WO2020088698A1

WO2020088698A1 - Homogeneous chemiluminescence analysis method and application thereof

Info

Publication number: WO2020088698A1
Application number: PCT/CN2019/128670
Authority: WO
Inventors: 章春奇; 金鑫; 李临
Original assignee: 博阳生物科技（上海）有限公司; 科美诊断技术股份有限公司
Priority date: 2018-10-31
Filing date: 2019-12-26
Publication date: 2020-05-07
Also published as: CN118010979A; CN111122852A

Abstract

A homogeneous chemiluminescence analysis method and an application thereof, the method comprising the following steps: in the presence of an anti-interference agent, detecting the strength of a chemiluminescence signal produced by a reaction of a receptor in a test sample with active oxygen, in order to determine whether the test sample contains test target molecules and/or the concentration of the test target molecules. The anti-interference agent comprises a carrier and active molecules, the carrier being a porous medium, the active molecules being filled in the carrier and capable of specifically binding to biotin molecules. The method can avoid false positive or false negative results caused by free biotin.

Description

A homogeneous chemiluminescence analysis method and its application

Cross-reference of related applications

This application requires the priority of the Chinese patent application CN201811285877.4 entitled "A homogeneous biochemiluminescence analysis method for anti-biotin interference and its application" filed on October 31, 2018. The entire contents of this application are approved by The reference is incorporated herein.

Technical field

The invention belongs to the technical field of chemiluminescence, and particularly relates to a homogeneous chemiluminescence analysis method and its application.

Background technique

Chemiluminescence analysis method is divided into homogeneous chemiluminescence analysis method and heterogeneous chemiluminescence analysis method according to separation or not. Heterogeneous chemiluminescence analysis requires multiple steps such as embedding, elution, and separation. The analysis process is cumbersome and the analysis time is long, which cannot meet the requirements of rapid detection and diagnosis. The homogeneous chemiluminescence analysis method effectively avoids the elaborate steps such as elution and separation, which greatly improves the analysis efficiency and cost performance, and is increasingly used. Biotin-Avidin-System (BAS) is a new type of biological reaction amplification system developed in the late 1970s. The BAS system has the advantages of high-affinity, high-sensitivity, and high-stability signal amplification and labeling technology. By combining the two, large-molecule biologically active substances such as antigens and antibodies can be coupled. Their combination is rapid, specific, stable, and has a multi-stage amplification effect. At present, the BAS system is mainly used in immunology, molecular biology and other fields. It has great advantages in the practical application of in vitro diagnosis. The biggest disadvantage of this method is the interference of biotin, which causes the detection error.

Biotin interference may produce false positives or false negatives. In general, the sandwich method produces false negatives and the competition method produces false positives. The current common solutions: 1. Replace the platform that does not use the avidin-biotin system; 2. Retest every other day or one week after discontinuing the relevant drugs / food; 3. Sample pretreatment: Streptavidin coating The particles remove the biotin in the sample.

However, there is currently no way to solve the problem of biotin interference in the avidin-biotin system.

Summary of the invention

The present invention provides a homogeneous chemiluminescence analysis method for the deficiencies of the prior art, which can solve the problem of biotin interference.

To this end, the first aspect of the present invention provides a homogeneous chemiluminescence analysis method, which includes the following steps: in the presence of an anti-interference agent, by detecting the luminescence signal intensity generated by the reaction of the receptor in the test sample with reactive oxygen species To analyze and determine whether the test sample contains the target molecule and / or the concentration of the target molecule;

Wherein, the anti-interference agent includes a carrier and an active molecule; the carrier is a porous medium; the active molecule is filled in the carrier and can specifically bind to biotin molecules.

In some embodiments of the invention, the anti-interference agent is capable of recognizing free biotin molecules and biotin markers.

In some embodiments of the invention, the anti-interference agent can selectively adsorb free biotin molecules.

In other specific embodiments of the present invention, the free biotin molecule can diffuse into the carrier and specifically bind to the active molecule therein.

In some embodiments of the present invention, the anti-interference agent is capable of restricting biological macromolecules larger than the size of the active molecule from entering its carrier.

In other embodiments of the present invention, the anti-interference agent can be evenly distributed in the liquid phase reaction system.

In some embodiments of the present invention, the porous medium is selected from one or more of porous metal materials, porous non-metallic materials, and porous polymer materials.

In other embodiments of the present invention, the carrier is mesoporous microspheres, preferably ordered mesoporous microspheres.

In some embodiments of the present invention, the pore size of the mesoporous microspheres is 2-50 nm, preferably 4-30 nm, and more preferably 5-15 nm.

In other embodiments of the present invention, the mesoporous microspheres are cage-like hollow mesoporous microspheres.

In some preferred embodiments of the present invention, the mesoporous microspheres are selected from Al ₂ O ₃ mesoporous materials, WO ₃ mesoporous materials, TiO ₂ mesoporous materials, ZrO ₂ mesoporous materials, silicon-based mesoporous materials And / or at least one of mesoporous carbon materials, preferably selected from silicon-based mesoporous materials.

In some embodiments of the invention, the active molecule is selected from avidin and / or streptavidin.

In other embodiments of the present invention, the active molecules are filled in the carrier by physical adsorption.

In some preferred embodiments of the present invention, the active molecule is filled in the carrier by contacting the carrier in a system containing a buffer.

In other preferred embodiments of the present invention, the pH value of the buffer-containing system is 7 to 9, preferably 7.1 to 8.0, more preferably 7.2 to 7.8, further preferably 7.3 to 7.6.

In some embodiments of the present invention, the active molecule is filled in the carrier by direct or indirect chemical crosslinking.

In some preferred embodiments of the present invention, the inner surface of the carrier is modified with a chemical group, and the active molecule is filled in the carrier by covalent coupling with the chemical group; wherein, the The chemical group is selected from one or more of carboxyl group, aldehyde group, amino group, mercapto group and hydroxyl group.

In some other preferred embodiments of the present invention, biotin molecules are connected to the inner surface of the carrier, and the active molecules are filled in the carrier by specific binding action with biotin molecules.

In some embodiments of the present invention, the anti-interference agent further includes a buffer solution, preferably a PBS buffer solution.

In some embodiments of the present invention, the preparation method of the anti-interference agent includes: step S1, contacting the carrier with the active molecule; preferably, the contacting is performed in the first buffer system.

In some preferred embodiments of the present invention, the preparation method of the anti-interference agent further includes step S0: the carrier is washed with a second buffer system, and step S0 is performed before step S1.

In other preferred embodiments of the present invention, the preparation method of the anti-interference agent further includes step S2: removing active molecules that are not filled into the carrier, step S2 is performed after step S1; preferably, by step S1 A third buffer system is added to the treated carrier, and then solid-liquid separation is performed to remove active molecules not filled in the carrier.

In some embodiments of the present invention, the receptor surface is directly or indirectly connected to a biomacromolecule, which can specifically bind to the target molecule to be tested.

In some other embodiments of the present invention, the substance in the sample to be tested contains a biotin marker, which includes a biomacromolecule that can be directly or indirectly bound to the target molecule to be tested bound to biotin.

In some preferred embodiments of the present invention, the biomacromolecule is selected from protein molecules, nucleic acid molecules, polysaccharide molecules and lipid molecules; preferably protein molecules; further preferably, the protein molecules are selected from antigens and / or Antibody; wherein, the antigen refers to a substance with immunogenicity; the antibody refers to an immunoglobulin produced by the body that can recognize a specific foreign object.

In some embodiments of the present invention, the sample to be tested further includes a donor; the surface of the donor directly or indirectly binds to a biotin-specific binding substance, and can generate active oxygen in an excited state.

In some specific embodiments of the present invention, the method includes the following steps:

S1, mixing the test sample with the reagent a containing the receptor, the reagent b containing the biotin marker, the reagent c containing the anti-interference agent, and the reagent d containing the donor to obtain the sample to be tested;

S2, using energy or an active compound to contact the sample to be tested obtained in step S1 to excite the donor to generate active oxygen;

S3. Analyze and determine whether the target molecule and / or the concentration of the target molecule in the test sample include the intensity of the luminescence signal generated by the reaction between the receptor and the active oxygen in the test sample.

In some preferred embodiments of the present invention, in step S1, the sample to be tested is mixed with the reagent a containing the receptor, the reagent b containing the biotin marker, and the reagent c containing the anti-interference agent, and then mixed with the The reagent d of the body is mixed to obtain a sample to be tested.

In some embodiments of the present invention, in step S2, the sample to be tested obtained in step S1 is irradiated with red excitation light of 600-700 nm to excite it to generate chemiluminescence.

In some embodiments of the present invention, in step S3, the detection wavelength at which the light-emitting signal value is recorded is 520-620 nm.

A second aspect of the present invention provides a homogeneous chemiluminescence detection device, which uses the method as described in the first aspect of the invention to perform homogeneous chemiluminescence detection.

A third aspect of the present invention provides a control method for a homogeneous chemiluminescence detection device according to the second aspect of the present invention.

The fourth aspect of the present invention provides a method according to the first aspect of the present invention or a detection device according to the second aspect of the present invention or a control method according to the third aspect of the present invention in biotin-streptavidin The application in the detection system of Hesu; preferably the application in the detection of thyroid function; further preferably the application in the detection of triiodothyronine and / or tetraiodothyronine.

The beneficial effects of the present invention are: the method of the present invention is carried out in the presence of an anti-interference agent, which uses active molecules such as SA or avidin protein molecules as "guest molecules" to fill the porous in an appropriate manner Within the pores of the medium, a "mesoporous assembly host-guest" system is formed, which can effectively distinguish free biotin molecules from biotin markers, thereby enabling the method of the present invention to eliminate the interference of free biotin and avoid chemiluminescence immunoassay False positive and / or false negative results. In addition, the method of the present invention is also practical and versatile, can be applied on different technical platforms, and has little effect on the performance of the reagents.

detailed description

In order to make the present invention easy to understand, the present invention will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for describing particular embodiments only, and is not meant to be limiting.

Where a range of values is provided, it should be understood that each intervening value between the upper and lower limits of the range and any other specified or intervening values in the stated range is encompassed within the invention. The upper and lower limits of these smaller ranges can be independently included in the smaller range, and are also covered by the present invention, subject to any explicitly excluded limit in the specified range. In the case where the specified range includes one or two limits, a range excluding either or both of those included limits is also included in the present invention.

Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those 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.

I. Terminology

The term "homogeneous chemiluminescence reaction" in the present invention means that under homogeneous conditions, the acceptor, the donor and the target molecule to be measured form a complex, and the complex formed under the irradiation of excitation light generates chemiluminescence the process of.

The term "carrier" in the present invention refers to a substance that can carry active molecules to participate in chemical or physical processes together. The chemical composition of the carrier is not particularly limited in the present invention, and may be organic materials or inorganic materials, such as high molecular polymers, metals, glass, mineral salts, diatoms, phospholipid vesicles, silicon particles, microcrystalline dyes and many more.

The term "porous medium" used in the present invention refers to a substance composed of a skeleton composed of a solid substance and a minute gap formed by the skeleton divided into a large number of densely packed groups.

The term "active molecule" as used in the present invention refers to a molecule having specific binding ability to biotin molecule. Exemplary active molecules are avidin and streptavidin.

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

The term "target molecule to be detected" in the present invention refers to the substance in the sample to be detected during detection. One or more substances with specific binding affinity to the target molecule to be tested will be used to detect the target molecule. The target molecule to be tested may be a protein, a peptide, an antibody, or a hapten that can bind the antibody.

The term "sample to be tested" in the present invention refers to a multi-component mixed liquid to be tested including a sample to be tested, a reagent containing a donor, a reagent containing an acceptor, and a reagent containing an anti-interference agent, etc. .

The term "antibody" in the present invention is used in the broadest sense and includes any isotype of antibody, antibody fragments that retain specific binding to antigen, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies , Humanized antibodies, single-chain antibodies, bispecific antibodies, and fusion proteins containing the antigen-binding portion of the antibody and non-antibody proteins.

The term "antigen" as used in the present invention refers to a substance that can stimulate the body to produce an immune response, and can combine with antibodies and sensitized lymphocytes of immune response products in vivo and in vivo to produce immune effects.

The term "binding" in the present invention refers to the direct union between two molecules due to interactions such as covalent, electrostatic, hydrophobic, ionic, and / or hydrogen bonds, including but not limited to, such as salt bridges and water bridges. .

The term "specific binding" in the present invention refers to the mutual discrimination and selective binding reaction between two substances. From the perspective of stereostructure, it is the conformational correspondence between the corresponding reactants.

The term "active oxygen" in the present invention refers to a general term for substances in the body or in the natural environment that are composed of oxygen and contain oxygen and are active in nature. It is mainly an excited state of oxygen molecules, including superoxide, an electron reduction product of oxygen Anions (O ₂ ·-), two-electron reduction product hydrogen peroxide (H ₂ O ₂ ), three-electron reduction product hydroxyl radical (· OH), nitric oxide and singlet oxygen (1O ₂ ), etc.

In the present invention, the term "receptor" refers to a substance that can react with reactive oxygen to generate a detectable signal. The donor is activated by energy or an active compound and releases active oxygen in a high-energy state, which is captured by a close-range acceptor, thereby transferring energy to activate the acceptor. In some specific embodiments of the present invention, the receptor is a substance that undergoes a chemical reaction with active oxygen (eg, singlet oxygen) to form an unstable metastable intermediate, the metastable intermediate The body can decompose and emit light simultaneously or subsequently. Typical examples of these substances include, but are not limited to: enol ethers, enamines, 9-alkylidene xanthan gum, 9-alkylidene-N-alkylacridine, aromatic vinyl ethers, diethylene oxide, dimethyl Thiophene, aromatic imidazole or gloss. In other specific embodiments of the present invention, the receptor is capable of reacting with active oxygen (eg, singlet oxygen) to form a hydroperoxide or dioxetane which can be decomposed into ketones or carboxylic acid derivatives Olefins; stable dioxetane that can be decomposed by the action of light; acetylenes that can react with active oxygen (such as singlet oxygen) to form diketones; hydrazones that can form azo compounds or azocarbonyl compounds Or hydrazides, such as luminol; and aromatic compounds that can form internal peroxides. Specific, non-limiting examples of receptors that can be utilized in accordance with this disclosure and the claimed invention are described in US Patent No. US5340716 (the patent document is hereby incorporated by reference in its entirety). In some other specific embodiments of the present invention, the receptor includes an olefin compound and a metal chelate compound, which is non-particulate and soluble in an aqueous medium. For the situation of this receptor, see Patent PCT / US2010 / 025433 (This patent document is hereby incorporated by reference in its entirety). .

In the present invention, the "chemiluminescent compound" refers to a compound called a marker that can undergo a chemical reaction to cause luminescence, for example, by being converted to another compound formed under an electronically excited state. The excited state can be a singlet state or a triplet excited state. The excited state can relax to the ground state to directly emit light, or it can restore itself to the ground state by transferring the excitation energy to the emission energy acceptor. In this process, the energy acceptor microspheres will be transitioned to an excited state and emit light.

The term "can directly or indirectly bind" means that the specified physical object can specifically bind to the physical object (directly), or the specified physical object can specifically bind to the specific binding pair member, or have two or More complexes (indirectly) with specific binding partners capable of binding other entities.

The "specific binding pairing member" of the present invention is selected from (1) small molecules and binding partners for the small molecules, and (2) large molecules and binding partners for the large molecules

In the present invention, the active oxygen may be provided by a "donor". The term "donor" in the present invention refers to a sensitizer capable of generating an active intermediate such as singlet oxygen that reacts with an acceptor after activation by energy or an active compound. The donor can be photoactivated (such as dyes and aromatic compounds) or chemically activated (such as enzymes, metal salts, etc.). In some specific embodiments of the present invention, the donor is a photosensitizer, and the photosensitizer may be a photosensitizer known in the art, preferably a compound that is relatively light stable and does not effectively react with singlet oxygen, which is not limited Examples include compounds such as methylene blue, rose red, porphyrin, phthalocyanine, and chlorophyll disclosed in US Pat. No. 5,999,994, which is incorporated herein by reference in its entirety, and derivatives of these compounds having 1-50 atom substituent The substituents are used to make these compounds more lipophilic or more hydrophilic, and / or as linking groups to specific binding partners. Other examples of photosensitizers known to those skilled in the art can also be used in the present invention, for example, the content described in US Pat. No. 6,406,913, which is incorporated herein by reference. In other specific embodiments of the present invention, the donor is a chemically activated other sensitizer. Non-limiting examples are certain compounds that catalyze the conversion of hydrogen peroxide into singlet oxygen and water. Examples of other donors include: 1,4-dicarboxyethyl-1,4-naphthalene endoperoxide, 9,10-diphenylanthracene-9,10-endoperoxide, etc. Heating these compounds or These compounds directly absorb light and release active oxygen (eg singlet oxygen).

The photosensitizer generally activates the chemiluminescent compound by irradiating a medium containing the above reactants. The medium must be irradiated with light having a certain wavelength and sufficient energy to convert the photosensitizer to an excited state, thereby enabling it to activate molecular oxygen into singlet oxygen. The excited state of a photosensitizer capable of exciting molecular oxygen is generally in the triplet state, which has an energy higher than the photosensitizer in the ground state by about 20 Kcal / mol, usually at least 23 Kcal / mol. Although shorter wavelengths can be used, such as 230-950 nm, preferably, light with a wavelength of approximately 450-950 nm is used to illuminate the medium. The light generated can be measured in any conventional manner, such as photography, visual inspection, photometer, etc., in order to determine the amount related to the analyte content in the medium. The photosensitizer is preferably relatively non-polar to ensure solubility in lipophilic members.

The photosensitizer and / or chemiluminescent compound can be selected to dissolve, or non-covalently bind to the surface of the particles. In this case, these compounds are preferably hydrophobic to reduce their ability to dissociate from the particles so that both compounds can bind to the same particles.

II. Specific implementation plan

The present invention will be explained in more detail below.

In the method of the present invention, by adding an anti-interference agent, the anti-interference agent is filled with a carrier in an appropriate manner by using an active molecule that can specifically bind to biotin molecules as a "guest molecule" to form a "mesoporous assembly "Host-guest" system, so that the homogeneous chemiluminescence analysis method of the present invention avoids false positive or false negative results due to free biotin.

The homogeneous chemiluminescence analysis method according to the first aspect of the present invention includes the following steps: in the presence of an anti-interference agent, analysis and judgment is made by detecting the intensity of the luminescence signal generated by the reaction of the receptor in the sample to be tested with active oxygen Whether the sample to be tested contains the target molecule to be tested and / or the concentration of the target molecule to be tested;

Wherein, the anti-interference agent includes a carrier and an active molecule; the carrier is a porous medium; the active molecule is filled in the carrier and can specifically bind to biotin molecules. Here, "the active molecules are filled in the carrier" means that the active molecules are located in the gaps in the carrier, and may or may not be in contact with the framework.

In some embodiments of the invention, the anti-interference agent is capable of recognizing free biotin molecules and biotin markers. In the present invention, "recognition" may mean that the active molecules in the anti-interference agent and the free biotin molecules and / or biotin markers achieve mutual binding through the synergistic effect of intermolecular forces.

In other specific embodiments of the present invention, the free biotin molecule can diffuse into the carrier and specifically bind to the active molecule therein. In the present invention, the "diffusion" may mean that free biotin molecules are dispersed into the carrier due to the random movement of the molecules.

In some embodiments of the present invention, the carrier satisfies at least one of the following conditions: a) The inner pores of the carrier have a sufficiently large surface area (far beyond the carrier surface area), and the voids only allow active molecules to enter, but limit activity Proteins with larger molecules, such as antibodies or large antigens; b) Active molecules such as SA or Avdin can be filled into the carrier by chemical or physical adsorption methods, such as inside the void; c) The carrier can be stably and uniformly distributed in the solution ( Such as aqueous solution), without precipitation.

In some embodiments of the present invention, the inner surface area of the carrier is greater than its outer surface area; preferably, the inner surface area of the carrier is more than 5 times, preferably more than 10 times, more preferably more than 20 times . In some preferred embodiments of the present invention, the internal surface area of the carrier is a multiple of its external surface area including but not limited to: 5 times, 6 times, 8 times, 10 times, 12 times, 16 times, 18 times, 20 times , 22 times, 24 times, 26 times, 28 times or 30 times.

In other embodiments of the present invention, the particle size of the carrier is 15-300nm, such as 15nm, 20nm, 25nm, 30nm, 40nm, 50nm, 100nm, 250nm, 300nm, etc., preferably 30-250nm, more preferably 50-200nm. If the particle size of the carrier is too large, it will cause the carrier to settle too quickly, which is not conducive to the formation of a stable uniform solution.

In some embodiments of the present invention, the specific surface area of the carrier 200m ² / g or more, for example, ^{^{200m 2 / g, 400m 2 /}} g, 600m 2 / g, 800m 2 / g, 1000m 2 / g, 1200m 2 / g, 1500 m ² / g, etc., preferably 400 m ² / g or more, more preferably 600 m ² / g or more, and most preferably 1000 m ² / g or more.

In other embodiments of the present invention, the minimum porosity of the support is greater than 40%, preferably greater than 50%, and more preferably greater than 60%.

In some embodiments of the present invention, the pore diameter of the mesoporous microspheres is 2-50 nm, such as 2 nm, 5 nm, 10, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 50 nm, etc., preferably 4-30 nm, More preferably, it is 5-15 nm.

Silicon-based mesoporous materials are a type of periodic mesoporous materials composed of SiO ₂ (CH ₂ ) ₂ tetrahedral structural units. Mesoporous silica materials can be divided into two categories from the microscopic view: one is disordered mesoporous solids represented by silica xerogels and aerogels. The disordered mesoporous silica can be powder, bulk, flake or thin film. The other type is ordered mesoporous silica represented by MCM41. The structural characteristic of ordered mesoporous silica is that the pore size is uniform, arranged in an orderly manner, and the mesoporous pore size can be adjusted between 2-10 nm. Due to the thin wall of the hole, the exchange degree of the silicon-based unit is not high, and the hydrothermal stability is not good. Its specific surface area can reach 1000m ² / g. There are SBA series, HMM series, TUD series, FSM series, KIT series, CMK series, FDU series, starbon and so on. Among them, there are also more SBA-15 studies, and the hydrothermal stability of the material is better than that of the MCM series. The aperture is adjustable between 5-30nm. HMM is a spherical mesoporous material with an aperture of 4-15nm and an adjustable outer diameter of 20-80nm.

In some embodiments of the invention, the active molecule is selected from avidin and / or streptavidin. Avidin is a glycoprotein that can be extracted from egg white. Its molecular weight is about 60kD. Each molecule is composed of 4 subunits and can be intimately combined with 4 biotin molecules. The avidin includes but is not limited to: avidin, streptavidin, yolk avidin and avidin. Streptavidin (SA) is a protein with similar biological characteristics as avidin (A). It is a protein product secreted by Streptomyces avidin bacteria during the cultivation process. SA can also be genetically engineered produce. The molecular weight of SA is 65000, which is composed of four peptide chains with the same sequence. Each SA peptide chain can be combined with one biotin molecule. Therefore, like avidin, each SA molecule also has four binding sites for biotin molecules, and the binding constant is the same as avidin, which is 1015mol / L.

In other embodiments of the present invention, the active molecules are filled in the carrier by physical adsorption. Physical adsorption is also called van der Waals adsorption. It is caused by the force between the adsorbate and the adsorbent molecules. This force is also called van der Waals force.

In other embodiments of the present invention, the total concentration of the carrier and the active molecules filled in the carrier in the anti-interference agent is 5-50ug / mL, such as 5ug / mL, 10ug / mL, 15ug / mL , 20ug / mL, 25ug / mL, 30ug / mL, 35ug / mL, 40ug / mL, 45ug / mL, 50ug / mL, etc., preferably 8-30ug / mL, more preferably 10-20ug / mL.

In some embodiments of the present invention, the preparation method of the anti-interference agent includes: step S1, contacting the carrier with the active molecule; preferably, the contacting is performed in the first buffer system. In some embodiments of the above method, the pH of the first buffer system is 7.0 to 9, preferably 7.1 to 8.0, more preferably 7.2 to 7.8, still more preferably 7.3 to 7.7, further preferably 7.35 to 7.50, and most preferably 7.40 . In this embodiment, examples of the pH value of the first buffer system include 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 9.0, and the like.

In some other embodiments of the above method, the pH value of the first buffer system is 3.0 to 7.0, preferably 3.5 to 6.8, more preferably 4.0 to 6.5, still more preferably 5.0 to 6.4, further preferably 5.5 to 6.3, and most preferably 6.0. In this embodiment, examples of the pH value of the first buffer system include 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.1, 6.2, 6.3, 6.4 , 6.5, 6.6, etc.

According to some specific embodiments, the method further includes step S0: the carrier is washed with a second buffer system, and step S0 is performed before step S1. In some embodiments of the above method, the pH value of the second buffer system is 7.0 to 9, preferably 7.1 to 8.0, more preferably 7.2 to 7.8, still more preferably 7.3 to 7.7, further preferably 7.35 to 7.50, most preferably 7.40 . In this embodiment, examples of the pH value of the first buffer system include 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 9.0, and the like. In some other embodiments of the above method, the pH value of the second buffer system is 3.0 to 7.0, preferably 3.5 to 6.8, more preferably 4.0 to 6.5, still more preferably 5.0 to 6.4, further preferably 5.5 to 6.3, and most preferably 6.0. In this embodiment, examples of the pH value of the first buffer system include 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.1, 6.2, 6.3, 6.4 , 6.5, 6.6, etc.

According to some specific embodiments, the method further includes step S2: removing active molecules that are not filled into the carrier, and step S2 is performed after step S1. Preferably, by adding a third buffer system to the carrier treated in step S1, and then performing solid-liquid separation, the active molecules not filled in the carrier are removed.

In some embodiments of the above method, the pH value of the third buffer system is 7.0 to 9, preferably 7.1 to 8.0, more preferably 7.2 to 7.8, still more preferably 7.3 to 7.7, further preferably 7.35 to 7.50, and most preferably 7.40 . In this embodiment, examples of the pH value of the third buffer system include 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 9.0, and the like. In some other embodiments of the above method, the pH value of the third buffer system is 3.0 to 7.0, preferably 3.5 to 6.8, more preferably 4.0 to 6.5, still more preferably 5.0 to 6.4, further preferably 5.5 to 6.3, and most preferably 6.0. In this embodiment, examples of the pH value of the third buffer system include 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.1, 6.2, 6.3, 6.4 , 6.5, 6.6, etc.

In some embodiments of the above method, the first buffer system comprises a phosphate buffer, piperazine-1,4-diethanesulfonic acid buffer, 3-morpholinepropanesulfonic acid buffer, 4- One or more of hydroxyethylpiperazine ethanesulfonic acid buffer and 3- (hydroxyethylpiperazine) -2-hydroxypropanesulfonic acid buffer. In some embodiments of the above method, the second buffer system comprises a phosphate buffer, piperazine-1,4-diethanesulfonic acid buffer, 3-morpholinepropanesulfonic acid buffer, 4- One or more of hydroxyethylpiperazine ethanesulfonic acid buffer and 3- (hydroxyethylpiperazine) -2-hydroxypropanesulfonic acid buffer. In some embodiments of the above method, the third buffer system comprises a phosphate buffer, piperazine-1,4-diethylsulfonic acid buffer, 3-morpholinepropanesulfonic acid buffer, 4- One or more of hydroxyethylpiperazine ethanesulfonic acid buffer and 3- (hydroxyethylpiperazine) -2-hydroxypropanesulfonic acid buffer.

In some embodiments of the above method, the third buffer system further includes a surfactant. According to some embodiments, the surfactant comprises one or more selected from Tween-20, Tween-80, Triton X-405, Triton X-100, BRIJ 35, and Pluronic L64. According to some embodiments, the surfactant comprises Tween-20.

In some embodiments of the above method, in step S1, the temperature of the contact is 0-50 ° C, preferably 20-40 ° C, such as 25-30 ° C (ie room temperature); and / or, the contact time is 6- 24 hours, preferably 8-12 hours, such as 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, etc. In some other embodiments of the above method, the temperature of the contact is 0-50 ° C, preferably 20-40 ° C, for example 25-30 ° C (ie room temperature); and / or, the contact time is 1-10 hours It is preferably 2-6 hours, for example, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, etc.

In some embodiments of the above method, step S3 is further included, and a fourth buffer system is added. Preferably, the fourth buffer system comprises a phosphate buffer, piperazine-1,4-diethylsulfonic acid buffer, 3-morpholinepropanesulfonic acid buffer, 4-hydroxyethylpiperazine ethanesulfonate One or more of acid buffer and 3- (hydroxyethylpiperazine) -2-hydroxypropanesulfonic acid buffer.

In some embodiments of the present invention, the sample to be tested further includes a donor; the surface of the donor directly or indirectly binds to a biotin-specific binding substance, and can generate active oxygen in an excited state. Preferably, the biotin-specific conjugate is streptavidin.

In some preferred embodiments of the present invention, in step S1, the sample to be tested is mixed with the reagent a containing the receptor, the reagent b containing the biotin marker, and the reagent c containing the anti-interference agent, and then mixed with the donor The reagent d is mixed to obtain the sample to be tested.

In some other embodiments of the present invention, in step S3, the detection wavelength at which the light-emitting signal value is recorded is 520-620 nm.

It is worth noting that the method of the present invention is not limited to the sandwich method detection, but can also be used for the detection of the capture method, the competition method and the like.

The second aspect of the present invention relates to a homogeneous chemiluminescence detection device which uses the method as described in the first aspect of the present invention to perform homogeneous chemiluminescence detection; preferably, performs homogeneous chemiluminescence detection against biotin interference.

The third aspect of the present invention relates to a control method of a homogeneous chemiluminescence detection device according to the second aspect of the present invention.

The fourth aspect of the invention relates to a method according to the first aspect of the invention or a detection device according to the second aspect of the invention or a control method according to the third aspect of the invention in biotin-streptavidin affinity The application in the detection of hormone system; preferably the application in the detection of thyroid function; further preferably the application in the detection of triiodothyronine and / or tetraiodothyronine.

Ⅲ. Examples

In order to make the present invention easier to understand, the present invention will be further described in detail in conjunction with the embodiments below. These embodiments are merely illustrative and are not limited to the scope of application of the present invention. Unless otherwise specified, the raw materials or components used in the present invention can be prepared through commercial channels or conventional methods.

Reagents and instruments:

SA (Sigma Aldrich), carboxyl functionalized silicon-based microspheres (particle size 15-200nm, pore size 2-15nm, Sigma Aldrich), phosphate buffer (0.02M PBS, pH7.4), 1- (3- Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDAC (Thermo fisher), Tween-20, 0.1M MES buffer (pH 6.0), biotin (D-biotin), hormone-free serum, Triiodothyronine (T3) detection kit (Boyang Biotechnology (Shanghai) Co., Ltd.), universal solution for photoexcitation chemiluminescence analysis system (photosensitive bead solution / streptavidin-labeled donor solution). LiCA HT (Boyang Biotechnology (Shanghai) Co., Ltd.), Hitachi high-speed refrigerated centrifuge.

Preparation of the anti-interference agent of the present invention by physical adsorption

Example 1

In the first step, take 10mg of carboxyl functionalized silicon-based microspheres (particle size 15nm, pore size 2nm) in a 2mL centrifuge tube, add 0.02M PBS (pH7.4) buffer, centrifuge at 10000rpm at 4 ℃, and wash once at 15min.

In the second step, add 200uL of PBS buffer solution to disperse ultrasonically evenly, then add 150uL of 10mg / mL SA aqueous solution, supplement PBS buffer solution to the microsphere reaction concentration of 20mg / mL, and stir at room temperature overnight.

In the third step, the SA microspheres were centrifuged with 0.02M PBS (pH7.4) buffer solution containing 0.5% Tween-20, centrifuged at 10000rpm at 4 ° C for 15min, washed three times to remove unadsorbed SA, and finally used 0.02M PBS (pH 7.4) Make the buffer volume to 10 mg / mL.

Example 2-7

The preparation method is the same as that in Example 1, except that each example uses carboxyl functionalized silicon-based microspheres with different particle sizes and / or pore sizes (see Table 1).

Preparation of the anti-interference agent of the present invention by covalent coupling

Example 8 (Covalent coupling method)

In the first step, take 10mg of carboxyl functionalized silicon-based microspheres in a 2mL centrifuge tube, centrifuge at 10000rpm at 4 ° C with 0.1M MES (pH 6.0) buffer solution, and wash once at 15min.

In the second step, add 200uL 0.1M MES (pH 6.0) buffer solution to disperse ultrasonically, then add 150uL 10mg / mL SA aqueous solution, then add 100uL 10mg / mLEDAC (0.1M MES) solution, and stir at room temperature for 4h.

In the third step, the SA microspheres were centrifuged and washed three times with 0.02M PBS (pH7.4) buffer solution containing 0.5% Tween-20 to remove unadsorbed SA. Finally, the volume was adjusted to 10 mg / mL with PBS buffer.

Example 9: Evaluation of the effect of the homogeneous chemiluminescence analysis method of anti-biotin interference of the present invention

Experimental steps:

1. Add T3 concentrated solution to hormone-free serum to prepare T3 solution with concentration of 1 nmol / L and 2 nmol / L.

2. Prepare 40ug / mL photosensitive bead solution, and use the anti-interference agent prepared in Examples 1-7 to prepare solutions of different concentrations (diluted with PBS), see Table 1.

3. Add biotin to the above T3 solution to prepare sample solutions with biotin concentrations of 0, 128 ng / ml, respectively.

4. Add 25ul of sample solution, and then sequentially add 25uL of Reagent 1 (containing diiodothyronine-coated receptor) and Reagent 2 (containing biotin-labeled anti-triiodothyronine antibody) in the T3 kit. (Manually add 25ul sample solution and 25uL reagent one and 25uL reagent two according to the reaction mode), and then add the 25uL anti-interference agent solution prepared in step 2 according to Table 1 below, in which conditions 1, 2 do not add anti-interference agent.

5. Put in LICA HT for the first stage of incubation: incubate at 37 ° C for 17 minutes.

6. Manually add 175ul of universal solution (containing streptavidin-labeled donor).

7. Carry out the second stage of incubation: incubate at 37 ° C for 15 minutes, and obtain the corresponding sample to be tested after the incubation.

8. Use energy to excite the sample to be tested and read the luminescence signal generated. See Tables 2 and 3 for reading results.

Table 1

data analysis:

When the T3 concentration was 1 nM and the biotin concentration was 128 ng / mL, the signal of the chemiluminescent immune response dropped by 89%, and biotin interference was more serious. When adding microspheres with a pore size of 2nm (No. 3 in Table 1), the luminescence signal showed almost no significant change. When using a particle size of 50nm, pore size 5nm, and 10nm (Nos. 4 and 5 in Table 1), the luminescence signal showed a certain Increase, the decline range is 50% -70%.

When the particle size and pore size are unchanged, increase the concentration of the anti-interference agent to 10ug / mL (compared with the serial numbers 5 and 6 in Table 1), the luminous signal is further improved, and the decline range is about 25%. When the pore diameter is 10nm, the concentration is 10ug / ml, and the particle diameter of the microspheres is increased to 100nm (Sequence Nos. 6 and 7 in Table 1), the deviation of the drop amplitude is within 10%, and the phenomenon of biotin interference disappears. When the concentration is increased to 20ug / ml (sequence numbers 7 and 8 in Table 1), the signal drop deviation is within 10%. When the concentration of 20ug / mL is unchanged, and the particle size and pore size are increased, the signal drops to a certain extent, with a drop of 20% -40%.

Experimental results:

When the anti-interference agent added by the method of the present invention is a microsphere filled with SA (streptavidin) with a particle size of 100 nm, a pore size of 10 nm, and a concentration of 10-20 ug / mL, the anti-interference ability of the method is the most Strong. When the pore size of the added anti-interference agent is small, 2 nm, the method has no anti-biotin interference capability. When the particle size of the added anti-interference agent is greater than 100 nm and the pore size is 10 nm, and the particle size and pore size continue to be increased, the anti-biotin interference capability of the method will decrease.

Example 10: Effect evaluation II of the homogeneous chemiluminescence analysis method of anti-biotin interference of the present invention

Experimental steps:

2. Prepare 40ug / mL photosensitive bead solution, and use the anti-interference agents prepared in Examples 5 and 8 to prepare solutions of different concentrations (diluted with PBS), see Table 4 (diluted with PBS) 10ug / mL, 20ug / mL.

3. Add biotin to the above T3 solution to prepare sample solutions with biotin concentrations of 0 and 128 ng / ml, respectively.

4. Add 25ul of sample solution, and then add 25uL of reagent 1 and reagent 2 in the T3 kit in sequence (manually add 25ul sample solution and 25uL reagent 1 and 25uL reagent 2 according to the reaction mode), then add 25uL in step 2 according to the following table The prepared anti-interference agent solution, conditions 1 and 2 do not add anti-interference agent.

6. Manually add 175ul universal liquid.

8. Use energy to excite the above-mentioned sample to be tested and read the luminescence signal generated. See Tables 5 and 6 for reading results.

Table 4

data analysis:

From the serial numbers 3 and 5 in Table 4, the anti-interference agent prepared by physical adsorption method has strong anti-biotin interference ability, the luminescence signal drops by about 10%, and the luminescence signal of the anti-interference agent prepared by covalent coupling method drops by about 50% . The numbers 4 and 6 in Table 4, the anti-interference ability of the anti-interference agent prepared by physical adsorption method is also superior to that prepared by covalent coupling method.

Experimental results:

For the same particle size, pore size, concentration, specific surface area, the photo-induced chemiluminescence analysis method of adding an anti-interference agent prepared by physical adsorption method is superior to the light of adding anti-interference agent prepared by covalent coupling method. Excitation chemiluminescence analysis method.

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

Claims

A homogeneous chemiluminescence analysis method, which includes the following steps: in the presence of an anti-interference agent, analyze and determine whether the sample to be tested contains whether the sample to be tested contains The concentration of the target molecule to be measured and / or the target molecule to be measured;

Wherein, the anti-interference agent includes a carrier and an active molecule; the carrier is a porous medium; the active molecule is filled in the carrier and can specifically bind to biotin molecules.
The method of claim 1, wherein the anti-interference agent is capable of recognizing free biotin molecules and biotin markers.
The method according to claim 1 or 2, wherein the anti-interference agent can selectively adsorb free biotin molecules.
The method according to claim 3, wherein the free biotin molecule can diffuse into the carrier and specifically bind to the active molecule therein.
The method according to any one of claims 1 to 4, characterized in that the anti-interference agent can restrict biological macromolecules larger than the size of the active molecule from entering its carrier.
The method according to any one of claims 1 to 5, wherein the anti-interference agent can be uniformly distributed in the liquid phase reaction system.
The method according to any one of claims 1-6, wherein the porous medium is selected from one or more of porous metal materials, porous non-metallic materials, and porous polymer materials.
The method according to any one of claims 1-7, wherein the carrier is mesoporous microspheres, preferably ordered mesoporous microspheres.
The method according to claim 8, wherein the pore diameter of the mesoporous microspheres is 2-50 nm, preferably 4-30 nm, and more preferably 5-15 nm.
The method according to claim 8 or 9, wherein the mesoporous microspheres are cage-like hollow mesoporous microspheres.
The method according to any one of claims 8-10, wherein the mesoporous microspheres are selected from Al 2 O 3 mesoporous materials, WO 3 mesoporous materials, TiO 2 mesoporous materials, ZrO 2 mesoporous materials At least one of the porous material, the silicon-based mesoporous material and / or the mesoporous carbon material is preferably selected from silicon-based mesoporous materials.
The method according to any one of claims 1-11, wherein the active molecule is selected from avidin and / or streptavidin.
The method according to any one of claims 1-12, wherein the active molecule is filled in the carrier by physical adsorption.
The method according to any one of claims 1 to 13, wherein the active molecule is filled in the carrier by contacting the carrier in a system containing a buffer solution.
The method according to claim 14, wherein the pH value of the buffer-containing system is from 7 to 9, preferably from 7.1 to 8.0, more preferably from 7.2 to 7.8, further preferably from 7.3 to 7.6.
The method according to any one of claims 1-12, wherein the active molecule is filled in the carrier by direct or indirect chemical crosslinking.
The method according to claim 16, wherein the inner surface of the carrier is modified with a chemical group, and the active molecule is filled in the carrier by covalent coupling with the chemical group; wherein The chemical group is selected from one or more of carboxyl group, aldehyde group, amino group, mercapto group and hydroxyl group.
The method according to any one of claims 1-17, wherein a biotin molecule is connected to the inner surface of the carrier, and the active molecule is filled in the carrier by a specific binding action with the biotin molecule in.
The method according to any one of claims 1-18, wherein the anti-interference agent further comprises a buffer solution, preferably a PBS buffer solution.
The method according to any one of claims 1-19, wherein the preparation method of the anti-interference agent comprises: step S1, contacting the carrier with the active molecule; preferably, the contact is in the first buffer In the liquid system.
The method according to claim 20, wherein the preparation method of the anti-interference agent further includes step S0: washing the carrier with a second buffer system, and step S0 is performed before step S1.
The method according to claim 20 or 21, characterized in that the method for preparing the anti-interference agent further comprises step S2: removing active molecules not filled into the carrier, step S2 is performed after step S1; preferably, by The third buffer system is added to the carrier treated in step S1, and then solid-liquid separation is performed to remove active molecules not filled in the carrier.
The method according to any one of claims 1-22, wherein the surface of the receptor is directly or indirectly connected to a biomacromolecule, and the biomacromolecule can specifically bind to the target molecule to be tested.
The method according to any one of claims 1 to 23, wherein the substance in the sample to be tested contains a biotin marker, which includes a biotin-bound molecule capable of directly or indirectly binding to the target molecule to be tested Biomacromolecule.
The method according to claim 23 or 24, wherein the biomacromolecule is selected from protein molecules, nucleic acid molecules, polysaccharide molecules and lipid molecules; preferably protein molecules; further preferably, the protein molecules are selected from Antigen and / or antibody; wherein, the antigen refers to an immunogenic substance; the antibody refers to an immunoglobulin produced by the body that can recognize a specific foreign object.
The method according to any one of claims 1-25, wherein the sample to be tested further comprises a donor; the surface of the donor directly or indirectly binds to a biotin-specific conjugate, which is capable of exciting Active oxygen is generated in the state.
The method according to any one of claims 1-26, characterized in that the method comprises the following steps:

S1, mixing the test sample with the reagent a containing the receptor, the reagent b containing the biotin marker, the reagent c containing the anti-interference agent, and the reagent d containing the donor to obtain the sample to be tested;

S2, using energy or an active compound to contact the sample to be tested obtained in step S1 to excite the donor to generate active oxygen;

S3. Analyze and determine whether the target molecule and / or the concentration of the target molecule in the test sample include the intensity of the luminescence signal generated by the reaction between the receptor and the active oxygen in the test sample.
The method according to claim 27, characterized in that in step S1, the sample to be tested is mixed with a reagent a containing a receptor, a reagent b containing a biotin marker, and a reagent c containing an anti-interference agent, and then mixed with The reagent d containing the donor is mixed to obtain a sample to be tested.
The method according to claim 27 or 28, wherein in step S2, the sample to be tested obtained in step S1 is irradiated with red excitation light of 600-700 nm to excite it to generate chemiluminescence.
The method according to any one of claims 27 to 29, wherein in step S3, the detection wavelength at which the light emission signal value is recorded is 520 to 620 nm.
A homogeneous chemiluminescence detection device, which uses the method of any one of claims 1-30 to perform homogeneous chemiluminescence detection.
A control method of a homogeneous chemiluminescence detection device according to claim 31.
An application of the method according to any one of claims 1-30 or the detection device according to claim 31 or the control method according to claim 32 in the detection of a biotin-streptavidin system ; Preferably used in the detection of thyroid function; further preferably used in the detection of triiodothyronine and / or tetraiodothyronine.