CN110736737A

CN110736737A - microsphere composition for chemiluminescence detection and application thereof

Info

Publication number: CN110736737A
Application number: CN201910652148.6A
Authority: CN
Inventors: 杨阳; 赵卫国; 刘宇卉; 李临
Original assignee: Boyang Biotechnology (Shanghai) Co Ltd; Chemclin Diagnostics Corp
Current assignee: Shanghai Suoxin Biological Technology Co ltd
Priority date: 2018-07-18
Filing date: 2019-07-18
Publication date: 2020-01-31
Also published as: CN116577319A

Abstract

The invention relates to microsphere compositions for chemiluminescence detection and application thereof in the technical field of chemiluminescence, wherein the microsphere compositions comprise respective donor microspheres which can generate active oxygen in an excited state, the surfaces of the donor microspheres are coated with markers, receptor microspheres which can react with the active oxygen to generate detectable chemiluminescence signals, the surfaces of the receptor microspheres are coated with biomolecules which can be specifically combined with target molecules to be detected, and the particle size of the donor microspheres is equal to that of the receptor microspheres.

Description

microsphere composition for chemiluminescence detection and application thereof

Technical Field

The invention belongs to the technical field of chemiluminescence, and particularly relates to microsphere compositions for chemiluminescence detection and application thereof.

Background

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

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

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

Although the detection sensitivity of the chemiluminescence detection method can be improved at degree by the method in the field, the detection range or linear range is narrow, therefore, methods which not only have higher sensitivity but also have wider detection range need to be developed.

Disclosure of Invention

The invention provides microsphere compositions for chemiluminescence detection, which have high sensitivity and wide detection range when used for chemiluminescence detection.

To this end, the present invention provides in an th aspect microsphere compositions for use in chemiluminescent detection comprising:

a donor microsphere capable of generating active oxygen in an excited state, the donor microsphere having a label coated on the surface thereof; and the combination of (a) and (b),

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

wherein the particle size of the donor microspheres is equal to the particle size of the acceptor microspheres.

In embodiments of the present invention, the donor and acceptor microspheres each have an average particle size of 20nm to 350nm, preferably 50nm to 300nm, more preferably 100nm to 250nm, and most preferably 180nm to 220 nm.

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

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

In embodiments of the invention, the label is avidin.

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

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

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

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

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

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

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

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

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

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

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

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

In a second aspect, the invention provides reagent compositions for chemiluminescence detection, which comprises a donor reagent acceptor reagent, wherein the acceptor reagent comprises acceptor microspheres in the microsphere composition according to the aspect of the invention, the acceptor microspheres have a C.V value of variation coefficient of particle size distribution in the acceptor reagent of 5% or more, and the donor reagent comprises donor microspheres in the microsphere composition according to the aspect of the invention, wherein the donor microspheres have a C.V value of variation coefficient of particle size distribution in the donor reagent of 5% or more.

In a third aspect of the present invention, there is provided chemiluminescent detection method comprising the step of contacting a sample to be tested with the microsphere composition according to the aspect of the present invention or the reagent composition according to the second aspect of the present invention.

In a fourth aspect, the present invention provides chemiluminescent detection systems for detecting a target molecule in a sample to be detected by using the microsphere composition according to the aspect, the reagent composition according to the second aspect, and/or the method according to the third aspect.

In embodiments of the present invention, the apparatus includes the following:

the reaction device is used for carrying out chemical reaction on a sample to be detected, donor microspheres and acceptor microspheres;

the excitation and reading device excites the donor microsphere to generate active oxygen by using excitation light with the wavelength of 600-700nm, the acceptor microsphere reacts with the received active oxygen to generate emission light with the wavelength of 520-620nm, and the optical signal of the emission light is recorded;

and the processor judges whether the target molecules to be detected exist in the sample to be detected or not and/or determines the content of the target molecules to be detected according to the recorded existence and/or intensity of the optical signals of the emitted light.

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

Drawings

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

FIG. 1 is a Gaussian distribution diagram of aldehyde-based polystyrene latex microspheres prepared in example 11.

FIG. 2 is a Nicomp distribution plot of aldehyde-based polystyrene latex microspheres prepared in example 11.

FIG. 3 is a Gaussian distribution diagram of donor microspheres prepared in example 11.

FIG. 4 is a Gaussian distribution plot of dextran-coated microspheres prepared in example 12

FIG. 5 is a Gaussian distribution plot of donor microspheres prepared in example 12.

FIG. 6 is a graph showing the Gaussian distribution of aldehyde-based polystyrene latex microspheres prepared in example 13.

Fig. 7 is a Gaussian distribution graph of aldehyde-based polystyrene latex microspheres embedded with a light-emitting composition prepared in example 13.

FIG. 8 is a Gaussian distribution diagram of aldehyde-based polystyrene latex microspheres with embedded luminescent composition coated with dextran prepared in example 13.

FIG. 9 is a Gaussian distribution diagram of acceptor microspheres prepared in example 13 with an average particle size around 250 nm.

Detailed Description

In order that the invention may be readily understood, a detailed description of the invention is provided below. However, before the invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

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

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

Term (I)

In the present invention, the "donor microsphere" can be a polymeric microparticle coated on a carrier via a functional group to form a photosensitizer filled polymer microsphere capable of generating active oxygen (e.g., singlet oxygen) upon photoexcitation, in which case the donor microsphere can also be referred to as a photosensitive microsphere or photosensitive microparticle, the photosensitizer is filled inside the donor microsphere, the photosensitizer can be a photosensitizer known in the art, preferably a compound that is relatively light stable and does not react efficiently with singlet oxygen, non-limiting examples of which include compounds such as methylene blue, rose bengal, porphyrin, and phthalocyanine, and derivatives of these compounds having 1 to 50 atom substituents that are used to render these compounds more lipophilic or hydrophilic and/or as a linking group to a specific binding partner.

The "acceptor microsphere" may be a polymer particle coated with a functional group on a support to form a polymer particle filled with a luminescent compound, which may be referred to as a luminescent microsphere or a luminescent particle, the acceptor microsphere of the luminescent microsphere has hydrophilic carboxyl dextran on the surface and a chemical composition filled therein and capable of reacting with active oxygen (e.g., singlet oxygen). in some embodiments of the present invention, the chemical composition undergoes a chemical reaction with singlet oxygen to form an unstable metastable intermediate that can decompose and emit light simultaneously or subsequently³⁺。

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

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

The term "antibody" as used herein is used in its broadest sense and includes antibodies of any isotype, antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, bispecific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein.

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

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

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

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

Detailed description of the preferred embodiments

The present invention will be described in more detail below.

The microsphere composition for chemiluminescence detection according to the aspect of the present invention comprises:

In the invention, the advantages that the particle sizes of the donor microsphere and the acceptor microsphere are the same are as follows:

1. when the particle sizes of the donor microsphere and the acceptor microsphere are the same, batches of polystyrene microsphere filling dye can be used, and the density of carboxyl or aldehyde functional groups on the surfaces of the microspheres can be ensured to be the same.

2. Chemical reaction collision theory indicates that reactant molecules must collide with each other to be possible to react, and the collision between the receptor microsphere coated with biomolecules and the donor microsphere coated with labels in the detection is random collision generated by brownian motion. Brownian motion is related to particle size, and the collision probability of microspheres with the same particle size is the same.

3. The principle of detecting the luminescent signal is that the photosensitizer in the donor microsphere is irradiated by 680nm laser to release singlet oxygen, the existence time of the singlet oxygen is microsecond level, the propagation distance is only 200nm, and the luminescent efficiency of the microsphere with smaller particle size is high.

In , the donor and acceptor microspheres each have an average particle size of 20nm to 350nm, preferably 50nm to 300nm, more preferably 100nm to 250nm, and most preferably 180nm to 220nm, e.g., in embodiments of the invention, the average particle size of the donor and acceptor microspheres can be 20nm, 50nm, 70nm, 90nm, 100nm, 120nm, 140nm, 160nm, 180nm, 200nm, 220nm, 240nm, or 250 nm.

In the preferred embodiments of the present invention, the particle size of the donor microsphere and the particle size of the acceptor microsphere are both 200nm, and the luminescence signal value of the detection is optimal, and the sensitivity of the detection is the best.

In the embodiments of the invention, the label is avidin, in which case, when the microsphere composition is used to detect a target molecule to be detected, the detection reagent includes a biotin-labeled biomolecule in addition to the microsphere composition.

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

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

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

Formula I

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

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

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

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

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

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

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

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

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

Thus, the second aspect of the present invention relates to reagent compositions for use in chemiluminescent detection comprising a donor reagent acceptor reagent comprising acceptor microspheres of the microsphere composition according to the aspect of the present invention, wherein the acceptor microspheres have a C.V value or more of the variation coefficient of particle size distribution in the acceptor reagent of 5% or more, and the donor reagent comprising donor microspheres of the microsphere composition according to the aspect of the present invention, wherein the donor microspheres have a C.V value or more of the variation coefficient of particle size distribution in the donor reagent of 5% or more.

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

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

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

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

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

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

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

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

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

The third aspect of the present invention relates to chemiluminescent assay methods, which comprises the step of contacting a sample to be tested with the microsphere composition according to or the reagent composition according to the second aspect of the present invention, wherein the intensity of a chemiluminescent signal generated by the reaction of the acceptor microsphere and singlet oxygen is detected to analyze and determine whether the sample to be tested contains a target molecule to be tested and/or the concentration of the target molecule to be tested.

The fourth aspect of the present invention relates to chemiluminescent detection systems for detecting a target molecule to be detected in a sample to be detected using the microsphere composition according to the aspect of the present invention or the reagent composition according to the second aspect of the present invention and/or the method according to the third aspect of the present invention.

In embodiments of the present invention, the apparatus includes the following:

Example III

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

Example 1: preparation of acceptor microspheres

1. A25 mL round-bottom flask was prepared, 0.1g of europium (III) complex and 10mL of 95% ethanol were added, magnetic stirring was performed, and the temperature in the water bath was raised to 70 ℃ to obtain a europium (III) complex solution.

2. A100 mL three-necked flask was prepared, 10mL 95% ethanol, 10mL water and 10mL 10% polystyrene microspheres coated with carboxyl dextran hydrogel having a particle size of 200nm were added, and the mixture was magnetically stirred and heated to 70 ℃ in a water bath.

3. Slowly and dropwise adding the europium (III) 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 to obtain the emulsion.

4. The emulsion was centrifuged for 1 hour at 30000g, and the supernatant was discarded after centrifugation and then resuspended in 50% ethanol. After repeating the centrifugal washing 3 times, the mixture was resuspended in 50mM CB buffer solution having a pH of 10 to a final concentration of 20mg/mL to obtain a receptor microsphere solution having a particle size of 200 nm.

5. The same method is used for preparing acceptor microsphere solutions with the grain diameters of 100nm, 150nm, 250nm and 350nm respectively.

Example 2: receptor microsphere coated antibody

1. Weighing 10mg of receptor microsphere coated with carboxyl dextran hydrogel in a centrifugal tube according to the preparation amount, and centrifuging at 10000rpm for 60 min.

2. The supernatant was discarded, and 2mg of Anti-PCT antibody I (which may be the antibody examples for any other assay (Anti-cTnI antibody I and Anti-PCT antibody I), 50. mu.L of Tween-20(50mg/mL) was added to the pellet, which was supplemented with volumetric amounts of 0.05M MES pH 6.0 to give a final concentration of 10mg/mL of receptor microspheres.

3. And (5) quickly mixing by ultrasound.

4. Add 50. mu.L of NaBH to the centrifuge tube₃CN (50mg/mL, 0.05M MES pH 6.0) was mixed well and reacted in a rotary mixer at 37 ℃ for 36-48 h.

5. And (3) sealing: 1mL of BSA (50mg/mL, 0.05M MES pH 6.0) was added and reacted in a rotary mixer at 37 ℃ for 12-16 h.

6. Cleaning: washed 3 times with 0.05M MES buffer.

7. And sampling and measuring the concentration, the grain diameter and the signal value of the washed receptor microsphere coated with the antibody.

Example 3: preparation of Donor microspheres

1. A25 mL round-bottomed flask was prepared, and 0.1g of copper (II) phthalocyanine and 10mL of DMF were added thereto, and stirred magnetically, and the temperature in a water bath was raised to 70 ℃ to obtain a copper (II) phthalocyanine solution.

2. Preparing a 100mL three-neck flask, adding 10mL 95% ethanol, 10mL water and 10mL polystyrene microspheres which are 10% in concentration and 200nm in particle size and coated with aldehyde dextran hydrogel, magnetically stirring, and heating in a water bath to 70 ℃.

3. And (3) slowly dropwise adding the copper (II) phthalocyanine solution obtained in the step (1) into the three-neck flask obtained in the step (2), reacting for 2 hours at 70 ℃, stopping stirring, and naturally cooling to obtain the emulsion.

4. The emulsion was centrifuged for 1 hour at 30000g, and after centrifugation the supernatant was discarded and resuspended in 50% ethanol. After repeating the centrifugal washing 3 times, the mixture was resuspended in 50mM CB buffer solution having a pH of 10 to a final concentration of 20mg/mL to obtain a donor microsphere solution having a particle size of 200 nm.

5. The same method is used to prepare donor microsphere solutions with the particle sizes of 80nm, 100nm, 150nm, 250nm and 350nm respectively.

Example 4: donor microsphere coated avidin

1. And (3) processing the donor microsphere suspension, namely sucking quantitative donor microspheres into a high-speed refrigerated centrifuge for centrifugation, removing the supernatant, adding quantitative MES buffer solution, performing ultrasonic treatment on an ultrasonic cell disruptor until the particles are resuspended, and adding MES buffer solution to adjust the concentration of the donor microspheres to 100 mg/mL.

2. Avidin solution preparation-weighing quantitative avidin (streptavidin or neutralized avidin is also available), adding MES buffer to dissolve to 8 mg/mL.

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

4. 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.

5. 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.

6. Cleaning: adding MES buffer solution into the reacted solution, centrifuging by a high-speed refrigerated centrifuge, discarding the supernatant, adding fresh MES buffer solution, resuspending by an ultrasonic method, centrifuging again, cleaning for 3 times, finally suspending by a small amount of buffer solution, measuring the solid content, and adjusting the concentration to 10mg/mL by the buffer solution.

Example 5 preparation of Biotin-labeled antibody (reagent )

1. Antibody treatment: Anti-PCT antibody II (which may be the antibody example corresponding to any other assay item) (Anti-cTnI antibody II, Anti-NT-proBNP antibody II, Anti-PCT antibody II and Anti-IL-6 antibody II)) was dialyzed against 0.1M NaHCO₃Solution, antibody concentration was determined and adjusted to 1 mg/mL.

2. A16.17 mg/mL biotin solution was prepared in DMSO.

3. Marking: mixing the treated Anti-PCT antibody II (antibody examples corresponding to any other analysis item (Anti-cTnI antibody II and Anti-PCT antibody II)) at the concentration of 1mg/mL with the prepared biotin solution according to the volume ratio of 10000:54, quickly mixing, and standing at the temperature of 2-8 ℃ for reaction for 12-16 hours.

4. And (3) dialysis: the reacted biotin-labeled antibody was dialyzed against biotin-labeled dialysis buffer (pH 8.00).

5. Dialyzed biotinylated antibody was aspirated and transferred to a clean centrifuge tube, and samples were taken to determine antibody concentration. The concentration of the biotin labeled antibody which is qualified for quality inspection is adjusted to 0.5 mg/mL.

Example 6: detection of microsphere compositions for chemiluminescence analysis.

The detection process comprises the steps of adding 25 mu L of a sample to be detected, 25 mu L of a biotin-labeled antibody, 175 mu L of a mixed solution of avidin-coated donor microspheres and 25 mu L of an acceptor microsphere reagent into a sample hole to be detected, th reagent hole, a donor reagent hole and an acceptor reagent hole of a reagent card respectively, placing the reagent card into a liquid-phase POCT analyzer developed by Boyang biotechnology (Shanghai) company, adding a sample adding needle in a reagent adding module into the sample adding module to obtain a corresponding volume of the sample to be detected, adding the biotin-labeled antibody and the avidin-coated donor microspheres, vibrating, incubating at 37 ℃ for 10 minutes to form a mixed liquid, continuously adding the mixed liquid into the acceptor reagent hole, vibrating, incubating at 37 ℃ for 10 minutes to form a mixture, irradiating the acceptor reagent hole with laser excited to emit, reacting for a certain time through , and detecting the light intensity emitted by the mixture by using an optical signal detection module.

Example 7: detection of luminescence signal quantity

The method described in example 6 and microsphere compositions with different particle sizes (taking Anti-PCT antibody i coated receptor microspheres as an example) were used to detect PCT to be detected in a sample, and the detection results are shown in table 1.

TABLE 1

As can be seen from table 1, under the condition that the particle size of the donor microsphere is fixed, the detected luminescence signal value gradually decreases with the increase of the particle size of the acceptor microsphere, and when the particle size of the acceptor microsphere is equal to the particle size of the donor microsphere, the detected luminescence signal value is the largest. And when the particle size of the donor microsphere and the particle size of the acceptor microsphere are both 200nm, the detected light-emitting signal value is optimal, and the detection sensitivity is best.

Example 8: preparation of quality control product and calibrator

1. Preparation of quality control product

And (3) taking the newborn bovine serum as a diluent, and respectively diluting the pure antigen products into 2 working solutions with different concentrations, wherein the working solutions are quality control products Q1 and Q2. And taking the quality control products Q1 and Q2 to be detected to perform three times of repeated calibration on the instrument system of the company. And measuring 10 holes each time, and calculating the overall mean value and SD, wherein the mean value +/-3 SD is the allowable range of the concentration measurement of the quality control substance.

2. Preparation of calibrator

The pure antigen is diluted into a series of concentrations by calf serum (containing preservative), and is frozen and stored for later use after being calibrated by national standard for immunoassay. The shelf life is 2 years when the product is stored at-20 ℃.

Simultaneously analyzing and measuring the calibrator and the national standard with corresponding concentration, and fitting by using 4 parameters or other models, wherein the absolute value of the correlation coefficient (r) of the calibrator dose-response curve is required to be not lower than 0.9900; while the two dose-response curves did not deviate significantly from parallelism (t-test); the ratio of the measured titer of the calibrator to the calibrated titer is 0.90-1.10 by taking the national standard as a standard.

Example 9: detection of precision between batches of microsphere compositions for chemiluminescence detection

A sample to be detected: quality control product Q1 prepared in example 8;

the process is as follows: repeating the detection 20 times to obtain a light intensity value (RLU)

Criterion for outlier determination: not less than 3SD

Reagents used in the detection:

(1) receptor microspheres prepared in example 2 (the particle size of the oxygen-receiving microspheres is 200nm, and the oxygen-receiving microspheres are respectively connected with anti-cTnI antibody I and anti-PCT antibody I);

(2) th reagent prepared in example 5 (biotin-labeled antibody, anti-cTnI antibody II, anti-PCT antibody II, respectively);

(3) example 4 preparation of donor microspheres (avidin coated donor microspheres of different particle sizes (80nm, 200 nm)).

The reagents (1) - (3) were combined to form a reagent set 1 and a reagent set 2 shown in table 2, cTnI and PCT antigen were detected respectively (after dilution to appropriate concentrations, the same samples were detected simultaneously by the reagent sets 1 and 2), and the detection was repeated for 20 wells, as described in example 6, and the detection results are shown in table 3.

Table 2:

reagent grouping	Reagent set 1	Reagent set 2
			Particle size/nm of acceptor microspheres	200nm	200nm
Donor microsphere particle size/nm	80nm	200nm

Table 3:

as can be seen from Table 3, when the light intensity of reagent set 2 is increased as compared with reagent set 1, that is, when the particle size of the acceptor microsphere is equal to that of the donor microsphere, the light intensity detected by this method is increased. Meanwhile, compared with the reagent group 1, the reagent group 2 has a smaller Coefficient of Variation (CV), i.e., when the particle size of the acceptor microspheres is equal to that of the donor microspheres, the precision is higher.

Example 10: detection of analytical sensitivity of microsphere compositions for chemiluminescence detection

A sample to be detected: a zero value calibrator;

Sensitivity: RLU substitution calibration curve

The reagents and procedures used in the detection were the same as those in example 9, and the results are shown in Table 4.

Table 4:

as can be seen from table 4, the sensitivity of detection by reagent set 2 was better than that of reagent set 1, i.e., when the particle size of the acceptor microsphere was equal to that of the donor microsphere, the sensitivity of the reagent was improved.

Comparative example 1: preparation of comparative microsphere compositions

, coating antibody directly on the surface of receptor microsphere

1. anti-PCT antibody i was dialyzed into 50mM CB buffer at PH 10 to a measured concentration of 1 mg/mL.

2. 0.5mL of the receptor microsphere prepared in example 1 and 0.5mL of anti-PCT antibody I were added to a 2mL centrifuge tube, mixed and added with 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 a 100mg/mLBSA 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, followed by resuspension in 50mM MES buffer. And repeating the centrifugal washing for 4 times, and diluting to a final concentration of 100 mu g/mL to obtain the anti-PCT antibody I-coated receptor microspheres with the particle sizes of 100nm, 150nm, 200nm, 250nm and 350nm respectively.

Second, the surface of the donor microsphere is directly coated with the antibody

1. The anti-PCT antibody ii was dialyzed into 50mM CB buffer at PH 10 to a measured concentration of 1 mg/mL.

2. Adding 0.5mL of photosensitive microsphere and 0.5mL of conjugated antibody II into a 2mL centrifuge tube, uniformly mixing, and adding 100 mu L of 10mg/mL of 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 2-8 ℃ 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 4 times and diluted to a final concentration of 100. mu.g/mL. Obtaining the donor microspheres coated with the anti-PCT antibody II with the grain sizes of 100nm, 150nm, 200nm, 250nm and 350nm respectively.

Comparative example 2: detection of luminescent signal content of contrast microsphere compositions

The procedure of the test was the same as in example 7 except that the microsphere composition used was replaced with the comparative microsphere composition prepared in comparative example 1, and the test results are shown in Table 5.

TABLE 5

As can be seen from table 5, the comparative microsphere composition detected a significantly lower amount of luminescence signal than the microsphere compositions described herein, and the detection sensitivity was significantly reduced. And when the particle size of the acceptor microsphere is equal to that of the donor microsphere in the comparison microsphere composition, the detected luminescence signal value is not optimal.

Example 11: donor microsphere with average particle size of 250nm and surface not coated or connected with polysaccharide and preparation of donor reagent

() preparation of aldehyde polystyrene latex microspheres

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 thereinto₂30min。

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 N₂30min。

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 latex microspheres had a Gaussian distribution with an average particle diameter of 201.3nm, a coefficient of variation (C.V.) -8.0%, a Gaussian distribution as shown in fig. 1, and a Nicomp distribution as shown in fig. 2, as measured by a nano-particle sizer. The aldehyde group content of the latex microsphere is 260nmol/mg measured by an electric conductivity titration method.

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

b) The preparation of the avidin solution comprises weighing quantitative streptavidin, adding MES buffer solution to dissolve 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, discarding the supernatant, adding fresh MES buffer solution, resuspending by an ultrasonic method, centrifuging again, washing for 3 times, finally suspending by a small amount of donor microsphere buffer solution, measuring the solid content, and adjusting the concentration to 150 mu g/ml by the donor microsphere buffer solution to obtain the donor reagent containing the donor microspheres.

The average gaussian distribution particle size of the donor microspheres was 227.7nm as measured by a nanometer particle sizer, and the coefficient of variation (C.V.) -6.5%, as shown in fig. 3.

Example 12: preparation of polysaccharide-coated donor microspheres with average particle size of 250nm and donor reagent

The preparation of aldehyde-based polystyrene latex microspheres and the filling process of the sensitizer were the same as those in () and (ii) of example 11.

() preparation of 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.

k) The amino group content was found to be 0.83mmol/g by TNBSA method.

Preparation of (di) aldehyde dextran

a) 10g of dextran with a mean molecular weight distribution of 500000kDa were weighed into a 250 beaker, 100ml of 0.1M phosphate buffer pH 6.0 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.

f) The aldehyde group content was measured by using the BCA Kit to be 0.94 mmol/g.

(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 microspheres 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) Adding the microspheres into an aldehyde dextran solution, and stirring 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%, as shown in fig. 4.

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

g) And (3) processing microsphere suspension, namely sucking the microspheres prepared in the quantitative step (III) into a high-speed refrigerated centrifuge for centrifugation, removing supernatant, adding quantitative MES buffer solution, performing ultrasound on an ultrasonic cell disruption instrument until the microspheres are resuspended, and adding MES buffer solution to adjust the concentration of the donor microspheres to 100 mg/ml.

h) Preparing avidin solution, weighing quantitative neutral avidin, adding MES buffer solution to dissolve to 8 mg/ml.

i) 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.

j) 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.

k) 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.

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

The average particle size of the donor microspheres in gaussian distribution was 249.9nm as measured by a nanometer particle sizer, and the coefficient of variation (C.V.) -11.6%, as shown in fig. 5.

Example 13: preparation of acceptor microspheres with average particle size of 250nm

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 thereinto₂30min；

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 N₂30min；

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 a Gaussian distribution measured by a nanometer particle sizer was 202.2nm, the coefficient of variation (C.V.) -4.60%, and the Gaussian distribution curve is shown in fig. 6. The aldehyde group content of the latex microsphere is 280nmol/mg measured by an electric conductivity titration method.

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 the microspheres in a Gaussian distribution measured by a nanometer particle sizer was 204.9nm, and the coefficient of variation (C.V.) (see FIG. 7) was 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 at this time was 241.6nm as measured by a nanometer particle sizer, and the coefficient of variation (C.V.) (see fig. 8) 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 a PCT antibody-coupled receptor microsphere solution.

The mean particle size of the Gaussian distribution of the particle sizes of the receptor microspheres measured by a nano-particle sizer was 253.5nm, and the coefficient of variation (C.V value) was 9.60% (as shown in fig. 9).

Example 14: test results and analysis on computer (test substance: PCT antigen)

The PCT homogeneous chemiluminescent assay kit (photo-activated chemiluminescent assay) used in this example consisted of reagent 1(R1 ') containing th anti-PCT antibody-coated acceptor microsphere, reagent 2(R2 ') containing biotin-labeled second anti-PCT antibody, and further contained a universal solution (R3 ') containing donor microsphere, where R1 ' was the acceptor reagent prepared using the acceptor microsphere (particle size distribution variation coefficient C.V ═ 9.6%) in example 13, and R3 ' was the donor reagent prepared using the donor microspheres in examples 11 and 12.

The detection process is completed on a full-automatic light-activated chemiluminescence analysis system (LiCA HT) developed by Boyang Biotechnology (Shanghai) Inc., and the detection results are output, and the specific detection results are shown in the following table 6.

TABLE 6

From the results in table 6, it is clear that both the sensitivity and the upper limit of detection of the microsphere composition of the present application are excellent. And the sensitivity and the upper limit of detection using the donor microsphere in example 11 are superior to those of the donor microsphere in example 12. Therefore, the performance of the donor microsphere without the polysaccharide coating on the surface is more excellent.

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

A microsphere composition for use in chemiluminescent detection comprising:

a donor microsphere capable of generating active oxygen in an excited state, the donor microsphere having a label coated on the surface thereof; and the combination of (a) and (b),

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

wherein the particle size of the donor microspheres is equal to the particle size of the acceptor microspheres.
2. Microsphere composition according to claim 1, wherein the average particle size of the donor and acceptor microspheres is 20nm to 350nm, preferably 50nm to 300nm, more preferably 100nm to 250nm, most preferably 180nm to 220 nm.
3. The microsphere composition of claim 1 or 2, wherein the donor microsphere comprises an th carrier, wherein the th carrier is filled with a sensitizer, and wherein the th carrier has a label chemically bonded to its surface.
4. The microsphere composition of claim 3, wherein the surface of said th carrier is not coated or linked with a polysaccharide substance, which directly chemically bonds to the label.
5. A microsphere composition according to claim 3 or 4, wherein said label is avidin, preferably said avidin is selected from the group consisting of ovalbumin, streptavidin, vitellin, neutravidin and avidin, and further wherein is preferably selected from the group consisting of neutravidin and streptavidin.
6. The microsphere composition of claim 5, wherein the avidin is chemically bonded to the surface of the carrier by reacting an amino group with an aldehyde group on the surface of the carrier to form a Schiff base.
7. The microsphere composition of any of claims 4 to 6, wherein the surface of the th carrier carries a bonding functional group for chemically bonding a label to the surface of the th carrier.
8. The microsphere composition of claim 7, wherein said bonding functional group is selected from the group consisting of amine group, amide group, hydroxyl group, aldehyde group, carboxyl group, maleimide group and thiol group; preferably selected from aldehyde groups and/or carboxyl groups.
9. A microsphere composition according to claim 7 or 8, wherein the bonded functional group content on the surface of the th carrier is 100 to 500nmol/mg, preferably 200 to 400 nmol/mg.
10. The microsphere composition of claim 3, wherein the surface of the th carrier is coated with hydrophilic aldehyde dextran, and the aldehyde group of the aldehyde dextran is chemically bonded with a label.
11. A microsphere composition according to any , wherein the photosensitizer is selected from the group consisting of methylene blue, rose bengal, porphyrin and phthalocyanine .
12. The microsphere composition of any of claims 1-11, wherein the acceptor microsphere comprises a second support, wherein the interior of the second support is filled with the luminescent composition, and wherein the surface of the second support is coated with at least layers of polysaccharide, wherein the surface of the polysaccharide layer is linked to a biomolecule.
13. The microsphere composition of claim 12, wherein the surface of the second carrier is coated with hydrophilic carboxydextran.
14. A microsphere composition according to claim 12 or 13, wherein said luminescent composition comprises europium complex, further preferably said europium complex is MTTA-EU³⁺。
15. The microsphere composition of any , wherein the material of the th vector and/or the second vector 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 from polystyrene, polypropylene, poly (4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate, or polyacrylate.
16. The microsphere composition of any of claims 1 to 15, wherein said active oxygen is singlet oxygen.
17. a reagent composition for use in chemiluminescent detection comprising a donor reagent acceptor reagent comprising acceptor microspheres from the microsphere composition of any of claims 1-16 wherein said acceptor microspheres have a particle size distribution variation coefficient of C.V value ≥ 5% in said acceptor reagent and donor microspheres from the microsphere composition of any of claims 1-16 wherein said donor microspheres have a particle size distribution variation coefficient of C.V value ≥ 5% in said donor reagent.
A chemiluminescent assay method of comprising the step of contacting a sample to be tested with the microsphere composition of any of claims 1-16 or the reagent composition of claim 17.
19, chemiluminescent detection system for detecting a target molecule to be detected in a sample to be detected using the microsphere composition of any of claims 1-16 or the reagent composition of claim 17 and/or the method of claim 18.
20. The system of claim 19, wherein the means comprises:

the reaction device is used for carrying out chemical reaction on a sample to be detected, donor microspheres and acceptor microspheres;

the excitation and reading device excites the donor microsphere to generate active oxygen by using excitation light with the wavelength of 600-700nm, the acceptor microsphere reacts with the received active oxygen to generate emission light with the wavelength of 520-620nm, and the optical signal of the emission light is recorded;

and the processor judges whether the target molecules to be detected exist in the sample to be detected or not and/or determines the content of the target molecules to be detected according to the recorded existence and/or intensity of the optical signals of the emitted light.