Light-excited chemiluminescence lateral flow immunodetection method and reagent

Abstract

The invention relates to a light-excited chemiluminescence lateral flow immunodetection method and reagent. The method comprises the following steps: S1, adding a sample to a sample area of a chip from a sample opening of a substrate; s2, enabling a sample to sequentially flow through a first marking region, a detection region and a reference region of the chip to enter a waste liquid chamber, forming a to-be-detected luminous compound in the detection region, and forming a reference luminous compound in the reference region; and S3, detecting chemiluminescence signals of the to-be-detected luminescent compound in the detection region and the reference luminescent compound in the reference region. According to the scheme provided by the invention, a simple and rapid lateral flow immunomicrofluidic technology and a high-sensitivity light-excited chemiluminescence analysis technology can be fused, rapid detection of samples such as respiratory virus antigens is realized, and the detection sensitivity is improved.

Description

Technical Field

[0001] This application relates to the field of immunoassay technologies, and particularly to a lateral flow immunoassay method and reagent based on photochemiluminescence. Background Art

[0002] In recent years, rapid detection of respiratory-related viruses has received much attention. Compared with gene detection technologies that detect viral genetic material, immunological detection methods based on antigen-antibody binding are simple and rapid. Among immunological detection indicators, compared with the detection of specific antibodies such as IgM antibodies, viral antigen detection has a shorter window period, which is beneficial for the early isolation and treatment of infected individuals.

[0003] In related technologies, technologies based on colloidal gold immunochromatography, latex agglutination chromatography, and even fluorescence microfluidic chips have been widely used in the rapid detection of respiratory virus antigens. However, due to low detection sensitivity, false negative results often occur, leading to missed detections. Therefore, there is an urgent need to improve the detection sensitivity for the rapid detection of respiratory virus antigens. Summary of the Invention

[0004] To solve or partially solve the problems existing in related technologies, this application provides a lateral flow immunoassay method and reagent based on photochemiluminescence, which can integrate the simple and rapid lateral flow immuno-microfluidic technology and the highly sensitive photochemiluminescence analysis technology to achieve rapid detection of samples such as respiratory virus antigens and improve detection sensitivity.

[0005] The first aspect of this application provides a lateral flow immunoassay method based on photochemiluminescence, including:

[0006] S1: Adding a sample from the sample port of a substrate to the sample area of a chip;

[0007] S2: The sample sequentially flows through the first labeling area, detection area, reference area of the chip and enters the waste liquid chamber, and a test luminescent complex is formed in the detection area, and a reference luminescent complex is formed in the reference area;

[0008] S3: Detecting the chemiluminescence signals of the test luminescent complex in the detection area and the reference luminescent complex in the reference area.

[0009] As an optional embodiment, first labeling particles are pre-set in the first labeling area, and when the sample flows, the target molecule to be detected in the sample binds to the first labeling particles to form a labeled complex; the labeled complex forms a test complex in the detection area and a reference complex in the reference area.

[0010] As an alternative embodiment, a first specific binding substance and a second specific binding substance are respectively immobilized in the detection area and the reference area; the first labeled particle can specifically bind to the first specific binding substance through the target molecule to be detected, and can also specifically bind to the second specific binding substance.

[0011] As an alternative embodiment, the first labeled particle comprises a luminescent particle labeled with a first labeling molecule and a second labeling molecule;

[0012] The complex to be detected includes a complex formed by the binding of the first specific binding substance to the first labeling molecule on the luminescent particle in the labeled complex through an immune reaction mediated by the target molecule to be detected; and / or,

[0013] The reference complex includes a complex formed by the binding of the first labeling molecule on the luminescent particle in the labeled complex to the second specific binding substance.

[0014] As an alternative embodiment, step S2 further includes:

[0015] S21: Adding the dissolution solution from the reagent port of the substrate to the second labeling area of the chip to dissolve the second labeled particles pre-placed in the second labeling area;

[0016] S22: The second labeled particles flow through the detection area and the reference area of the chip in sequence and enter the waste liquid chamber, and a complex to be detected luminescence is formed in the detection area, and a reference luminescence complex is formed in the reference area.

[0017] As an alternative embodiment, the second labeled particle comprises a photosensitive particle labeled with a third labeling molecule, and the third labeling molecule can specifically bind to the second labeling molecule.

