specific binding pair.

25. The kit according to any one of claims 16 to 24, wherein the polysaccharide is selected from the group consisting of carbohydrates comprising three or more unmodified or modified monosaccharide units; preferably selected from the group consisting of dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably selected from dextran, starch, glycogen and polyribose.

26. The kit according to any one of claims 1 to 25, wherein the sugar content is detected by an anthrone method;

27. A homogeneous chemiluminescent POCT assay kit comprising the kit of any one of claims 1 to 26, wherein POCT is a point of care test or clinical assay performed in the vicinity of a patient.

28. The kit according to claim 27, wherein the kit comprises a reagent strip, wherein a plurality of holes for containing reagents are provided on the reagent strip, and the holes at least comprise:

a first reagent well site for holding said donor reagent;

a second reagent well site for holding said acceptor reagent.

29. A homogeneous chemiluminescent POCT assay for detecting a target molecule in a test sample using the kit of claim 27 or 28.

30. A homogeneous chemiluminescent POCT assay device for detecting a target molecule in a sample using the kit of claim 27 or 28 or the method of claim 29.

31. The homogeneous chemiluminescent POCT assay device of claim 30, comprising:

an incubation module for controlling the temperature of the reagent strip of claim 28;

32. A method of performing homogeneous chemiluminescent assay using the POCT test device of claim 30 or 31 comprising the steps of: