CN111665236A

CN111665236A - Preparation method and application of light-emitting microarray chip

Info

Publication number: CN111665236A
Application number: CN201910176398.7A
Authority: CN
Inventors: 刘宇卉; 杨阳; 李临
Original assignee: Shanghai Suoxin Biological Technology Co ltd
Current assignee: Shanghai Suoxin Biological Technology Co ltd
Priority date: 2019-03-08
Filing date: 2019-03-08
Publication date: 2020-09-15

Abstract

The invention relates to a preparation method and application of a luminescence microarray chip, belonging to the technical field of chemiluminescence analysis. The preparation method comprises the step of arranging one of chemiluminescent paired microspheres on the surface of a solid support, wherein the microsphere can generate active oxygen under the excitation of energy or active compounds, and the active oxygen can react with the other one of chemiluminescent paired microspheres contacted with the active oxygen to generate a chemiluminescent signal. The homogeneous phase chemiluminescence detection method of the microarray chip prepared by the method can effectively eliminate the matrix effect in homogeneous phase detection, improves the sensitivity and accuracy of detection, has high reaction speed and short detection time, can be applied to the determination of various biomolecules, and has the advantages of high speed, high efficiency, low cost and the like.

Description

Preparation method and application of light-emitting microarray chip

Technical Field

The invention belongs to the technical field of chemiluminescence analysis, and particularly relates to a preparation method and application of a luminescence microarray chip.

Background

Chemiluminescence is a specific chemical reaction, and organic molecules absorb chemical energy and then undergo energy level transition to generate an intermediate with high-energy electronic excited state instability, and when the intermediate returns to a ground state to emit photons, the intermediate is chemiluminescence. The immunoassay technique formed by combining chemiluminescence with antigen antibodies is chemiluminescence immunoassay. Chemiluminescence Immunoassay (CLIA) is a non-radioactive Immunoassay that has developed very rapidly worldwide in the last decade. The method has the advantages of high sensitivity, wide detection range, simple and rapid operation, good marker stability, no pollution, simple and economical instrument and the like. It is a substitute for radioimmunoassay and common enzyme immunoassay, and is an important development direction of immunoassay. CLIA is a rapid development, has already taken the first place of various immunoassays, and is the best substitute for radioimmunoassay and enzyme-linked immunoassay at present.

The immunoassay can be classified into heterogeneous immunoassay and homogeneous immunoassay according to whether a substance to be detected is to be separated from a reaction system in the determination process; heterogeneous immunoassay refers to the procedure of introducing a probe for labeling, in which various related reagents are required to be separated after mixing and reacting, and an object to be detected is separated from a reaction system and then detected, and is the mainstream direction in the existing immunoassay, such as: enzymatic chemiluminescence analysis, magnetic microsphere chemiluminescence immunoassay. Homogeneous immunoassay refers to direct measurement after mixing and reacting an analyte with a relevant reagent in a reaction system in the measurement process, such as: light-activated chemiluminescence analysis and electrochemiluminescence analysis.

Although homogeneous immunoassays do not require a separate washing step, some substances in serum or plasma can interfere with label signal detection or with chemiluminescent reactions. Meanwhile, in the homogeneous labeling immunoassay technology, the chemiluminescence field is involved, and some of the homogeneous labeling immunoassay technology relates to redox reactions, for example, blood samples containing some oxidation-reduction drugs (such as vitamin C) also interfere with the generation process of optical signals, so that the accuracy of detection results is seriously influenced. In addition, each reaction of the existing homogeneous immunoassay can only aim at one molecule to be detected, and time and labor are wasted.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a preparation method of a chemiluminescence microarray chip, the homogeneous phase chemiluminescence detection method of the microarray chip prepared by the method can simultaneously detect a plurality of molecules to be detected, the reaction speed is high, the detection time is short, the matrix effect in homogeneous phase immunoassay is effectively eliminated, and the detection accuracy is improved.

To this end, the present invention provides in a first aspect a method of preparing a luminescent microarray chip comprising the step of disposing one of chemiluminescent paired microspheres on a surface of a solid support, said microsphere being capable of generating reactive oxygen species upon excitation by energy or a reactive compound, said reactive oxygen species being capable of reacting with another of the chemiluminescent paired microspheres with which it is in contact to generate a chemiluminescent signal.

In some embodiments of the invention, one of the chemiluminescent paired microspheres is a donor microsphere and the other microsphere is an acceptor microsphere.

In other embodiments of the present invention, the solid support is selected from the group consisting of a glass chip, a quartz chip, a plastic chip, a polymer microsphere, a magnetic microsphere, a microplate, an NC membrane, and a PDVF membrane.

In some embodiments of the invention, the donor microspheres are distributed in an array on different regions of the surface of the solid support.

In some embodiments of the invention, donor microspheres are attached to magnetic microspheres and the donor microspheres are immobilized on the surface of a solid support by a magnetic field.

In other embodiments of the present invention, molecules are modified on the surface of the donor microsphere, and chemical groups are modified on the surface of the solid support, and the solid microsphere is immobilized on the surface of the solid support through the chemical bond interaction between the molecules modified on the surface of the donor microsphere and the chemical groups modified on the surface of the solid support; preferably, the chemical bond is selected from the group consisting of an ionic bond, a covalent bond, a metallic bond, and a coordinate bond; further preferably a covalent bond.

In some embodiments of the invention, the surface of the donor microsphere and the surface of the solid support are modified with molecules, and the donor microsphere is immobilized on the surface of the solid support by intermolecular forces between the surface modified molecules of the donor microsphere and the surface modified molecules of the solid support; preferably, the intermolecular forces are selected from hydrogen bonds, van der waals forces, ionic bonds, hydrophobic forces, aromatic ring stacking effects, and halogen bonds; further preferred are hydrogen bonds.

In some embodiments of the invention, 4 to 200 reaction wells are arranged in an array on the surface of a solid support.

In some embodiments of the invention, 4 to 200 microwells are disposed in an array on the surface of a solid support, with at least 1 reaction well disposed in each microwell.

In some embodiments of the invention, the diameter of the reaction pores is less than the particle size of the donor microspheres.

In some embodiments of the invention, donor microspheres are recessed in the reaction wells.

In some embodiments of the present invention, the surface of the donor microsphere and/or the acceptor microsphere is coated with dextran, and the dextran surface is modified with a chemical group selected from aldehyde group, carboxyl group, sulfhydryl group, amino group and hydroxyl group.

In other embodiments of the invention, a positional reference point is provided on the surface of the solid support.

In some embodiments of the invention, the method further comprises the step of directly or indirectly attaching a first specific binding substance to the surface of the donor microsphere, said first specific binding substance being capable of specifically binding to the target molecule to be detected.

In some embodiments of the invention, the solid support is contacted with a first specific binding substance, and the first specific binding substance is directly or indirectly attached to the surface of the donor microsphere through interaction with a specific partner.

In some embodiments of the invention, the donor microsphere is a photoactivated or chemically activated sensitizer.

In other embodiments of the present invention, the donor microsphere is a polymeric microsphere filled with a photosensitizer, which is capable of generating active oxygen under light excitation.

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

In other embodiments of the present invention, the particle size of the donor microsphere is 100-500nm, preferably 150-400nm, and more preferably 200-400 nm.

In some embodiments of the present invention, the acceptor microsphere includes a luminescent composition and a matrix, and the luminescent composition is filled in the matrix and/or coated on the surface of the matrix.

In other embodiments of the present invention, the luminescent composition is capable of reacting with reactive oxygen species to produce a detectable chemiluminescent signal comprising a chemiluminescent compound and a metal chelate.

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

In other 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 selected from europium.

In some embodiments of the invention, the metal chelate comprises a chelating agent selected from the group consisting of: NHA, BHHT, BHHCT, DPP, TTA, NPPTA, NTA, TOPO, TPPO, BFTA, 2-dimethyl-4-perfluorobutanoyl-3-butanone (fod), 2' -bipyridine (bpy), bipyridylcarboxylic acids, azacrown ethers, azacryptands and trioctylphosphine oxides and derivatives thereof.

In some preferred embodiments of the invention, the chemiluminescent compound is a derivative of dimethylthiophene and the metal chelate is a europium chelate.

In some embodiments of the invention, the matrix is selected from polymeric microspheres, preferably latex microspheres, more preferably polystyrene microspheres.

In other embodiments of the present invention, the particle size of the acceptor microsphere is 100-500nm, preferably 150-400nm, and more preferably 200-400 nm.

In a second aspect, the present invention provides a light-emitting microarray chip prepared according to the method of the first aspect of the present invention.

In a third aspect, the present invention provides a luminescent microarray chip prepared according to the method of the first aspect of the present invention for use in homogeneous chemiluminescent detection.

In a fourth aspect, the present invention provides a light-emitting microarray chip prepared by the method of the first aspect of the present invention for use in a POCT detection device.

In a fifth aspect, the present invention provides a POCT detection device for simultaneously detecting a plurality of markers of myocardial injury using the light-emitting microarray chip prepared according to the method of the first aspect of the present invention, wherein the markers of myocardial injury are selected from cTnI, cTnT, IL-6, CK-MB, MYO, NT-pro BNP, and PCT.

The invention has the beneficial effects that: the homogeneous phase chemiluminescence detection method of the microarray chip prepared by the method can effectively eliminate matrix effect in homogeneous phase detection, improves the sensitivity and accuracy of detection, has high reaction speed and short detection time, and can be applied to the determination of various biomolecules, including enzyme activity, receptor ligand reaction, low-affinity reaction, second messenger level, DNA, RNA, protein, polypeptide and carbohydrate. Meanwhile, the method can be used for simultaneously detecting a plurality of molecules to be detected, and has the advantages of high speed, high efficiency, low cost and the like.

Drawings

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

FIG. 1 is a schematic diagram of embodiment 3 of the present invention; the reference numerals in the figures have the following meanings: 1 a solid support; 2 donor microspheres; 3 a first specific binding substance (antibody I bound to a target molecule to be detected); 4 a second specific binding substance (antibody II bound to the target molecule to be detected); 5 acceptor microspheres.

FIG. 2 is a graph showing the results of detection in example 3 of the present invention.

FIG. 3 is a schematic diagram of embodiment 4 of the present invention; the reference numerals in the figures have the following meanings: 1 a solid support; 2 donor microspheres; 3 a first specific binding substance (antibody I bound to a target molecule to be detected); 4 a second specific binding substance (antibody II bound to the target molecule to be detected); 5 acceptor microspheres.

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

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

Term (I)

The term "homogeneous" as used herein is defined in english as "homogeneous" and means that the bound antigen-antibody complex and the remaining free antigen or antibody are detected without separation.

The term "specific binding substance" as used herein refers to a substance capable of specifically binding to a target molecule to be detected.

The term "specific pair" as used herein refers to a pair of substances capable of specifically binding to each other.

The term "binding" as used herein refers to direct association between two molecules due to interactions such as covalent, electrostatic, hydrophobic, ionic and/or hydrogen bonding, including but not limited to interactions such as salt and water bridges.

The term "specific binding" as used herein refers to the mutual discrimination and selective binding reaction between two substances, and is the conformation correspondence between the corresponding reactants in terms of the three-dimensional structure.

The term "chemiluminescent paired microspheres" as used herein refers to a pair of materials comprising an acceptor microsphere and a donor microsphere. When the donor microsphere is excited with energy or an activating compound, the photosensitizer on the surface of the donor microsphere decomposes oxygen in the surrounding environment to form active oxygen (singlet oxygen), which diffuses to the nearby acceptor microsphere, transferring energy to the acceptor microsphere, thereby generating excitation light.

The term "donor microsphere" as used herein refers to a sensitizer capable of generating a reactive intermediate, such as singlet oxygen, upon activation by energy or an active compound, which reacts with the acceptor microsphere. The donor microspheres may be light activated (e.g., dyes and aromatic compounds) or chemically activated (e.g., enzymes, metal salts, etc.). In some embodiments of the invention, the donor microspheres are polymeric microspheres filled with a photosensitizer, which may 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, porphyrins, phthalocyanines, and chlorophylls disclosed in, for example, U.S. patent No. 5709994, which is incorporated herein by reference in its entirety, and derivatives of these compounds having 1-50 atom substituents that are used to render these compounds more lipophilic or more hydrophilic and/or as a linker group to a member of a specific binding pair. Examples of other photosensitizers known to those skilled in the art may also be used in the present invention, such as those described in US patent No. US6406913, which is incorporated herein by reference.

The term "acceptor microsphere" as used herein refers to a compound that is capable of reacting with singlet oxygen to produce a detectable signal. The donor microsphere is induced by energy or an active compound to activate and release singlet oxygen in a high energy state that is captured by a nearby acceptor microsphere, thereby transferring energy to activate the acceptor microsphere. In some embodiments of the present invention, the acceptor microsphere comprises a luminescent composition and a matrix, wherein the luminescent composition is filled in the matrix and/or coated on the surface of the matrix. The "matrix" according to the present invention is microspheres or microparticles known to the skilled person, of any size, which may be organic or inorganic, which may be expandable or non-expandable, which may be porous or non-porous, which have any density, but preferably have a density close to that of water, preferably are capable of floating in water, and which are made of a transparent, partially transparent or opaque material. The substrate may or may not have a charge, and when charged, is preferably negatively charged. The matrix may be latex particles or other particles containing organic or inorganic polymers, lipid bilayers such as liposomes, phospholipid vesicles, oil droplets, silica particles, metal sols, cells and microcrystalline dyes.

The term "biotin" is widely present in animal and plant tissues, and has two cyclic structures on the molecule, namely, an imidazolone ring and a thiophene ring, wherein the imidazolone ring is the main part bound with streptavidin. Activated biotin can be conjugated to almost any biological macromolecule known to include proteins, nucleic acids, polysaccharides, lipids, and the like, mediated by a protein crosslinking agent. The "avidin" molecule consists of 4 identical peptide chains, each of which is capable of binding a biotin. Thus, each antigen or antibody can be conjugated to multiple biotin molecules simultaneously, thereby creating a "tentacle effect" that increases assay sensitivity.

The term "liquid phase reactant" as used herein refers to a reagent comprising a material capable of interacting or chemically reacting with a solid support surface.

The "target molecule to be detected" according to the present invention may be DNA, RNA, protein, polypeptide, carbohydrate, etc., preferably protein, more preferably immune molecule, such as antigen or antibody.

The term "test substance" as used herein refers to a mixture that may contain a target molecule to be detected. Typical test samples that may be used in the disclosed methods include body fluids such as blood, plasma, serum, urine, semen, saliva, and the like.

Detailed description of the preferred embodiments

The present invention will be described in detail below.

To this end, the method for preparing a luminescent microarray chip according to the first aspect of the present invention comprises the step of disposing one of chemiluminescent paired microspheres on a surface of a solid support, wherein the one of chemiluminescent paired microspheres is capable of generating active oxygen under excitation by energy or an active compound, and the active oxygen is capable of reacting with the other of chemiluminescent paired microspheres contacted therewith to generate a chemiluminescent signal.

In other embodiments of the present invention, the particle size of the donor microsphere is 100-500nm, preferably 150-400nm, and more preferably 200-400 nm. In some embodiments of the invention, the donor microspheres may have a particle size of 100nm, 150nm, 200nm, 250nm, 300nm, 400nm, and 500 nm.

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

In other embodiments of the present invention, the particle size of the acceptor microsphere is 100-500nm, preferably 150-400nm, and more preferably 200-400 nm. In some embodiments of the invention, the acceptor microspheres may have a particle size of 100nm, 150nm, 200nm, 250nm, 300nm, 400nm, and 500 nm.

In some embodiments of the present invention, the chemiluminescent microarray chip comprises a solid support having donor microspheres disposed on a surface thereof, wherein the donor microspheres are capable of generating reactive oxygen species upon excitation by energy or a reactive compound, and wherein the reactive oxygen species are capable of reacting with acceptor microspheres contacted therewith to generate a chemiluminescent signal.

In other embodiments of the invention, the donor microspheres are attached to magnetic microspheres that are immobilized on the surface of a solid support by a magnetic field.

In some embodiments of the invention, the donor microsphere is immobilized on the surface of the solid support by chemical bonding of its surface-modified molecule to a solid support surface-modified chemical group; preferably, the chemical bond is selected from the group consisting of an ionic bond, a covalent bond, a metallic bond, and a coordinate bond; further preferably a covalent bond.

In other embodiments of the invention, the donor microsphere is immobilized on the surface of the solid support by intermolecular forces between its surface-modified molecule and the solid support surface-modified molecule; preferably, the intermolecular forces are selected from hydrogen bonds, van der waals forces, ionic bonds, hydrophobic forces, aromatic ring stacking effects, and halogen bonds; further preferred are hydrogen bonds.

In some embodiments of the invention, the array of solid support surfaces comprises 4 to 200 reaction wells.

In other embodiments of the present invention, the array of the surface of the solid support comprises 4 to 200 microwells, and at least 1 reaction well is disposed in each microwell.

In some embodiments of the invention, the donor microspheres are recessed in the reaction wells.

In other embodiments of the present invention, the surface of the donor microsphere and/or the acceptor microsphere is coated with dextran, and the dextran surface is modified with a chemical group selected from aldehyde group, carboxyl group, sulfhydryl group, amino group and hydroxyl group.

In some embodiments of the invention, the surface of the solid support is further provided with a positioning datum.

In other embodiments of the present invention, the surface of the donor microsphere is directly or indirectly connected with a first specific binding substance, and the first specific binding substance can be specifically combined with a target molecule to be detected.

In some embodiments of the invention, the solid support is contacted with a first specific binding substance, and the first specific binding substance is attached to the surface of the donor microsphere through interaction with a specific partner.

In some embodiments of the invention, the homogeneous chemiluminescent detection method comprises the steps of:

step S1, a liquid-phase reactant containing the acceptor microspheres and a suspected substance to be detected containing the target molecules to be detected is contacted with the surface of a solid support for reaction, wherein donor microspheres are arranged on the surface of the solid support;

step S2, exciting the donor microsphere to generate active oxygen by using energy or active compounds, and reacting the acceptor microsphere with the active oxygen contacted with the acceptor microsphere to generate a chemiluminescent signal;

and step S3, detecting the intensity of the chemiluminescence signal in the step S2, and analyzing whether the target molecules to be detected exist in the object to be detected and/or the concentration of the target molecules to be detected.

In some embodiments of the present invention, the step S1 includes the following steps:

step S1-101, a liquid phase reactant of a to-be-detected substance suspected to contain a target molecule to be detected is contacted with a solid phase support for reaction;

step S1-102, the second specific binding substance is contacted and reacted with the surface of the solid phase support;

step S1-103, finally, the acceptor microsphere is contacted and reacted with the surface of the solid phase support;

the second specific binding substance can be specifically bound with a target molecule to be detected, and the second specific binding substance can be connected with the receptor microsphere through a specific counterpart.

In other embodiments of the present invention, the step S1 includes the following steps:

step S1-201, a liquid phase reactant containing the second specific binding substance and a to-be-detected substance suspected of containing a to-be-detected target molecule is contacted with the surface of the solid phase support for reaction;

step S1-202, the acceptor microballoon is contacted with the surface of the solid phase support for reaction;

step S1-301, a liquid phase reactant of a test substance suspected to contain a target molecule to be tested is contacted with a solid phase support for reaction;

step S1-302, the acceptor microsphere connected with the second specific binding substance is contacted with the surface of the solid phase support for reaction;

the second specific binding substance is connected with the receptor microsphere through a specific pairing substance, and the second specific binding substance can be specifically combined with a target molecule to be detected.

step S1-401, the liquid phase reactant of the analyte suspected to contain the target molecule to be detected contacts and reacts with the solid phase support;

step S1-402, the mixture of the second specific binding substance and the acceptor microsphere is contacted with the surface of the solid phase support for reaction;

In some embodiments of the present invention, the step S1 includes the following steps: contacting and reacting a mixture of a liquid-phase reactant of a to-be-detected substance suspected to contain a target molecule to be detected, a second specific binding substance and an acceptor microsphere with the surface of a solid support;

In other embodiments of the present invention, the liquid-phase reactant suspected of containing the analyte of interest is flowed over the surface of the solid support and contacted with the second microspheres on the surface of the solid support in a flowing manner.

In the method of the present invention, the contact reaction may be incubated as necessary. Specifically, the temperature of the incubation can be 35-45 ℃ and the time can be 10-60 min; preferably, the temperature of the incubation may be selected from 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃ or 44 ℃; the incubation time may be selected from 10min, 20min, 30min, 35min, 40min, 45min, 50min, 55min or 60 min.

Examples

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

Example 1: and (3) preparing a chemiluminescence microarray chip for simultaneously detecting cTnI, CK-MB, MYO and NT-proBNP.

1. Modification of the surface of the solid support.

On a glass chip having a 4X 4 layout, 16 reaction regions were provided, and groups capable of binding to biotin molecules were modified on the surface of each reaction region.

(1) Preparing 20mg/mL biotin solution by DMSO,

(2) with 0.1M NaHCO₃Solution biotin solution was diluted to 1 mg/ml.

(3) Soaking the modified glass chip in the biotin solution, and standing and reacting at 2-8 ℃ for 12-16 hours.

(4) The glass chip was removed and washed with 0.1M NaHCO₃And (3) washing with the solution to obtain the solid phase support (glass chip) with the surface modified with the biotin.

2. Preparation of avidin-coated donor microspheres

1) 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.

2) Donor microsphere coated avidin

(1) The donor microspheres were centrifuged to remove the supernatant, and 0.05M MES buffer was added to adjust the microsphere concentration to 200 mg/ml.

(2) Preparing 10mg/ml avidin solution by using MES buffer solution, and mixing the treated donor microsphere suspension, the 10mg/ml avidin solution and the MES buffer solution according to a certain volume ratio to form reaction liquid.

(3) Reaction: the reaction solution was mixed by shaking at 37 ℃ for 30min, and then 25mg/ml NaBH3CN solution was added and reacted at 37 ℃ for 48 hours by shaking.

(4) And (3) sealing: the reaction was carried out by adding 4ul of 150mg/ml glycine per 1mg of donor beads, shaking at 37 ℃ for 16 hours.

(5) Cleaning: washing with PB buffer (containing 0.05% Tween-20) and adjusting the concentration to 10mg/ml to obtain the avidin-coated donor microsphere solution.

3. Immobilization of donor microspheres on the surface of a solid support

(1) The prepared biotin-modified glass chips were immersed in 50ug/ml PB buffer (containing 0.05% Tween-20) coated with avidin donor microspheres under a green light and incubated at 37 ℃ for 2 hours.

(2) The glass chip was taken out and washed in PB buffer (containing 0.05% Tween-20) to obtain a solid support (glass chip) having donor microspheres immobilized on its surface.

4. Preparation of biotinylated antibody I

(1) Antibody treatment: the cTnI antibody I 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 1mg/mL cTnI antibody I and the prepared biotin solution according to the volume ratio of 10000:54, quickly mixing uniformly, and standing for reaction for 12-16 hours at the temperature of 2-8 ℃.

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

(5) Dialyzed biotinylated antibody I was aspirated and transferred to a clean centrifuge tube, and the antibody concentration was determined by sampling. The concentration of the biotin labeled antibody I which is qualified for quality inspection is adjusted to 0.5 mg/mL.

Biotinylated CK-MB antibody I, MYO antibody I, and NT-proBNP antibody I were prepared as described above, respectively.

5. Immobilization of antibody I on the surface of a solid support (glass chip)

(1) Biotinylated antibody I was diluted to 2ug/ml with biotin reagent buffer.

(2) Respectively dispensing corresponding biotinylated antibodies I on the positions of a glass chip shown in the table 1 by using a machine automatic sample applicator, and incubating for 2 hours at 37 ℃;

(3) washing in a biotin reagent buffer solution, standing and drying at 37 ℃ for 12-16 hours to obtain a solid support (glass chip) with a corresponding antibody I fixed on the surface, namely a chemiluminescent microarray chip for simultaneously detecting cTnI, CK-MB, MYO and NT-proBNP.

TABLE 1

	1	2	3	4
					A	cTnI	cTnI	cTnI	cTnI
B	CK-MB	CK-MB	CK-MB	CK-MB
					C	MYO	MYO	MYO	MYO
D	NT-proBNP	NT-proBNP	NT-proBNP	NT-proBNP

Example 2: preparation of antibody II coated receptor microspheres

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 suspension was resuspended in 50mM CB buffer solution having a pH of 10 to a final concentration of 20mg/mL to obtain a bead acceptor microsphere solution.

2) Receptor microsphere coated antibody II

(1)0.05M CB buffer (pH9.6) dialysis treatment of cTnI antibody II.

(2) And (3) mixing the receptor microsphere and the cTnI antibody II according to a certain proportion, putting the mixture on a multi-tube adjustable rotary mixer in a constant-temperature incubator, uniformly mixing at 37 ℃ and 25-40 rpm for 14-18 hours to form a reaction solution.

(3) And adding 8mg/ml NaBH4 solution, measuring NaBH4 solution, quickly adding the NaBH4 solution into the reaction solution, and reacting for 2 hours at the temperature of 2-8 ℃ in a multi-tube adjustable rotary mixer at the rpm of 25-40.

(4) And (4) washing, and performing constant volume to obtain a receptor microsphere solution coated with a cTnI antibody II.

Receptor microsphere solutions coated with CK-MB antibody II, MYO antibody II and NT-proBNP antibody II were prepared as described above.

Example 3: the microarray chip prepared by the method of the invention is used for simultaneously detecting cTnI, CK-MB, MYO and NT-proBNP in a substance to be detected.

(1) 15ul of each of the 4 samples were spotted on

columns

1, 2, 3 and 4 shown in Table 1.

(2) The acceptor microspheres coated with cTnI antibody II, CK-MB antibody II, MYO antibody II and NT-proBNP antibody II were diluted to 50ug/ml with buffer solutions, 15ul of each was spotted onto A, B, C, D lines shown in Table 1, and incubated at 37 ℃ for 10 minutes.

(3) The glass chip is irradiated by excitation light with the wavelength of 680nm, the donor microspheres are induced and activated, and active oxygen in a high energy state is released. The active oxygen in the high energy state is trapped by the acceptor microsphere at a close distance, thereby transferring energy to activate the luminescent compound in the acceptor microsphere. After several microseconds, the luminescent compound in the acceptor microsphere releases 612nm high-level red light, and the detection result is displayed by using a CCD imaging technology. The detection principle is shown in FIG. 1, and the detection result is shown in FIG. 2. The optical signal is further converted into a digitized electrical signal by a converter, and the detection result is presented in numerical value, as shown in table 2.

TABLE 2

Signal value	1	2	3	4
					A	32270	38912	95	15
B	34501	36124	112	17
					C	528	321	79	169
D	639	511	85	127

The detection signal of the kit has a linear relation with the concentration of a sample, and concentration values of cTnI, CK-MB, MYO and NT-proBNP in 4 known samples are shown in Table 3.

TABLE 3

Known concentration	1	2	3	4
					A	975.12pg/ml	1034.71pg/ml	13.29pg/ml	<2pg/ml
B	87.36ng/ml	68.21ng/ml	2.46ng/ml	<0.1ng/ml
					C	6.9ng/ml	46.08ng/ml	10.45ng/ml	19.78ng/ml
D	73.55pg/ml	65.19pg/ml	9.33pg/ml	15.89pg/ml

The detection results of four clinical samples with known concentration can be known: the signal value of the microarray chip prepared by the method for homogeneous detection has a linear relation with the reference concentration, and the accurate concentration can be obtained through conversion. Meanwhile, the detection signal of a low-value sample is very low, and the method has high sensitivity. In addition, only a small amount of sample and a reagent are needed to be added, the concentration of 4 target molecules to be detected can be obtained after the reaction is carried out for 10 minutes, and the detection method is quick and simple.

Example 4: the microarray chip prepared by the method of the invention can be used for simultaneously detecting cTnI, CK-MB, MYO and NT-proBNP in a detected object.

The reaction scheme is shown in FIG. 3. Firstly, through an injection molding process, a microfluidic chip with a 4 x 4 layout is injection molded on the surface of PDMS by using a metal mold, 16 areas are arranged on the surface of the chip, 1 reaction hole is arranged in each area, the particle size of each reaction hole is 200nm, and a positioning datum point is arranged at the same time.

And (2) enabling a reagent containing the receptor microsphere with the particle size of 400nm to flow through the surface of the chip at a proper flow rate, and strictly controlling the flow rate of the liquid, so that the receptor microsphere is sunken in a reaction hole on the surface of the PDMS chip, and the surface of the receptor microsphere is connected with avidin. Then biotinylated antibodies I (biotinylated cTnI antibody I, CK-MB antibody I, MYO antibody I, NT-proBNP antibody I) were passed over the surface of the PDMS chip, incubated at 37 ℃ for 60min, and immobilized on the receptor microspheres on the surface of the PDMS chip. The distribution of the different biotinylated antibodies I on the PDMS chip surface is shown in Table 4.

TABLE 4

4 samples were distributed and passed through

columns

1, 2, 3 and 4 of the PDMS chip surface as shown in Table 4, and then solutions of donor microspheres coated with cTnI antibody II, CK-MB antibody II, MYO antibody II and NT-proBNP antibody II were passed through lines A, B, C, D of the PDMS chip surface as shown in Table 4, respectively, the concentration of the donor microspheres was 50ug/ml, and the mixture was incubated at 37 ℃ for 10 min.

And irradiating the PDMS chip by using excitation light with the wavelength of 680nm, inducing and activating the donor microspheres, and releasing active oxygen ions in a high energy state. The active oxygen ions in the high energy state are captured by the acceptor microsphere at a close distance, thereby transferring energy to activate the luminescent compound in the acceptor microsphere. After several microseconds, the luminescent compound in the acceptor microsphere releases 612nm high-level red light, the CCD imaging technology is used for displaying the detection result, and then the converter is used for converting the optical signal into a digital electric signal, wherein the numerical value is shown in the following table 5:

TABLE 5

Signal value	1	2	3	4
					A	236618	9284	2354	104
B	15902	1039	586	2677
					C	17321	1660	56814	31486
D	21473	15999	167835	2659

The detection signal values are also in linear relation with the concentration of the sample, and the concentration values of cTnI, CK-MB, MYO and NT-proBNP in 4 known samples are shown in Table 6.

TABLE 6

Known concentration	1	2	3	4
					A	13187.23pg/ml	116.94pg/ml	30.56pg/ml	7.88pg/ml
B	22.82ng/ml	2.16ng/ml	0.92ng/ml	5.7ng/ml
					C	122ng/ml	43.5ng/ml	337.56ng/ml	219.18ng/ml
D	1484.74pg/ml	1109.15pg/ml	25211.74pg/ml	141.07pg/ml

As can be seen from Table 6, the detection signal is linearly related to the control concentration, and the accurate concentration can be obtained by conversion. And the detection signal of the low-value sample is very low, the method has high sensitivity, the concentration of 4 target molecules to be detected can be obtained after reaction for 10 minutes, and the detection method is quick and simple.

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

Claims

1. A method of preparing a luminescent microarray chip comprising the step of disposing one of chemiluminescent paired microspheres on a surface of a solid support, said microsphere capable of generating reactive oxygen species upon excitation by energy or a reactive compound, said reactive oxygen species capable of reacting with another of the chemiluminescent paired microspheres with which it is in contact to produce a chemiluminescent signal.

2. The method of claim 1, wherein one of the chemiluminescent paired microspheres is a donor microsphere and the other microsphere is an acceptor microsphere.

3. The method of claim 2, wherein the solid support is selected from the group consisting of a glass chip, a quartz chip, a plastic chip, a polymer microsphere, a magnetic microsphere, a microplate, an NC membrane, and a PDVF membrane.

4. The method of claim 2 or 3, wherein the donor microspheres are distributed on different areas of the surface of the solid support in an array.

5. The method of claim 4, wherein the donor microspheres are attached to magnetic microspheres and the donor microspheres are immobilized on the surface of the solid support by a magnetic field.

6. The method of claim 4, wherein the surface of the donor microsphere is modified with molecules and the surface of the solid support is modified with chemical groups, and the solid microsphere is immobilized on the surface of the solid support by chemical bonding between the molecules modified on the surface of the donor microsphere and the chemical groups modified on the surface of the solid support; preferably, the chemical bond is selected from the group consisting of an ionic bond, a covalent bond, a metallic bond, and a coordinate bond; further preferably a covalent bond.

7. The method of claim 4, wherein the donor microspheres and the surface-modified molecules of the solid support are modified, and the donor microspheres are immobilized on the surface of the solid support by intermolecular forces between the surface-modified molecules of the donor microspheres and the surface-modified molecules of the solid support; preferably, the intermolecular forces are selected from hydrogen bonds, van der waals forces, ionic bonds, hydrophobic forces, aromatic ring stacking effects, and halogen bonds; further preferred are hydrogen bonds.

8. The method of claim 4, wherein 4 to 200 reaction wells are arrayed on the surface of the solid support.

9. The method of claim 4, wherein 4 to 200 microwells are arranged in an array on the surface of the solid support, and at least 1 reaction well is arranged in each microwell.

10. The method of claim 8 or 9, wherein the reaction pores have a diameter smaller than the particle size of the donor microspheres.

11. The method of any one of claims 8 to 10, wherein donor microspheres are recessed in the reaction wells.

12. The method according to any one of claims 2 to 11, wherein the surface of the donor microsphere and/or the acceptor microsphere is coated with dextran, and the dextran surface is modified with a chemical group selected from aldehyde group, carboxyl group, sulfhydryl group, amino group and hydroxyl group.

13. The method of any one of claims 1 to 12, wherein a positioning datum is provided on the surface of the solid support.

14. The method of any one of claims 1 to 13, further comprising the step of attaching a first specific binding substance directly or indirectly to the surface of the donor microsphere, said first specific binding substance being capable of specifically binding to the target molecule to be detected.

15. The method of claim 14, wherein the solid support is contacted with the first specific binding substance and the first specific binding substance is directly or indirectly attached to the surface of the donor microsphere through interaction of the specific partner.

16. The method of any one of claims 2 to 15, wherein the donor microspheres are photoactivated or chemically activated sensitizers.

17. The method of claim 16, wherein the donor microspheres are polymeric microspheres filled with a photosensitizer capable of generating reactive oxygen species upon optical excitation.

18. The method of claim 17, wherein said photosensitizer is selected from one of methylene blue, rose bengal, a porphyrin and a phthalocyanine.

19. The method according to any one of claims 2 to 18, wherein the particle size of the donor microsphere is 100-500nm, preferably 150-400nm, and more preferably 200-400 nm.

20. The chip of any one of claims 2 to 19, wherein the acceptor microspheres comprise a luminescent composition and a matrix, and the luminescent composition is filled in the matrix and/or coated on the surface of the matrix.

21. The method of claim 20, wherein the luminescent composition is capable of reacting with reactive oxygen species to produce a detectable chemiluminescent signal and comprises a chemiluminescent compound and a metal chelate.

22. The method according to claim 21, wherein the chemiluminescent compound is selected from the group consisting of olefinic compounds, preferably from the group consisting of dimethylthiophene, dibutyldione compounds, dioxines, enol ethers, enamines, 9-alkylidenexanthanes, 9-alkylene-N-9, 10 dihydroacridines, arylethyletherenes, arylimidazoles, and lucigenins and derivatives thereof, more preferably from the group consisting of dimethylthiophene and its derivatives.

23. The process according to claim 21 or 22, wherein 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 from europium.

24. The method of any one of claims 21 to 23, wherein the metal chelate comprises a chelating agent selected from the group consisting of: NHA, BHHT, BHHCT, DPP, TTA, NPPTA, NTA, TOPO, TPPO, BFTA, 2-dimethyl-4-perfluorobutanoyl-3-butanone (fod), 2' -bipyridine (bpy), bipyridylcarboxylic acids, azacrown ethers, azacryptands and trioctylphosphine oxides and derivatives thereof.

25. The method of any one of claims 21 to 24, wherein the chemiluminescent compound is a derivative of dimethylthiophene and the metal chelate is a europium chelate.

26. The method according to any one of claims 20 to 25, wherein the matrix is selected from polymeric microspheres, preferably latex microspheres, more preferably polystyrene microspheres.

27. The method according to any one of claims 2 to 26, wherein the particle size of the acceptor microsphere is 100-500nm, preferably 150-400nm, and more preferably 200-400 nm.

28. A luminescent microarray chip prepared according to the method of any one of claims 1 to 27.

29. Use of a luminescent microarray chip prepared according to any one of claims 1 to 27 in homogeneous chemiluminescence detection.

30. Use of a luminescent microarray chip prepared according to any one of claims 1 to 27 in a POCT detection device.

31. A POCT detection device for simultaneously detecting a plurality of markers of myocardial injury using the light-emitting microarray chip prepared according to any one of claims 1 to 27, wherein the markers of myocardial injury are selected from cTnI, cTnT, IL-6, CK-MB, MYO, NT-pro BNP, and PCT.