A homogeneous chemiluminescence detection kit and its application

Abstract

The present invention relates to a homogeneous chemiluminescence detection kit and its application. The kit includes a donor reagent and an acceptor reagent. The donor reagent contains donor particles, and the acceptor reagent contains acceptor particles. The donor particles include a first carrier, which is filled with a sensitizer and has a specific binding pair member directly or indirectly attached to its surface. The sugar content per milligram of the donor particles is no more than 25 μg. The acceptor particles include a second carrier, which is filled with a luminescent composition and coated with a coating layer. The coating layer is attached to a reporter molecule capable of specifically binding to a target molecule to be detected. The sugar content per milligram of the donor particles is no less than 40 μg. The POCT detection method performed using the kit has the characteristics of high sensitivity, high precision, wide range, rapidity, and portability.

Description

Technical Field

[0001] The present invention belongs to the technical field of homogeneous chemiluminescence, and in particular relates to a homogeneous chemiluminescence detection kit and application thereof. Background Art

[0002] Homogeneous chemiluminescence analysis refers to a method that can perform chemiluminescence detection without separating the complex formed after binding and the remaining free reactants.

[0003] Existing homogeneous chemiluminescence analysis has the following disadvantages:

[0004] A. The instrument system is bulky and occupies a large area. At the same time, due to the high test throughput, the reagent card is used in units of 100 tests, which places requirements on the laboratory sample size;

[0005] B. The instrument system and reagents are expensive and have high maintenance costs, making them unsuitable for primary medical institutions;

[0006] C. The instrument is large and cannot be carried into the diagnosis and treatment site;

[0007] D. Chemiluminescence systems mainly use serum and plasma as samples, and generally cannot use whole blood, which limits their scope of use.

[0008] At the same time, a new point-of-care testing (POCT) technology has emerged in recent years, specifically bedside testing, which involves clinical testing performed at the patient's side. The mainstream POCT technologies are fluorescent quantitative chromatography or colloidal gold, which primarily utilize fluorescent microspheres or colloidal gold coated with fluorescent substances for rapid immunoassays via membrane chromatography. However, since both technologies primarily perform release testing on NC membranes, the membrane's inherent CV (variable constant) (CV) is already over 5%. Therefore, solid-phase membrane-based POCT testing typically has a CV exceeding 10%, resulting in poor precision. This makes quantification extremely difficult for tests requiring high sensitivity, such as cTnI. Furthermore, while newer technologies, such as microfluidic chip-based POCT testing, offer advantages such as fast reaction speed and low sample volume, they also suffer from low sensitivity due to inadequate reaction times.

[0009] Therefore, there is an urgent need to provide a homogeneous chemiluminescence POCT detection kit and method with high sensitivity, high precision and wide range, as well as rapidity and portability. Summary of the Invention

[0010] The technical problem to be solved by the present invention is to provide a homogeneous chemiluminescence POCT detection kit and method. The POCT detection method performed using the kit combines the high sensitivity, high precision and wide range of chemiluminescence analysis technology with the characteristics of POCT detection technology such as rapidity and portability.

[0011] To this end, the first aspect of the present invention provides a homogeneous chemiluminescent detection kit, which includes a donor reagent and an acceptor reagent, wherein the donor reagent contains donor particles, which can generate reactive oxygen species after being excited, and the acceptor reagent contains a second buffer solution and acceptor particles, which can react with the received reactive oxygen species to generate a chemiluminescent signal.

[0012] The donor particles include a first carrier, the interior of the first carrier is filled with a sensitizer, a surface of the first carrier is directly or indirectly connected to one of the specific binding pair members, and the sugar content in each milligram of the donor particles is no more than 25 μg;

[0013] The acceptor particles include 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 to a reporter molecule, the reporter molecule can specifically bind to the target molecule to be detected, and the sugar content in each milligram of the donor particles is not less than 40ug.

[0014] In some embodiments of the present invention, one of the specific binding pair members is directly bonded to the surface of the first carrier.

[0015] In other embodiments of the present invention, the surface of the first carrier is not coated or linked with polysaccharide substances, and it directly bonds to one of the specific binding pair members.

[0016] In some embodiments of the present invention, the surface of the first carrier carries a bonding functional group, and the bonding functional group is used to directly bond one of the specific binding pair members to the surface of the first carrier.

[0017] In other embodiments of the present invention, the bonding functional group is selected from amine, amide, hydroxyl, aldehyde, carboxyl, maleimide and thiol; preferably selected from aldehyde and / or carboxyl.

[0018] In some embodiments of the present invention, the specific binding pair members are selected from a pair of substances that can specifically bind to each other, including antibodies, antibody fragments, ligands, oligonucleotides, oligonucleotide binding proteins, lectins, haptens, antigens, immunoglobulin binding proteins, avidin, avidin or biotin.

[0019] In other embodiments of the present invention, the specific binding pair member is avidin-biotin, and the avidin is selected from egg white avidin, streptavidin, egg yolk avidin, neutravidin and avidin-like, preferably neutravidin and / or streptavidin.

[0020] In some embodiments of the present invention, the avidin is chemically bonded to the surface of the first carrier by reacting the amino group with the aldehyde group on the surface of the first carrier to form a Schiff base.

[0021] In other embodiments of the present invention, the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is ≥5%.

[0022] In some preferred embodiments of the present invention, the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is ≥8%; preferably, the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is ≥10%.

[0023] In other preferred embodiments of the present invention, the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is ≤40%; more preferably, the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is ≤20%.

[0024] In some embodiments of the present invention, the particle size distribution of the receptor particles in the receptor reagent is polydisperse.

[0025] In other embodiments of the present invention, the coefficient of variation CV value of the particle size distribution is calculated using Gaussian distribution.

[0026] In some embodiments of the present invention, using Gaussian distribution analysis, the Gaussian distribution curve of the receptor particles in the receptor reagent presents two or more peaks.

[0027] In other embodiments of the present invention, the receptor reagent comprises receptor particles with at least two average particle size distributions.

[0028] In some embodiments of the present invention, the coating material in the coating layer is selected from polysaccharides, high molecular polymers or biomacromolecules, preferably polysaccharides;

[0029] Further preferably, the surface of the second carrier is coated with at least two continuous polysaccharide coatings, and one of the specific binding pair members is linked to the surface of the continuous polysaccharide coatings.

[0030] In other embodiments of the present invention, each polysaccharide layer in the continuous polysaccharide coating layer spontaneously associates with the previous polysaccharide layer.

[0031] In some embodiments of the present invention, the polysaccharide has pendant functional groups, and the pendant functional groups of any polysaccharide layer in the continuous polysaccharide layers have charges opposite to those of the pendant functional groups of the previous polysaccharide layer.

[0032] In other embodiments of the present invention, the polysaccharide has pendant functional groups, and any polysaccharide layer in the continuous polysaccharide coating is covalently linked to the previous polysaccharide layer through a chemical bonding reaction between the pendant functional groups and the pendant functional groups of the previous polysaccharide layer.

[0033] 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.

[0034] In other embodiments of the present invention, the amine-reactive functional group is an aldehyde group or a carboxyl group.

[0035] In some embodiments of the present invention, the outermost polysaccharide layer of the continuous polysaccharide coating has at least one pendant functional group.

[0036] In other embodiments of the present invention, the pendant functional group is selected from at least one of aldehyde, carboxyl, thiol, amino, hydroxyl and malein; preferably selected from aldehyde and / or carboxyl.

[0037] In some embodiments of the present invention, the pendant functional groups of the continuous polysaccharide coating are directly or indirectly chemically bonded to a member of a specific binding pair.

[0038] In other embodiments of the present invention, the polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from glucan, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxyglucan and aminoglucan; more preferably selected from glucan, starch, glycogen and polyribose.

[0039] In some embodiments of the present invention, the sugar content is detected by anthrone method;

[0040] Preferably, the sugar is selected from carbohydrates containing three or more unmodified or modified monosaccharide units, preferably selected from glucan, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxyglucan and aminoglucan; more preferably selected from glucan, starch, glycogen and polyribose;

[0041] More preferably, the molecular weight distribution Mw of the dextran is 1000-1000000 KDa, preferably 10000-800000 KDa, more preferably 30000-700000 KDa.

[0042] The second aspect of the present invention provides a homogeneous chemiluminescent POCT detection kit, which includes the kit as described in the first aspect of the present invention. The POCT refers to a point-of-care test or a clinical test performed at the patient's side.

[0043] In some embodiments of the present invention, the kit includes a reagent strip having a plurality of wells for containing reagents, the wells including at least:

[0044] The first reagent well is used to hold the donor reagent;

[0045] The second reagent well is used to contain the receptor reagent.

[0046] The third aspect of the present invention provides a homogeneous chemiluminescence POCT detection method, which uses the kit as described in the second aspect of the present invention to detect the target molecule in the sample to be tested.

[0047] The fourth aspect of the present invention provides a homogeneous chemiluminescence POCT detection device, which uses the kit described in the second aspect of the present invention or the method described in the third aspect of the present invention to detect the target molecule in the sample to be tested.

[0048] In some embodiments of the invention, the apparatus comprises:

[0049] an incubation module, for controlling the temperature of the reagent strip as described in the second aspect of the present invention;

[0050] The light excitation and detection module is arranged on one side of the incubation module, and is used to emit excitation light to the reagent strip to generate a photo-induced chemiluminescence reaction; and detect the chemiluminescence signal generated by the reagent.

[0051] A fifth aspect of the present invention provides a method for performing homogeneous chemiluminescence analysis using the POCT detection device according to the fourth aspect of the present invention, comprising the following steps:

[0052] S1, contacting the sample to be tested with the receptor reagent and the donor reagent to generate a test mixture after reaction;

[0053] S2, using excitation light with a wavelength of 600 to 700 nm to excite the test mixture to perform chemiluminescence, and detecting the signal intensity of the chemiluminescence; the detection wavelength of the chemiluminescence is 520 to 620 nm;

[0054] S3, judging whether the sample to be tested contains the target molecule to be tested and / or the concentration of the target molecule to be tested in the sample to be tested based on the analysis of the chemiluminescence signal intensity.

[0055] The beneficial effects of the present invention are as follows: the kit of the present invention contains a donor reagent containing specific donor particles and / or a receptor reagent containing specific receptor particles, the donor particles have high efficiency in generating reactive oxygen species, the reactive oxygen species are more easily transferred to the receptor particles in a homogeneous system, and are not easily interfered with by other substances, and the donor particles themselves have high stability, can stably exist in the donor reagent, and are not easily inactivated; at the same time, 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 coefficient of variation CV value of the particle size distribution of the receptor particles is ≥5%, so that the phase chemiluminescence POCT detection performed using the kit of the present invention combines the high sensitivity, high precision and wide range of chemiluminescence analysis technology, and at the same time has the characteristics of rapidity and portability of POCT detection technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The present invention will be further described below with reference to the accompanying drawings.

[0057] Figure 1 is the Gaussian distribution diagram of the donor particles prepared in Example 1.

[0058] Figure 2 This is the Gaussian distribution diagram of the donor particles prepared in Example 2.

[0059] Figure 3 This is the Gaussian distribution diagram of the receptor particles with an average particle size of about 250 nm prepared in Example 3.

[0060] Figure 4 This is the standard curve for determining the sugar content in Example 4.

[0061] Figure 5 Schematic diagram of the structure of the reagent strip in Example 5.

[0062] Figure 6 This is a correlation coefficient diagram for the detection of different concentrations of CRP in serum and whole blood in Example 9. DETAILED DESCRIPTION

[0063] To facilitate understanding of the present invention, the present invention will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is intended solely to describe specific embodiments and is not intended to be limiting. The practice of the present invention is not limited to the following examples, and any modifications and / or variations made to the present invention fall within the scope of the present invention.

[0064] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of the range and any other specified or intervening values ​​in the specified range is encompassed within the present invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the present invention, subject to any express exclusions in the specified range. Where a specified range includes one or both limits, ranges excluding either or both of those included limits are also encompassed within the present invention.

[0065] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present 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, preferred methods and materials are now described.

[0066] I. Terminology

[0067] The term "active oxygen species" as used in the present invention refers to a general term for substances composed of oxygen, containing oxygen and having active properties in the body or the natural environment, mainly an excited state of oxygen molecules, including the one-electron reduction product of oxygen, the superoxide anion (O2·-), the two-electron reduction product of oxygen, the three-electron reduction product of oxygen, the hydroxyl radical (·OH), as well as nitric oxide and singlet oxygen (1O2).

[0068] The term "donor particles" as used herein refers to particles containing a sensitizer that can generate active intermediates such as reactive oxygen species that react with receptor particles after being activated by energy or an active compound. The donor particles can be photoactivated (such as dyes and aromatic compounds) or chemically activated (such as enzymes, metal salts, etc.). In some specific embodiments of the present invention, the donor particles are polymer microspheres filled with a photosensitizer, which can be a photosensitizer known in the art, preferably a compound that is relatively stable to light and does not effectively react with singlet oxygen. Non-limiting examples include compounds such as methylene blue, rose bengal, porphyrin, phthalocyanine, and chlorophyll disclosed in U.S. Pat. No. 5,709,994 (which is incorporated herein by reference in its entirety), as well as derivatives of these compounds having 1-50 atom substituents, the substituents being used to make these compounds more lipophilic or more hydrophilic, and / or as linking groups for attachment to specific binding pair members. Examples of other photosensitizers known to those skilled in the art can also be used in the present invention, such as those described in U.S. Pat. No. 6,406,913, which is incorporated herein by reference.

[0069] As used herein, the term "acceptor particles" refers to particles containing a compound capable of reacting with reactive oxygen species to produce a detectable signal. Donor particles are activated by energy or the reactive compound and release high-energy reactive oxygen species. These high-energy reactive oxygen species are captured by nearby acceptor particles, transferring energy to activate the acceptor particles. In some embodiments of the present invention, the acceptor particles comprise a luminescent composition and a carrier, wherein the luminescent composition is filled in the carrier and / or coated on the surface of the carrier.

[0070] The "carrier" of the present invention is selected from strips, sheets, rods, tubes, wells, microtiter plates, beads, particles and microspheres. It can be microspheres or microparticles known to those skilled in the art. It can be of any size. It can be organic or inorganic. It can be expandable or non-expandable. It can be porous or non-porous. It can be magnetic or non-magnetic. It has any density, but preferably has a density close to that of water, preferably can float in water, and is made of transparent, partially transparent or opaque material.

[0071] In the present invention, the "chemiluminescent compound" is a compound, also known as a label, that can undergo a chemical reaction to induce luminescence, such as by being converted into another compound in an electronically excited state. The excited state can be a singlet or triplet excited state. The excited state can relax to the ground state directly, emitting light, or it can transfer the excitation energy to an emitting energy acceptor, thereby returning to the ground state. During this process, the energy acceptor particle will transition to an excited state, emitting light.

[0072] The "specific binding pair members" of the present invention refer to a pair of substances that can specifically bind to each other.

[0073] The "coefficient of variation of particle size distribution CV value" mentioned in the present invention refers to the coefficient of variation of particle size in Gaussian distribution in the detection results of nanoparticle size analyzer. The formula for calculating the coefficient of variation is: CV value = (standard deviation SD / mean value) × 100%.

[0074] The term "Nicomp distribution" as used herein refers to an algorithmic distribution used in the American PSS nanoparticle size analyzer NICOMP. Compared to the Gaussian unimodal algorithm, the Nicomp multimodal algorithm offers unique advantages for analyzing multicomponent liquid dispersions with uneven particle size distributions, as well as for analyzing the stability of colloidal systems.

[0075] The term "test sample" as used herein refers to a mixture containing or suspected of containing the target molecule to be tested. The test sample that can be used in the present invention includes body fluids, such as blood (which can be anticoagulated blood commonly seen in collected blood samples), plasma, serum, urine, semen, saliva, cell culture, tissue extracts, etc. Other types of test samples include solvents, seawater, industrial water samples, food samples, environmental samples such as soil or water, plant materials, eukaryotic cells, bacteria, plasmids, viruses, fungi, and cells from prokaryotes. The test sample can be diluted with diluent as needed before use. For example, in order to avoid the HOOK effect, the test sample can be diluted with diluent before being tested on the machine and then tested on the detection instrument.

[0076] As used herein, the term "target molecule to be detected" refers to a substance in a sample to be detected. One or more substances having a specific binding affinity for the target molecule to be detected are used to detect the target molecule. The target molecule to be detected can be a protein, peptide, antibody, or a hapten that can bind to an antibody. The target molecule to be detected can be a nucleic acid or oligonucleotide that binds to a complementary nucleic acid or oligonucleotide. The target molecule to be detected can be any other substance that can form a specific binding pair member. Other typical examples of target molecules to be detected include: drugs such as steroids, hormones, proteins, glycoproteins, mucins, nucleoproteins, phosphoproteins, drugs of abuse, vitamins, antibacterial drugs, antifungal drugs, antiviral drugs, purines, antitumor agents, amphetamines, nitrogen compounds, nucleic acids, and prostaglandins, as well as metabolites of any of these drugs; pesticides and their metabolites; and receptors. Analytes also include cells, viruses, bacteria, and fungi.

[0077] 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. Where desired, the antibody may be further conjugated to other moieties, such as a member of a specific binding pair, e.g., biotin or avidin (a member of a biotin-avidin specific binding pair), etc.

[0078] The term "antigen" as used in the present invention refers to a substance that can stimulate the body to produce an immune response and can combine with the immune response products antibodies and sensitized lymphocytes in vivo or in vitro to produce an immune effect.

[0079] As used herein, the term "binding" refers to the direct association between two molecules due to interactions such as covalent, electrostatic, hydrophobic, ionic, and / or hydrogen bonds, including but not limited to salt and water bridges. The term "specific binding" as used herein refers to the mutual recognition and selective binding reaction between two substances, which, from a stereostructural perspective, refers to the conformational correspondence between the corresponding reactants. Within the technical concepts disclosed herein, methods for detecting specific binding reactions include, but are not limited to, double antibody sandwich assays, competitive assays, neutralization competition assays, indirect assays, or capture assays.

[0080] II. Specific implementation plan

[0081] The present invention will be described in more detail below with reference to the embodiments.

[0082] The inventors of the present application studied the sugar content and particle size uniformity in the donor particles and the acceptor particles and found that the sugar content in the donor particles should not be too high, and the sugar content in each milligram of the donor particles should not be higher than 25ug, while the sugar content in the acceptor particles should not be too low, and the sugar content in each milligram of the donor particles should not be lower than 40ug. In addition, by using microspheres with appropriate particle size uniformity (such as the coefficient of variation of the microsphere particle size distribution is greater than 5%), the sensitivity of photochemiluminescence detection can be guaranteed and the detection range can be broadened.

[0083] Therefore, the homogeneous chemiluminescent detection kit involved in the first aspect of the present invention comprises: a donor reagent and an acceptor reagent, wherein the donor reagent contains donor particles, and the donor particles can generate reactive oxygen species after being excited; the acceptor reagent contains a second buffer solution and acceptor particles, and the acceptor particles can react with the received reactive oxygen species to generate a chemiluminescent signal;

[0084] The donor particles include a first carrier, the interior of the first carrier is filled with a sensitizer, a surface of the first carrier is directly or indirectly connected to one of the specific binding pair members, and the sugar content in each milligram of the donor particles is no more than 25 μg;

[0085] The acceptor particles include 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 to a reporter molecule, the reporter molecule can specifically bind to the target molecule to be detected, and the sugar content in each milligram of the donor particles is not less than 40ug.

[0086] In some embodiments of the present invention, one of the specific binding pair members is directly bonded to the surface of the first carrier.

[0087] In other embodiments of the present invention, the surface of the first carrier is not coated or linked with polysaccharide substances, and it directly bonds to one of the specific binding pair members.

[0088] In some embodiments of the present invention, the surface of the first carrier carries a bonding functional group, and the bonding functional group is used to directly bond one of the specific binding pair members to the surface of the first carrier.

[0089] In other embodiments of the present invention, the bonding functional group is selected from amine, amide, hydroxyl, aldehyde, carboxyl, maleimide and thiol; preferably selected from aldehyde and / or carboxyl.

[0090] In some embodiments of the present invention, the content of the bonding functional groups on the surface of the first carrier is 100 to 500 nmol / mg, preferably 200 to 400 nmol / mg.

[0091] In some embodiments of the present invention, the specific binding pair members are selected from a pair of substances that can specifically bind to each other, including antibodies, antibody fragments, ligands, oligonucleotides, oligonucleotide binding proteins, lectins, haptens, antigens, immunoglobulin binding proteins, avidin, avidin or biotin.

[0092] In other embodiments of the present invention, the specific binding pair member is avidin-biotin, and the avidin is selected from egg white avidin, streptavidin, egg yolk avidin, neutravidin and avidin-like, preferably neutravidin and / or streptavidin.

[0093] In some embodiments of the present invention, the avidin is chemically bonded to the surface of the first carrier by reacting the amino group with the aldehyde group on the surface of the first carrier to form a Schiff base.

[0094] In some embodiments of the present invention, the coefficient of variation (CV) value of the particle size distribution of the donor particles in the donor reagent is ≥5%.

[0095] In other embodiments of the present invention, the coefficient of variation CV value of the particle size distribution of the donor particles in the donor reagent is ≥8%; preferably, the coefficient of variation CV value of the particle size distribution of the donor particles in the donor reagent is ≥10%.

[0096] In some embodiments of the present invention, the coefficient of variation CV value of the particle size distribution of the donor particles in the donor reagent is ≤40%; more preferably, the coefficient of variation CV value of the particle size distribution of the donor particles in the donor reagent is ≤20%.

[0097] It is worth noting that the coefficient of variation CV value of the particle size distribution of the donor particles described in the present invention refers to the coefficient of variation CV value of the particle size distribution after the donor particles are coated with the desired substance.

[0098] In some specific embodiments of the present invention, the coefficient of variation (CV) value of the particle size distribution of the donor particles in the donor reagent can be 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.

[0099] In other embodiments of the present invention, the particle size distribution of the donor particles in the donor reagent is polydisperse.

[0100] In some specific embodiments of the present invention, the coefficient of variation CV value of the particle size distribution is calculated by Gaussian distribution.

[0101] In other specific embodiments of the present invention, using Gaussian distribution analysis, the Gaussian distribution curve of the receptor particles in the receptor reagent presents two or more peaks.

[0102] In some embodiments of the present invention, the concentration of the donor particles in the donor reagent is 10 μg / ml to 1 mg / ml, preferably 20 μg / ml to 500 μg / ml, and more preferably 50 μg / ml to 200 μg / ml.

[0103] In other embodiments of the present invention, the donor reagent further includes a buffer solution with a pH value of 7.0 to 9.0, and the donor particles are suspended in the buffer solution.

[0104] In some embodiments of the present invention, the buffer solution contains a polysaccharide, and the polysaccharide is 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.

[0105] In other embodiments of the present invention, the molecular weight distribution Mw of the dextran is selected from 10,000 to 1,000,000 KDa, preferably selected from 100,000 to 800,000 KDa, and more preferably selected from 300,000 to 700,000 KDa.

[0106] In some embodiments of the present invention, the content of dextran in the buffer solution is 0.01 to 1 wt%, preferably 0.05 to 0.5 wt%.

[0107] In other embodiments of the present invention, the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is ≥5%.

[0108] In some preferred embodiments of the present invention, the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is ≥8%; preferably, the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is ≥10%.

[0109] In other preferred embodiments of the present invention, the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is ≤40%; more preferably, the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is ≤20%.

[0110] It is worth noting that the coefficient of variation CV value of the particle size distribution of the receptor particles described in the present invention refers to the coefficient of variation CV value of the particle size distribution after the receptor particles are coated with the required substance.

[0111] In some specific embodiments of the present invention, the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent can be 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.

[0112] In some embodiments of the present invention, the particle size distribution of the receptor particles in the receptor reagent is polydisperse.

[0113] In other embodiments of the present invention, the coefficient of variation CV value of the particle size distribution is calculated using Gaussian distribution.

[0114] In some embodiments of the present invention, using Gaussian distribution analysis, the Gaussian distribution curve of the receptor particles in the receptor reagent presents two or more peaks.

[0115] In other embodiments of the present invention, the receptor reagent comprises receptor particles with at least two average particle size distributions.

[0116] In some embodiments of the present invention, the coating material in the coating layer is selected from polysaccharides, high molecular polymers or biomacromolecules, preferably polysaccharides;

[0117] Further preferably, the surface of the second carrier is coated with at least two continuous polysaccharide coatings, and one of the specific binding pair members is linked to the surface of the continuous polysaccharide coatings.

[0118] In other embodiments of the present invention, each polysaccharide layer in the continuous polysaccharide coating layer spontaneously associates with the previous polysaccharide layer.

[0119] In some embodiments of the present invention, the polysaccharide has pendant functional groups, and the pendant functional groups of any polysaccharide layer in the continuous polysaccharide layers have charges opposite to those of the pendant functional groups of the previous polysaccharide layer.

[0120] In other embodiments of the present invention, the polysaccharide has pendant functional groups, and any polysaccharide layer in the continuous polysaccharide coating is covalently linked to the previous polysaccharide layer through a chemical bonding reaction between the pendant functional groups and the pendant functional groups of the previous polysaccharide layer.

[0121] 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.

[0122] In other embodiments of the present invention, the amine-reactive functional group is an aldehyde group or a carboxyl group.

[0123] In some embodiments of the present invention, the outermost polysaccharide layer of the continuous polysaccharide coating has at least one pendant functional group.

[0124] In other embodiments of the present invention, the pendant functional group is selected from at least one of aldehyde, carboxyl, thiol, amino, hydroxyl and malein; preferably selected from aldehyde and / or carboxyl.

[0125] In some embodiments of the present invention, the pendant functional groups of the continuous polysaccharide coating are directly or indirectly chemically bonded to a member of a specific binding pair.

[0126] In other embodiments of the present invention, the polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from glucan, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxyglucan and aminoglucan; more preferably selected from glucan, starch, glycogen and polyribose.

[0127] In some embodiments of the present invention, the sugar content is detected by anthrone method;

[0128] Preferably, the sugar is selected from carbohydrates containing three or more unmodified or modified monosaccharide units, preferably selected from glucan, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxyglucan and aminoglucan; more preferably selected from glucan, starch, glycogen and polyribose;

[0129] More preferably, the molecular weight distribution Mw of the dextran is 1000-1000000 KDa, preferably 10000-800000 KDa, more preferably 30000-700000 KDa.

[0130] In some embodiments of the present invention, the material of the first carrier and / or the second carrier is selected from natural, synthetic or modified naturally occurring polymers; preferably, synthetic polymers.

[0131] 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.

[0132] In some embodiments of the present invention, the first carrier and / or the second carrier are polystyrene latex microspheres.

[0133] In some embodiments of the present 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, porphyrin, phthalocyanine and chlorophyll.

[0134] In some embodiments of the present invention, the luminescent composition is capable of reacting with reactive oxygen species to generate a detectable chemiluminescent signal, and comprises a chemiluminescent compound and a metal chelate.

[0135] In some embodiments of the present invention, the chemiluminescent compound is selected from olefin compounds, preferably selected from dimethylthiophene, dibutanedione compounds, dioxine, enol ether, enamine, 9-alkylene xanthane, 9-alkylene-N-9,10 dihydroacridine, aryl ether ene, aryl imidazole and lucigenin and their derivatives, more preferably selected from dimethylthiophene and its derivatives.

[0136] In some embodiments of the present invention, the metal of the metal chelate is a rare earth metal or a Group VIII metal, preferably selected from europium, terbium, dysprosium, samarium, osmium and ruthenium, more preferably europium.

[0137] In some embodiments of the present invention, the metal chelate comprises a chelating agent selected from the group consisting of: 4'-(10-methyl-9-anthryl)-2,2':6'2"-terpyridine-6,6"-dimethylamine]tetraacetic acid (MTTA), 2-(1',1',2',2',3',3'-heptafluoro-4',6'-hexanedione-6'-yl)-naphthalene (NHA), 4,4'-bis(2",3",3"-heptafluoro-4",6"-hexanedione-6"-yl)-o-terphenyl (BHHT), 4,4'-bis(1",1",1",2",2",3",3"-heptafluoro-4",6"-hexanedione-6 ”-yl)-chlorosulfonyl-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), 2,2-dimethyl-4-perfluorobutyryl-3-butanone (FOD), 2,2'-bipyridine (BPY), bipyridylcarboxylic acid, azacrown ether, azacryptand and trioctylphosphine oxide and their derivatives.

[0138] The homogeneous chemiluminescent POCT detection kit involved in the second aspect of the present invention comprises the kit as described in the first aspect of the present invention, wherein the POCT refers to a point-of-care test or a clinical test performed at the patient's side.

[0139] In some embodiments of the present invention, the kit includes a reagent strip having a plurality of wells for containing reagents, the wells including at least:

[0140] The first reagent well is used to hold the donor reagent;

[0141] The second reagent well is used to contain the receptor reagent.

[0142] The homogeneous chemiluminescence POCT detection method involved in the third aspect of the present invention utilizes the kit as described in the second aspect of the present invention to detect the target molecule in the sample to be tested.

[0143] The homogeneous chemiluminescence POCT detection device involved in the fourth aspect of the present invention detects the target molecule in the sample to be tested using the kit described in the second aspect of the present invention or the method described in the third aspect of the present invention.

[0144] In some embodiments of the invention, the apparatus comprises:

[0145] an incubation module, for controlling the temperature of the reagent strip as described in the second aspect of the present invention;

[0146] The light excitation and detection module is arranged on one side of the incubation module, and is used to emit excitation light to the reagent strip to generate a photo-induced chemiluminescence reaction; and detect the chemiluminescence signal generated by the reagent.

[0147] A fifth aspect of the present invention relates to a method for performing homogeneous chemiluminescence analysis using the POCT detection device according to the fourth aspect of the present invention, comprising the following steps:

[0148] S1, contacting the sample to be tested with the receptor reagent and the donor reagent to generate a test mixture after reaction;

[0149] S2, using excitation light with a wavelength of 600 to 700 nm to excite the test mixture to perform chemiluminescence, and detecting the signal intensity of the chemiluminescence; the detection wavelength of the chemiluminescence is 520 to 620 nm;

[0150] S3, judging whether the sample to be tested contains the target molecule to be tested and / or the concentration of the target molecule to be tested in the sample to be tested based on the analysis of the chemiluminescence signal intensity.

[0151] In some embodiments of the present invention, the sample to be tested is diluted with a diluent and then contacted with the receptor reagent.

[0152] In some embodiments of the present invention, the sample to be tested is selected from materials suspected of containing the target molecule to be tested, including but not limited to: blood, serum, plasma, sputum, lymph, semen, vaginal mucus, feces, urine or cerebrospinal fluid.

[0153] Example

[0154] To make the present invention easier to understand, the present invention will be further described in detail below with reference to the following examples. These examples are merely illustrative and are not intended to limit the scope of application of the present invention. The raw materials or components used in the present invention can be obtained by commercial routes or conventional methods unless otherwise specified.

[0155] Example 1: Preparation of donor reagent

[0156] (1) Preparation of formaldehyde polystyrene latex microspheres

[0157] a) Prepare a 100 ml three-necked flask, add 40 mmol of styrene, 5 mmol of acrolein, and 10 ml of water, stir for 10 min, and then pass N2 for 30 min.

[0158] b) Weigh 0.11 g of ammonium persulfate and 0.2 g of sodium chloride and dissolve them in 40 ml of water to prepare an aqueous solution. Add the aqueous solution to the reaction system in step a) and continue to flow N2 for 30 minutes.

[0159] c) The reaction system was heated to 70° C. and reacted for 15 hours.

[0160] d) The emulsion after the reaction is cooled to room temperature and filtered through a suitable filter cloth. The resulting emulsion is washed with deionized water by repeated centrifugation until the conductivity of the supernatant at the beginning of the centrifugation is close to that of deionized water. It is then diluted with water and stored as an emulsion.

[0161] e) The Gaussian distribution average particle size of the latex microspheres measured by a nanoparticle size analyzer was 201.3 nm, and the coefficient of variation (CV) was 8.0%.

[0162] (2) Filling of sensitizer

[0163] a) Prepare a 25 ml round-bottom flask, add 0.11 g copper phthalocyanine and 10 ml N,N-dimethylformamide, stir magnetically, and heat in a water bath to 75°C to obtain a photosensitizer solution.

[0164] b) Prepare a 100 ml three-necked flask, add 10 ml of 95% ethanol, 10 ml of water and 10 ml of 10% formaldehyde polystyrene latex microspheres obtained in (1), stir magnetically and heat in a water bath to 70°C.

[0165] c) slowly adding the solution in step a) dropwise to the three-necked flask in step b), reacting at 70° C. for 2 hours, then stopping stirring and cooling naturally to obtain an emulsion.

[0166] d) The emulsion was centrifuged at 30,000 g for 1 hour, the supernatant was discarded, and the suspension was resuspended in 50% ethanol. The suspension was washed by centrifugation three times and then resuspended in 50 mM CB buffer (pH 10) to a final concentration of 20 mg / ml.

[0167] (III) Modifying the microsphere surface with avidin to prepare donor reagent

[0168] a) Microsphere suspension treatment: A certain amount of microspheres prepared in step (ii) were centrifuged in a high-speed refrigerated centrifuge, the supernatant was discarded, a certain amount of MES buffer was added, and the suspension was sonicated on an ultrasonic cell disruptor until the particles were resuspended. MES buffer was added to adjust the microsphere concentration to 100 mg / ml.

[0169] b) Preparation of avidin solution: Weigh a certain amount of streptavidin and dissolve it in MES buffer to 8 mg / ml.

[0170] c) Mixing: The treated microsphere suspension, 8 mg / ml avidin, and MES buffer were mixed at a volume ratio of 2:5:1 and mixed rapidly to obtain a reaction solution.

[0171] d) Reaction: Prepare a 25 mg / ml NaBH3CN solution in MES buffer and add it to the reaction solution at a volume ratio of 1:25. Mix quickly and incubate at 37°C with rotation for 48 hours.

[0172] e) Blocking: Prepare a 75 mg / ml Gly solution and a 25 mg / ml NaBH3CN solution in MES buffer at a volume ratio of 2:1:10 to the reaction solution. Mix thoroughly and incubate at 37°C with rotation for 2 hours. Then, add a 200 mg / ml BSA solution (in MES buffer) at a volume ratio of 5:8 to the reaction solution. Mix rapidly and incubate at 37°C with rotation for 16 hours.

[0173] f) Washing: Add MES buffer to the reaction solution, centrifuge in a high-speed refrigerated centrifuge, discard the supernatant, add fresh MES buffer, resuspend by ultrasonication, and centrifuge again. Wash in this way three times. Finally, resuspend with a small amount of donor particle buffer, measure the solid content, and adjust the concentration to 150 μg / ml with donor particle buffer to obtain a donor reagent containing donor particles.

[0174] g) The Gaussian distribution average particle size of the donor particles measured by a nanoparticle size analyzer is 227.7 nm, and the coefficient of variation (CV) is 6.5%. Figure 1 shown.

[0175] Example 2: Preparation of donor reagents

[0176] The preparation of formaldehyde polystyrene latex microspheres and the filling process of the sensitizer are the same as the preparation steps (i) and (ii) in Example 1.

[0177] (1) Preparation of aminodextran

[0178] a) Place a 500 mL four-necked flask in an oil bath, install a condenser, and flow nitrogen.

[0179] b) 10 g of dextran with an average molecular weight distribution of 500,000 kDa, 100 ml of deionized water, 2 g of NaOH, and 10 g of N-(2,3-epoxypropyl)phthalimide were added in sequence with mechanical stirring.

[0180] c) After 2 hours in a 90°C oil bath, turn off the heat and allow the mixture to cool naturally while maintaining stirring.

[0181] d) The reaction mixture was added to 2 L of methanol to precipitate the main mixture, and the solid was collected and dried.

[0182] e) Place a 200 mL four-necked flask in an oil bath, install a condenser, and flow nitrogen.

[0183] f) The dried solid, 100 mL of deionized water, 1.8 g of sodium acetate, and 5 mL of 50% hydrazine hydrate were added in sequence, and the pH was adjusted to 4, with mechanical stirring.

[0184] g) After 1 hour in an 85°C oil bath, turn off the heat and allow the mixture to cool naturally while maintaining stirring.

[0185] h) The reaction solution was adjusted to a neutral pH and then filtered to collect the filtrate.

[0186] i) The filtrate was placed in a dialysis bag and dialyzed against deionized water at 4°C for 2 days, changing the water 3-4 times per day.

[0187] j) After dialysis, freeze-drying was performed to obtain 9.0 g of aminodextran solid.

[0188] (2) Preparation of Aldehyde Dextran

[0189] a) Weigh 10 g of dextran with an average molecular weight distribution of 500,000 kDa and place it in a 250 beaker. Add 100 mL of 0.1 M / pH 6.0 phosphate buffer and stir at room temperature to dissolve.

[0190] b) Weigh 1.8 g of sodium metaperiodate into a 50 mL beaker, add 10 mL of 0.1 M / pH 6.0 phosphate buffer, and stir at room temperature to dissolve.

[0191] c) Slowly add the sodium metaperiodate solution dropwise to the dextran solution, and continue stirring for 1 hour after the reaction stops producing bubbles.

[0192] d) The reaction mixture was placed in a dialysis bag and dialyzed against deionized water at 4°C for 2 days, with the water being changed 3-4 times per day.

[0193] e) After dialysis, freeze-drying was performed to obtain 9.6 g of aldehyde dextran solid.

[0194] (3) Microspheres coated with dextran

[0195] a) Place 50 mg of aminodextran in a 20 mL round-bottom flask, add 5 mL of 50 mM / pH 10 carbonate buffer, and dissolve by stirring at 30°C in the dark.

[0196] b) 100 mg of donor particles were added to the aminodextran solution and stirred for 2 hours.

[0197] c) Dissolve 10 mg of sodium borohydride in 0.5 mL of 50 mM / pH 10 carbonate buffer and add dropwise to the above reaction solution. Allow to react overnight at 30° C. in the dark.

[0198] d) The reaction mixture was centrifuged at 30,000 G, the supernatant discarded, and 50 mM / pH 10 carbonate buffer was added for ultrasonic dispersion. The mixture was washed by centrifugation three times, and then the volume was adjusted to 20 mg / ml with 50 mM / pH 10 carbonate buffer.

[0199] e) 100 mg of aldehyde dextran solid was placed in a 20 mL round-bottom flask, 5 mL of 50 mM / pH 10 carbonate buffer was added, and the mixture was stirred and dissolved at 30°C in the dark.

[0200] f) Add the above particles to the aldehyde dextran solution and stir for 2 hours.

[0201] g) Dissolve 15 mg of sodium borohydride in 0.5 mL of 50 mM / pH 10 carbonate buffer and add dropwise to the above reaction solution. Allow to react at 30° C. in the dark overnight.

[0202] h) The reaction mixture was centrifuged at 30,000 G, the supernatant discarded, and 50 mM / pH 10 carbonate buffer was added for ultrasonic dispersion. The mixture was washed by centrifugation three times, and then the volume was adjusted to 20 mg / ml with 50 mM / pH 10 carbonate buffer.

[0203] i) The Gaussian distribution average particle size of the microspheres measured by a nanoparticle size analyzer was 235.6 nm, with a coefficient of variation (CV) of 8.1%.

[0204] (IV) Modifying the microsphere surface with avidin to prepare donor reagent

[0205] h) Microsphere suspension treatment: A certain amount of microspheres prepared in step (3) were centrifuged in a high-speed refrigerated centrifuge, the supernatant was discarded, a certain amount of MES buffer was added, and the microspheres were sonicated on an ultrasonic cell disruptor until they were resuspended. MES buffer was added to adjust the donor particle concentration to 100 mg / ml.

[0206] i) Preparation of avidin solution: Weigh a certain amount of neutravidin and dissolve it in MES buffer to 8 mg / ml.

[0207] j) Mixing: The treated microsphere suspension, 8 mg / ml avidin, and MES buffer were mixed at a volume ratio of 2:5:1 and mixed rapidly to obtain a reaction solution.

[0208] k) Reaction: Prepare a 25 mg / ml NaBH3CN solution in MES buffer and add it to the reaction solution at a volume ratio of 1:25. Mix quickly. Incubate at 37°C with rotation for 48 hours.

[0209] 1) Blocking: Prepare a 75 mg / ml Gly solution and a 25 mg / ml NaBH3CN solution in MES buffer at a volume ratio of 2:1:10 to the reaction solution. Mix thoroughly and incubate at 37°C with rotation for 2 hours. Then, add a 200 mg / ml BSA solution (in MES buffer) at a volume ratio of 5:8 to the reaction solution. Mix rapidly and incubate at 37°C with rotation for 16 hours.

[0210] m) Washing: Add MES buffer to the reaction solution, centrifuge in a high-speed refrigerated centrifuge, discard the supernatant, add fresh MES buffer, resuspend by ultrasonication, and centrifuge again. Wash in this way three times. Finally, resuspend with a small amount of donor particle buffer, measure the solid content, and adjust the concentration to 150 μg / ml with donor particle buffer to obtain a donor reagent containing donor particles.

[0211] n) The Gaussian distribution average particle size of the donor particles measured by a nanoparticle size analyzer is 249.9 nm, and the coefficient of variation (CV) is 11.6%. Figure 2 shown.

[0212] Example 3: Preparation of receptor reagents

[0213] 1. Preparation and characterization of formaldehyde-based polystyrene latex microspheres

[0214] 1) Prepare a 100ml three-necked flask, add 40mmol of styrene, 5mmol of acrolein, and 10ml of water, stir for 10min, and then pass N2 for 30min;

[0215] 2) Weigh 0.11g of ammonium persulfate and 0.2g of sodium chloride and dissolve them in 40ml of water to prepare an aqueous solution. Add this aqueous solution to the reaction system in step 1 and continue to flow N2 for 30 minutes.

[0216] 3) The reaction system was heated to 70°C and reacted for 15 hours;

[0217] 4) Cool the emulsion after the reaction to room temperature and filter it through a suitable filter cloth. Wash the resulting emulsion with deionized water by centrifugation until the conductivity of the supernatant is close to that of deionized water. Then dilute it with water and store it as an emulsion.

[0218] 5) The average particle size of the latex microspheres measured by the nanoparticle size analyzer is 202.2 nm in Gaussian distribution, and the coefficient of variation (CV) is 4.60%.

[0219] 2. Process and Characterization of Filling the Microspheres with Luminescent Composition

[0220] 1) Prepare a 25 ml round bottom flask, add 0.1 g of dimethylthiophene derivative and 0.1 g of europium (III) complex (MTTA-EU 3+ ), 10 ml of 95% ethanol, magnetic stirring, and heating in a water bath to 70°C to obtain a complex solution;

[0221] 2) Prepare a 100 ml three-necked flask, add 10 ml of 95% ethanol, 10 ml of water, and 10 ml of 10% formaldehyde polystyrene latex microspheres obtained in step 1, stir magnetically, and heat in a water bath to 70°C;

[0222] 3) slowly adding the complex solution in step 1) dropwise to the three-necked flask in step 2), reacting at 70° C. for 2 hours, then stopping stirring and cooling naturally;

[0223] 4) Centrifuging the emulsion for 1 hour at 30,000 G, discarding the supernatant to obtain formaldehyde-based polystyrene microspheres embedded with the luminescent composition.

[0224] 5) The Gaussian distribution average particle size of the microspheres measured by a nanoparticle size analyzer is 204.9 nm, and the coefficient of variation (CV) is 5.00%.

[0225] 3. Process and Characterization of Polysaccharide Coating on Microsphere Surface

[0226] 1) Place 50 mg of aminodextran in a 20 mL round-bottom flask, add 5 mL of 50 mM / pH 10 carbonate buffer, and dissolve by stirring at 30°C in the dark.

[0227] 2) Take 100 mg of the aldehyde-based polystyrene microspheres filled with the luminescent composition prepared in step 2, add them to the aminodextran solution and stir for 2 hours;

[0228] 3) Dissolve 10 mg of sodium borohydride in 0.5 mL of 50 mM / pH 10 carbonate buffer and add dropwise to the above reaction solution. React at 30°C in the dark overnight.

[0229] 4) Centrifuge the reaction mixture at 30,000g, discard the supernatant, and add 50mM / pH 10 carbonate buffer for ultrasonic dispersion. Repeat the centrifugation and washing three times, then adjust the volume with 50mM / pH 10 carbonate buffer to a final concentration of 20mg / ml.

[0230] 5) Place 100 mg of aldehyde dextran solid in a 20 mL round-bottom flask, add 5 mL of 50 mM / pH 10 carbonate buffer, and dissolve by stirring at 30°C in the dark.

[0231] 6) Add the above microspheres to the aldehyde dextran solution and stir for 2 hours;

[0232] 7) Dissolve 15 mg of sodium borohydride in 0.5 mL of 50 mM / pH 10 carbonate buffer and add dropwise to the above reaction solution. React at 30°C in the dark overnight.

[0233] 8) The reaction mixture was centrifuged at 30,000 G, the supernatant discarded, and 50 mM / pH 10 carbonate buffer was added for ultrasonic dispersion. Repeat the centrifugation and washing three times, and then the volume was adjusted to 20 mg / ml with 50 mM / pH 10 carbonate buffer.

[0234] 9) The Gaussian distribution average particle size of the microspheres measured by a nanoparticle size analyzer is 241.6 nm, and the coefficient of variation (CV) is 12.90%.

[0235] 4. PCT Antibody Conjugation Process

[0236] 1) The paired PCT antibody was dialyzed into 50 mM CB buffer at pH 10, and the concentration was measured to be 1 mg / ml.

[0237] 2) Add 0.5 ml of the microspheres obtained in step 3 and 0.5 ml of paired antibody I obtained in step 1) to a 2 ml centrifuge tube, mix well, add 100 μl of 10 mg / ml NaBH4 solution (50 mM CB buffer), and react at 2-8°C for 4 hours.

[0238] 3) After the reaction is complete, add 0.5 ml of 100 mg / ml BSA solution (50 mM CB buffer) and react at 2-8°C for 2 hours.

[0239] 4) After the reaction is complete, centrifuge at 30,000g for 45 minutes, discard the supernatant, and resuspend in 50mM MES buffer. Repeat the centrifugation and washing process four times, and dilute with buffer to a final concentration of 50μg / ml to obtain the receptor reagent conjugated to Antibody I.

[0240] 5) The Gaussian distribution average particle size of the receptor particles measured by the nanoparticle size analyzer is 253.5 nm, and the coefficient of variation (CV) is 9.60%. Figure 3 shown).

[0241] Example 4: Detection of sugar content of microspheres using the anthrone method

[0242] a) Microsphere sample pretreatment:

[0243] Donor reagent A containing 1 mg of donor microspheres a in Example 1, donor reagent B containing 1 mg of donor microspheres b in Example 2, and acceptor reagent containing 1 mg of acceptor microspheres in Example 3 were centrifuged at 20,000 g for 40 min. The supernatant was discarded and ultrasonically dispersed with purified water. The centrifugal dispersion was repeated three times and the volume was adjusted to 1 mg / mL with purified water.

[0244] b) Preparation of glucose standard solution:

[0245] Use purified water to prepare 1 mg / mL glucose stock solution into 0 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.075 mg / mL, 0.10 mg / mL, and 0.15 mg / mL standard solutions.

[0246] c) Preparation of anthrone solution: Prepare 2 mg / mL using 80% sulfuric acid solution (this solution is stable at room temperature for 24 hours and should be prepared immediately before use).

[0247] d) Add 0.1 mL of each concentration of glucose standard solution and the sample to be tested to the centrifuge tubes, and add 1 mL of anthrone test solution to each tube.

[0248] e) Incubate at 85°C for 30 min.

[0249] f) Centrifuge the sample reaction tube at 15,000 g for 40 minutes. Aspirate the clear liquid from the bottom of the tube with a pipette tip to measure the absorbance, avoiding aspirating the suspended matter above.

[0250] g) Return to room temperature and measure the absorbance at 620 nm (measurement should preferably be performed within 2 h).

[0251] h) With the concentration of the standard solution as the X value and the absorbance as the Y value, a linear regression was performed. The test results and the obtained standard curve are shown in Table 1 and Table 2, respectively. Figure 4 As shown, the sugar concentration of the sample to be tested is detected.

[0252] Table 1

[0253] Serial number Concentration mg / mL Absorbance A Absorbance B Average absorbance 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

[0254] Test results:

[0255] Sugar content of donor microspheres a in Example 1: 11.3 μg / mg

[0256] Sugar content of donor microspheres b in Example 2: 40.8 μg / mg

[0257] Sugar content of the acceptor microspheres in Example 3: 60.4 μg / mg

[0258] Example 5: Preparation of reagent strips and kits

[0259] The prepared reagent strip 11 is as follows Figure 5 As shown, there are several holes 111 for containing reagents, including:

[0260] The first reagent well is used to contain the donor reagent prepared in Example 1 or Example 2, wherein the concentration of the donor particles is 150 ug / ml.

[0261] The second reagent well is used to hold the receptor reagent prepared in Example 3, wherein the concentration of the receptor particles is 50 μg / ml;

[0262] A sample hole, which is used to hold the sample to be tested; and

[0263] Signal detection hole position.

[0264] Optionally, the cross section of the well 111 may be circular, elliptical or rectangular. Preferably, the cross sections of the plurality of wells are different from each other to distinguish different reagents contained therein.

[0265] Optionally, the reagent strip 11 may also be provided with a third reagent well and / or a fourth reagent well for carrying a diluent and / or an additional reagent. In the case of being provided with wells for carrying a diluent and an additional reagent, the order of adding the diluent and the additional reagent is as follows. First, the sample to be tested is added to the third reagent well carrying the diluent to mix it with the diluent, thereby performing a dilution operation. After the dilution is completed, a certain volume of the diluted sample to be tested is taken and added to the fifth reagent well carrying the additional reagent to mix it with the additional reagent. Afterwards, a certain volume of the mixed liquid is continued to be taken and added to the well carrying the first reagent. After a certain period of reaction, a certain volume of the mixed liquid is continued to be taken and added to the well carrying the second reagent to carry out the subsequent process.

[0266] In this embodiment, the end faces of the holes 111 are coated to seal their openings, wherein the coating can be a disposable or reusable sealing film.

[0267] In this embodiment, a barcode area 112 is provided on the side of the reagent strip. The barcode area 112 contains information about the reagent strip. For example, the barcode area 112 is provided with a barcode, which can be a one-dimensional code or a two-dimensional code.

[0268] 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 detection kit described in the present application.

[0269] Example 6: A homogeneous chemiluminescence POCT detection device

[0270] The principle of the homogeneous chemiluminescent POCT detection device described in this embodiment is as follows: the target molecule to be tested in the test sample reacts with the donor particles and the acceptor particles to form an immune complex. This interaction will bring the donor particles and the acceptor particles closer together. Under the irradiation of laser (wavelength of 680nm), the sensitizer in the donor particles converts the oxygen in the surrounding environment into more active monomeric oxygen. Monomeric oxygen diffuses to the acceptor particles and reacts with the chemiluminescent agent in the acceptor particles, further activating the luminescent groups also on the acceptor particles, causing them to emit light with a wavelength of 520-620nm. The half-life of monomeric oxygen is 4μSec, and the diffusion distance in the solution is about 200nm. If there is no interaction between the biomolecules, singlet oxygen cannot diffuse to the acceptor particles, and no light signal will be generated. Therefore, by measuring the light intensity emitted by the mixture, the concentration of the target molecule to be tested in the test sample can be calculated. The donor particles include a first carrier, the interior of the first carrier is filled with a sensitizer, and the surface of the first carrier is connected to one of the specific binding pair members; the acceptor particles include 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, and the surface of the coating layer is connected to a reporter molecule, and the reporter molecule can specifically bind to the target molecule to be detected.

[0271] A preferred structure of the photochemiluminescence immunoassay instrument described in this embodiment includes the following modules:

[0272] A reagent loading module, which is used to add a sample to be tested, a receptor reagent and / or a donor reagent; wherein the donor reagent includes donor particles, and the coefficient of variation (CV) of the particle size distribution of the donor particles in the donor reagent is ≥5%; and the coefficient of variation (CV) of the particle size distribution of the receptor particles in the receptor reagent is ≥5%;

[0273] An incubation module, used to control the temperature of the reagent strip; the incubation module can be in the form of a metal bath, a water bath, or an oil bath;

[0274] The light excitation and detection module is arranged on one side of the incubation module, and is used to emit excitation light to the reagent strip to generate a photo-induced chemiluminescence reaction; and detect the chemiluminescence signal generated by the reagent.

[0275] Example 7: On-machine test results and analysis (test substance: PCT antigen)

[0276] (1) Using the detection device in Example 5, the donor reagents in Examples 1 and 2 were simultaneously loaded onto the machine with the acceptor reagent in Example 3 to detect PCT antigen. The detection results are shown in Table 2. The PCT quantitative determination detection kit (photochemiluminescence method) used in this example consists of reagent 1 (R1') containing acceptor particles coated with a first anti-PCT antibody, reagent 2 (R2') containing a second anti-PCT antibody labeled with biotin, and a universal solution (R3') containing donor particles. R1' is an acceptor reagent prepared using the acceptor particles in Example 3 (particle size distribution coefficient of variation CV value = 9.6%); R3' is a donor reagent prepared using the donor particles in Examples 1 and 2.

[0277] Table 2

[0278]

[0279] As shown in Table 2, the sensitivity and upper detection limit of the kit provided by this application are both excellent. Furthermore, the sensitivity and upper detection limit of the kit containing the donor reagent of Example 1 are both superior to those of the kit containing the donor reagent of Example 2. This shows that the performance of the kit using donor particles that are not coated with polysaccharides is even better.

[0280] Example 8:

[0281] According to the methods given in Examples 1 to 3, a series of kits containing donor microspheres and acceptor microspheres with different sugar contents were prepared (as shown in the table below). Then, they were cross-assembled into the kit shown in Example 5. The signal levels of each kit for the same batch of samples were then tested on a phase chemiluminescence POCT detection device shown in Example 6. The detection process of the POCT device is as follows: 25 μL of the sample to be tested, 25 μL of biotin-labeled antibody, 25 μL of cytochrome P-30 ... After adding 175 μL of donor reagent and 25 μL of receptor reagent, the reagent strip was placed in a POCT analyzer developed by Boyang Biotechnology (Shanghai) Co., Ltd. The sample adding mechanism took the corresponding volume of the sample to be tested, added it to the additional reagent well, vibrated, and incubated at 37°C for 10 minutes; the liquid incubated in the additional reagent well was added to the first reagent well and vibrated, incubated at 37°C for 10 minutes to form a mixed liquid; the mixed liquid was continued to be added to the second reagent well, vibrated, and incubated at 37°C for 10 minutes to form a mixture to be tested, and then moved to the signal detection well. The laser emitted by the exciter in the light excitation and detection module was used to illuminate the mixed liquid to be tested in the signal detection well, and the generated chemiluminescent signal was detected. The results are shown in Table 3.