[0018] As an alternative embodiment, the complex to be detected luminescence includes a complex formed by the binding of the second labeling molecule on the luminescent particle in the complex to be detected to the third labeling molecule on the photosensitive particle; and / or,

[0019] The reference luminescence complex includes a complex formed by the binding of the second labeling molecule on the luminescent particle in the reference complex to the third labeling molecule on the photosensitive particle.

[0020] As an alternative embodiment, the detection area includes a plurality of detection points, and different kinds of first specific binding substances are immobilized in the plurality of detection points, and the different kinds of first specific binding substances can bind to different kinds of target molecules to be detected.

[0021] The second aspect of the present application provides a reagent for the lateral flow immunoassay method of the above-mentioned photochemiluminescence, including:

[0022] A second labeled particle, a powder, a first labeled particle, a first specific binding substance, and a second specific binding substance. The second labeled particle, the powder, and the first labeled particle are respectively pre-placed in the second labeled area, the sample area, and the first labeled area of the chip. The first specific binding substance and the second specific binding substance are respectively fixed in the detection area and the reference area;

[0023] Among them, the first labeled particle includes a luminescent particle labeled with a first labeled molecule and a second labeled molecule. The second labeled particle includes a photosensitive particle labeled with a third labeled molecule. The first labeled molecule can specifically bind to the first specific binding substance through the target molecule to be detected and can also specifically bind to the second specific binding substance. The second labeled molecule can specifically bind to the third labeled molecule.

[0024] As an optional embodiment, the particle size of the photosensitive particle is not greater than that of the luminescent particle.

[0025] As an optional embodiment, the particle size of the luminescent particle is 200nm - 400nm, and the particle size of the photosensitive particle is 50nm - 200nm.

[0026] The technical solution provided by the present application may include the following beneficial effects:

[0027] The present application can integrate the simple and rapid lateral flow immunoassay microfluidic technology and the highly sensitive photochemiluminescence analysis technology, and innovate the photochemiluminescence analysis to make it more in line with the requirements of lateral flow analysis. First, use lateral flow to achieve the washing and separation of the first labeled particles in the unbound or free first labeled area; then form the luminescent complex to be detected in the detection area and the reference luminescent complex in the reference area. The laser beam excites the detection area and the reference area, inducing the photochemiluminescence process of the luminescent complex to be detected and the reference luminescent complex, generating optical signals and performing detection, so as to achieve rapid detection of samples such as respiratory virus antigens and improve the detection sensitivity.

[0028] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] By describing the exemplary embodiments of the present application in more detail in conjunction with the drawings, the above and other objects, features, and advantages of the present application will become more obvious. Among them, in the exemplary embodiments of the present application, the same reference numerals generally represent the same components.

[0030] Figure 1 It is a schematic diagram of pre-setting reagents in the photo-induced chemiluminescence lateral flow immunoassay method shown in the embodiment of the present application;

[0031] Figure 2 It is a schematic diagram of adding samples in the photoinduced chemiluminescence lateral flow immunoassay method shown in the embodiment of the present application;

[0032] Figure 3 It is a schematic diagram of dripping a dissolving solution in the photoinduced chemiluminescence lateral flow immunoassay method shown in the embodiment of the present application;

[0033] Figure 4 It is a schematic diagram of the assembly structure of the photoinduced chemiluminescence lateral flow immunoassay chip shown in the embodiment of the present application;

[0034] Figure 5 yes Figure 4 A top view of the chip shown in FIG.

[0035] Figure 6 yes Figure 4 A bottom view of the substrate is shown in FIG.

[0036] In the figure: 1, substrate; 10, injection port; S, sample port; R, reagent port; 11, guide groove; 12, waste liquid chamber; 13, receiving groove; 2, chip; 20, second labeling area; 21, sample area; 22, first labeling area; 23, detection area; 230, detection point; 24, reference area; A, second labeling particle; a, third labeling molecule; GG, photosensitive particle; B, powder; C, first labeling particle; c1, first labeling molecule; c2, second labeling molecule; FG, luminescent particle; D, first specific binding substance; E, second specific binding substance; T, specific pair; W, target molecule to be detected; X, complex to be detected; Y, reference complex; X', luminescent complex to be detected; Y', reference luminescent complex. DETAILED DESCRIPTION

[0037] The embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although the embodiments of the present application are shown in the accompanying drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to make the present application more thorough and complete, and to fully convey the scope of the present application to those skilled in the art.

[0038] The terms used in this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The singular forms "a", "the", and "said" used in this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term "and / or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0039] It should be understood that although the terms "first", "second", "third", etc. may be used in this application to describe various information, such information should not be limited to these terms. These terms are only used to distinguish the same type of information from each other. For example, without departing from the scope of this application, the first information may also be referred to as the second information, and similarly, the second information may also be referred to as the first information. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. In the description of this application, "a plurality of" means two or more unless otherwise specifically defined.

[0040] In the related art, technologies based on colloidal gold immunochromatography, latex agglutination chromatography, and even fluorescence microfluidic chips have been widely used in the rapid detection of respiratory virus antigens. However, due to the low detection sensitivity, false negative results often occur, resulting in missed detections. Therefore, there is an urgent need to improve the detection sensitivity of the rapid detection of respiratory virus antigens.

[0041] To address the above problems, the embodiments of this application provide a lateral flow immunoassay method based on photochemiluminescence, which can integrate the simple and rapid lateral flow immuno-microfluidic technology and the highly sensitive photochemiluminescence analysis technology to achieve the rapid detection of samples such as respiratory virus antigens and improve the detection sensitivity.

[0042] The term "heterogeneous" as used in the embodiments of this application, also known as heterogeneous phase, refers to the need to separate unbound labels from the reaction system before signal detection.

[0043] The term "homogeneous" as used in the embodiments of this application has the corresponding English definition of "homogeneous", which means that the detection can be carried out without separating the bound antigen-antibody complex and the remaining free antigen or antibody.

[0044] The term "target molecule to be detected" used in the embodiments of this application can be DNA, RNA, protein, polypeptide, carbohydrate, etc., preferably protein, and more preferably an immune molecule, such as an antigen or an antibody.

[0045] As used in the embodiments of the present application, the term "sample" refers to a mixture that may contain a target molecule to be detected. Typical samples to be tested that can be used in the methods disclosed in the present application include body fluids such as blood, plasma, serum, urine, semen, saliva, nasal swabs, nasopharyngeal swabs, oropharyngeal swabs, etc.

[0046] As used in the embodiments of the present application, the term "binding" refers to the direct association between two molecules caused by interactions such as covalent, electrostatic, hydrophobic, ionic, and / or hydrogen bonding, including but not limited to interactions such as salt bridges and water bridges.

[0047] As used in the embodiments of the present application, the term "specific binding" refers to the mutual discrimination and selective binding reaction between two substances. From the perspective of three-dimensional structure, it is the conformational correspondence between the corresponding reactants.

[0048] As used in the embodiments of the present application, the term "photosensitive particle" refers to a sensitizer that can generate reactive intermediates such as singlet oxygen that can react with receptor microspheres after activation by energy or an active compound. The photosensitive particle can be photoactivated (such as dyes and aromatic compounds) or chemically activated (such as enzymes, metal salts, etc.).

[0049] As used in the embodiments of the present application, the term "luminescent particle" refers to a compound that can react with singlet oxygen to generate a detectable signal. The photosensitive particle is induced and activated by energy or an active compound and releases high-energy singlet oxygen. The high-energy singlet oxygen is captured by the nearby luminescent particle, thereby transferring energy to activate the luminescent particle. In some specific embodiments of the embodiments of the present application, the luminescent particle 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.

[0050] As used in the embodiments of the present application, the term "biotin" is widely present in animal and plant tissues. It has two ring structures on its molecule, namely an imidazolone ring and a thiophene ring, and the imidazolone ring is the main site for binding to streptavidin. Activated biotin can be conjugated to almost all known biological macromolecules under the mediation of a protein cross-linking agent, including proteins, nucleic acids, polysaccharides, and lipids, etc. The "avidin" molecule is composed of 4 identical peptide chains, and each peptide chain can bind one biotin. Therefore, each antigen or antibody can be conjugated with multiple biotin molecules at the same time, thereby producing a "tentacle effect" to improve the analysis sensitivity.

[0051] The technical solutions of the embodiments of the present application will be described in detail below with reference to the accompanying drawings.

[0052] See Figures 1 to 3 , the embodiments of the present application provide a lateral flow immunoassay method based on photoactivated chemiluminescence, including:

[0053] S1: Add the sample from the sample port S of the substrate 1 to the sample area 21 of the chip 2;

[0054] S2: The sample flows through the first labeling area 22, the detection area 23, and the reference area 24 of the chip 2 in sequence and enters the waste liquid chamber 12, and a to-be-detected luminescent complex X' is formed in the detection area 23, and a reference luminescent complex Y' is formed in the reference area 24.

[0055] S3: Detect the chemiluminescence signals of the to-be-detected luminescent complex X' in the detection area 23 and the reference luminescent complex Y' in the reference area 24.

[0056] In the embodiment of the present application, a lateral flow immunoassay chip based on photoinduced chemiluminescence is used for immunoassay of respiratory viruses. Among them, referring to Figure 4 and Figure 5 , the immunoassay chip may include a substrate 1 and a chip 2. The substrate 1 is provided with a sample port S, a diversion groove 11, a receiving groove 13, and a waste liquid chamber 12; the chip 2 is embedded in the receiving groove 13 and seals the diversion groove 11 and the waste liquid chamber 12; and a functional area is provided on the surface of the chip 2 located in the diversion groove 11. The functional area includes a sample area 21, a first labeling area 22, a detection area 23, and a reference area 24 arranged in sequence along the liquid flow direction. The first labeling area 22 may be pre-set with first labeling microparticles C. Before the to-be-detected luminescent complex X' is formed in the detection area 23 and the reference luminescent complex Y' is formed in the reference area 24 in the embodiment of the present application, the sample in the sample area 21 may first flow through the first labeling area 22, the detection area 23, and the reference area 24 along the liquid flow direction and enter the waste liquid chamber 12, and the unbound or free labeling microparticles in the first labeling area 22 are separated from the reaction system before signal detection.

[0057] Based on this, the immunoassay of the "lateral flow immunoassay chip based on photoinduced chemiluminescence" proposed in the embodiment of the present application no longer belongs to homogeneous immunoassay technology, but belongs to heterogeneous immunoassay technology. The main analysis is as above: before the to-be-detected luminescent complex X' is formed in the detection area 23 and the reference luminescent complex Y' is formed in the reference area 24, a lateral flow process is required to wash away the unreacted and free target molecules and the first labeling microparticles C and collect them in the waste liquid chamber 12. This washing mode is mainly realized by the lateral flow movement caused by the capillary force of microfluidics. Therefore, the embodiment of the present application is innovated based on photoinduced chemiluminescence analysis technology to make it more suitable for the lateral flow microfluidics technology platform.

[0058] On the one hand, in the embodiments of the present application, due to the adoption of the photochemiluminescence immunoassay technology, the photochemiluminescence process includes three - stage chemical reactions: exciting phthalocyanine to initiate the generation of reactive oxygen species; the reactive oxygen species release energy to induce the oxidation reaction of dimethylthiophene; dimethylthiophene is oxidized to release a light signal, and then lanthanide element europium (Eu) is excited to emit a fluorescence signal. In addition, the photochemiluminescence process is excited by a long wavelength such as 680 nm, and the emission wavelength such as 610 nm is shorter than the excitation light. This process is relatively rare in nature, which is why photochemiluminescence has an extremely low background. These two factors make photochemiluminescence have a very high sensitivity.

[0059] On the other hand, the simplicity, rapidity, and convenience of the embodiments of the present application are provided by the immunomicrofluidic technology. The lateral flow immuno - microparticle pore chip inherits and develops the simple and rapid characteristics of the colloidal gold chromatography technology. The microfluidic chip has extremely strong integration characteristics. The relevant reagent components are arranged in the microfluidic channel according to a preset program, and the dry - powder reagents are convenient for storage and transportation. At the same time, immunomicrofluidics belongs to the new POCT mode, with simple operation, no need for complex operations, and even less need for complex analytical instruments, which is an upgrade of POCT. In addition, the antigen - antibody binding in the lateral flow mode is different from the static reaction cup mode, and the reaction is faster.

[0060] In summary, the embodiments of the present application can integrate the simple and rapid lateral flow immuno - microfluidic technology and the highly sensitive photochemiluminescence analysis technology, and innovate the photochemiluminescence analysis to make it more in line with the requirements of lateral flow analysis. First, use the lateral flow to achieve the washing and separation of the first labeled microparticles C in the unbound or free first labeled region 22; then form the test luminescent complex X' in the detection region 23 and the reference luminescent complex Y' in the reference region 24. The laser beam excites the detection region 23 and the reference region 24 to induce the photochemiluminescence process of the test luminescent complex X' and the reference luminescent complex Y', generate light signals and perform detection, so as to achieve the rapid detection of samples such as respiratory virus antigens and improve the detection sensitivity.