[0282] Table 3

[0283]

[0284]

[0285] Example 9: Detection of clinical samples with different CRP concentrations

[0286] In the detection device of Example 6, 50 μL of clinical samples (including serum and whole blood) with different CRP concentrations were added to the reagent strip, and the average of the parallel tubes in each well was taken. 50 μL of biotinylated anti-CRP antibody and 50 μL of receptor reagent containing CRP receptor particles coupled to the reagent were added (the sugar content per mg of receptor particles was 59.3 μg, the average particle size of the receptor particles in the Gaussian distribution curve was 253.6 nm, and the coefficient of variation of the particle size distribution was CV value = 10.9%). The reaction was carried out at 37° C. for 7.5 min. Then, 50 μL of donor reagent was added (the sugar content per mg of donor particles was 9.5 μg / mg of particles, the average particle size of the donor particles in the Gaussian distribution curve was 226.7 nm, and the coefficient of variation of the particle size distribution was CV value = 8.1%). The reaction was carried out at 37° C. for 5 min, and light excitation detection was performed. The experimental results are shown in Tables 4 and 5. Figure 6 As shown in Table 4 and Figure 6 As can be seen, the correlation coefficient using serum and whole blood reached 0.9988. These experimental results demonstrate that the donor particles of the present invention significantly reduce nonspecific adsorption in samples, resulting in excellent correlation between the measurement results for serum and whole blood. This donor reagent has greatly enhanced its adaptability to clinical samples and can be directly used for testing clinical whole blood samples.

[0287] Table 4

[0288]

[0289]

[0290] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation of the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein. On the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. A homogeneous chemiluminescent detection kit comprising a donor reagent and an acceptor reagent, wherein the donor reagent comprises donor particles, which are capable of generating reactive oxygen species upon excitation; and the acceptor reagent comprises a second buffer solution and acceptor particles, which are capable of reacting with the received reactive oxygen species to generate a chemiluminescent signal, characterized in that: The donor particles include a first carrier, the interior of the first carrier is filled with a sensitizer, a surface of the first carrier is directly or indirectly connected to one of the specific binding pair members, and the sugar content in each milligram of the donor particles is no more than 25 μg; The receptor particle includes 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 to a reporter molecule, the reporter molecule can specifically bind to the target molecule to be detected, the coating in the coating layer is selected from dextran, and the sugar content per milligram of the receptor particle is not less than 40ug; The coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is ≥5%.

2. The kit according to claim 1, wherein One of the specific binding pair members is directly bonded to the surface of the first carrier.

3. The kit according to claim 2, wherein The surface of the first carrier is not coated or connected with polysaccharide substances, and is directly bonded to one of the specific binding pair members.

4. The kit according to claim 3, wherein The surface of the first carrier carries a bonding functional group, and the bonding functional group is used to directly bond one of the specific binding pair members to the surface of the first carrier.

5. The kit according to claim 4, characterized in that The bonding functional group is selected from an amino group, an amide group, a hydroxyl group, an aldehyde group, a carboxyl group, a maleimide group and a thiol group.

6. The kit according to claim 4, wherein The bonding functional group is selected from aldehyde group and / or carboxyl group.

7. The kit according to claim 1, wherein The specific binding pair members are selected from antibodies, antibody fragments, ligands, oligonucleotides, oligonucleotide binding proteins, lectins, haptens, antigens, immunoglobulin binding proteins, avidin, avidin or biotin, which are a pair of substances that can specifically bind to each other.

8. The kit according to claim 7, characterized in that The specific binding pair member is avidin-biotin, and the avidin is selected from the group consisting of avidin, streptavidin, vitellogenin, neutravidin and avidin-like.

9. The kit according to claim 7, characterized in that The avidin is selected from neutravidin and / or streptavidin.

10. The kit according to claim 8, characterized in that The avidin is chemically bonded to the surface of the first carrier by reacting the amino group with the aldehyde group on the surface of the first carrier to form a Schiff base.

11. The kit according to claim 1, wherein The coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is ≥8%.

12. The kit according to claim 1, wherein The coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is ≥10%.

13. The kit according to claim 1, wherein The coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is ≤40%.

14. The kit according to claim 1, wherein The coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is ≤20%.

15. The kit according to claim 1, wherein The particle size distribution of the receptor particles in the receptor reagent is polydisperse.

16. The kit according to claim 1, wherein The coefficient of variation CV value of the particle size distribution is calculated through Gaussian distribution.

17. The kit according to claim 1, wherein Using the Gaussian distribution analysis method, the Gaussian distribution curve of the receptor particles in the receptor reagent presents two or more peaks.

18. The kit according to claim 1, wherein The receptor reagent comprises receptor particles with at least two average particle size distributions.

19. The kit according to any one of claims 1 to 18, characterized in that The surface of the second carrier is coated with at least two continuous polysaccharide coatings, and one of the specific binding pair members is connected to the surface of the continuous polysaccharide coatings.

20. The kit according to claim 19, characterized in that Each polysaccharide layer in the continuous polysaccharide coating is spontaneously associated with the previous polysaccharide layer.

21. The kit according to claim 19, wherein The polysaccharide has pendant functional groups, and the pendant functional groups of any polysaccharide layer in the continuous polysaccharide coating layer have charges opposite to those of the pendant functional groups of the previous polysaccharide layer.

22. The kit according to claim 19, characterized in that The polysaccharide has pendant functional groups, and any polysaccharide layer in the continuous polysaccharide coating layer is covalently linked to the previous polysaccharide layer through a chemical bonding reaction between the pendant functional groups and the pendant functional groups of the previous polysaccharide layer.

23. The kit according to claim 21 or 22, characterized in that The pendant functional groups of the continuous polysaccharide coating alternate between amine functional groups and amine-reactive functional groups.

24. The kit according to claim 23, characterized in that The amine-reactive functional group is an aldehyde group or a carboxyl group.

25. The kit according to claim 19, wherein The outermost polysaccharide layer of the continuous polysaccharide coating has at least one pendant functional group.

26. The kit according to claim 21 or 22, characterized in that The pendant functional group is selected from at least one of an aldehyde group, a carboxyl group, a thiol group, an amino group, a hydroxyl group and a malein group.

27. The kit according to claim 26, characterized in that The pendant functional groups are selected from aldehyde groups and / or carboxyl groups.

28. The kit according to claim 21 or 22, characterized in that The pendant functional groups of the continuous polysaccharide coating are directly or indirectly chemically bonded to a member of the specific binding pair.

29. The kit according to claim 1, wherein The sugar content was determined by the anthrone method.

30. The kit according to claim 1, wherein The molecular weight distribution Mw of the dextran is 1000-1000000 KDa.

31. The kit according to claim 1, wherein The molecular weight distribution Mw of the dextran is 10,000 to 800,000 KDa.

32. The kit according to claim 1, wherein The molecular weight distribution Mw of the dextran is 30,000 to 700,000 KDa.

33. A homogeneous chemiluminescent POCT detection kit comprising the kit according to any one of claims 1 to 32, wherein the POCT refers to a point-of-care test or a clinical test performed at the patient's side.

34. The kit according to claim 33, wherein The reagent kit includes a reagent strip, on which a plurality of holes for containing reagents are provided, and the holes include at least: The first reagent well is used to hold the donor reagent; The second reagent well is used to contain the receptor reagent.

35. A homogeneous chemiluminescence POCT detection method, which utilizes the kit according to claim 33 or 34 to detect a target molecule in a sample.

36. A homogeneous chemiluminescent POCT detection device, which uses the kit according to claim 33 or 34 or the detection method according to claim 35 to detect a target molecule in a sample to be tested.

37. The homogeneous chemiluminescence POCT detection device according to claim 36, characterized in that: The device comprises: Incubation module, used to control the temperature of the reagent strip; The light excitation and detection module is arranged on one side of the incubation module, and is used to emit excitation light to the reagent strip to generate a photo-induced chemiluminescence reaction; and detect the chemiluminescence signal generated by the reagent.

38. A method for performing homogeneous chemiluminescence analysis using the POCT detection device according to claim 36 or 37, comprising the following steps: S1, contacting the sample to be tested with the receptor reagent and the donor reagent to generate a test mixture after reaction; S2, using excitation light with a wavelength of 600 to 700 nm to excite the test mixture to perform chemiluminescence, and detecting the signal intensity of the chemiluminescence; the detection wavelength of the chemiluminescence is 520 to 620 nm; S3, judging whether the sample to be tested contains the target molecule to be tested and / or the concentration of the target molecule to be tested in the sample to be tested based on the analysis of the chemiluminescence signal intensity.

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