[0061] As an optional embodiment, the first labeled microparticles C are pre - placed in the first labeled region 22. When the sample flows, the target molecule W to be detected in the sample binds to the first labeled microparticles C to form a labeled complex (not shown in the figure); the labeled complex forms the test complex X in the detection region 23 and the reference complex Y in the reference region 24.

[0062] In the embodiments of the present application, molecules such as antigens or antibodies that specifically bind to the target molecule W to be detected can be labeled on the microparticles of the first labeled microparticles C, so that the target molecule W to be detected can bind to the first labeled microparticles C to form a labeled complex before the sample flows through the first labeled region 22 to the detection region 23.

[0063] As an alternative embodiment, a first specific binding agent D and a second specific binding agent E are respectively fixed in the detection area 23 and the reference area 24; the first labeled particle C can specifically bind to the first specific binding agent D through the target molecule W to be detected, and can specifically bind to the second specific binding agent E.

[0064] First, the differences between "pre-set" and "fixed" in the embodiments of the present application need to be explained:

[0065] "Pre-set" means that the reagent components are pre-placed at positions such as the second labeling area 20, the sample area 21, and the first labeling area 22, without interacting with the surface of the chip 2. After reconstitution, the pre-set reagent components flow downstream with the liquid; the "pre-set" method can be placing pre-dried and formed powder at specific positions, or adding liquid reagents at specific positions and then dehydrating.

[0066] "Fixed" means that the reagent components are connected to the chip 2 at specific positions such as the detection area 23 and the reference area 24, and will not flow with the liquid, and can capture specific components in the flowing liquid and retain them at that position. In the embodiments of the present application, the first specific binding agent D and the second specific binding agent E can be respectively fixed in the detection area 23 and the reference area 24 through the interaction of the specific pairing agent T. For example, the specific pairing agent T can be biotin and streptavidin. Biotin is labeled on the first specific binding agent D and the second specific binding agent E, and streptavidin is labeled on the surfaces of the detection area 23 and the reference area 24, so that the test luminescent complex X' and the reference luminescent complex Y' generated in the detection area 23 and the reference area 24 can be fixed in the detection area 23 and the reference area 24 and will not flow downstream with the liquid.

[0067] When the sample flows, the target molecule W to be detected in the sample binds to the first labeled particle C to form a labeled complex. When the liquid continues to flow downstream, the sample, the first labeled particle C, and the labeled complex all continue to flow downstream. In the detection area 23, the labeled complex participates in the reaction, and the first labeled particle C in the labeled complex can specifically bind to the first specific binding agent D fixed on the surface of the chip in the detection area 23 through the target molecule W to be detected to form a test complex X and stay in the detection area 23. In the reference area 24, the first labeled particle C participates in the reaction, and the first labeled particle C directly specifically binds to the second specific binding agent E fixed on the surface of the chip in the reference area 24 to form a reference complex Y and stay in the reference area 24. The remaining unreacted and free target molecules and the first labeled particle C are collected in the waste liquid chamber 12.

[0068] In the embodiment of the present application, the first labeled particle C includes a luminescent particle FG labeled jointly by a first labeled molecule c1 and a second labeled molecule c2. The first labeled molecule c1 can be a molecule such as an antigen or an antibody that can bind to a target molecule W to be detected, such as an antibody or an antigen, so that the target molecule W to be detected can bind to the first labeled particle C in the first labeled region 22 to form a labeled complex, and the bound target molecule W to be detected, such as an antibody or an antigen, in the labeled complex can bind to a first specific binding substance D, such as an antigen or an antibody, in the detection region 23 to form a complex X to be detected.

[0069] In the embodiment of the present application, the first labeled particle C includes a luminescent particle FG labeled jointly by a first labeled molecule c1 and a second labeled molecule c2. The first labeled molecule c1 can be a molecule such as an antigen or an antibody that can bind to a target molecule W to be detected, such as an antibody or an antigen. In the reference region 24, the first labeled particle C can directly bind to a second specific binding substance E, such as a secondary antibody, through the first labeled molecule c1, such as an antibody, to form a reference complex Y.

[0070] As an optional embodiment, step S2 further includes:

[0071] S21: Add the dissolving solution from the reagent port R of the substrate 1 to the second labeled region 20 of the chip 2 to dissolve the second labeled particle A pre-set in the second labeled region 20;

[0072] S22: The second labeled particle A flows through the detection region 23 and the reference region 24 of the chip 2 in sequence and enters the waste liquid chamber 12, and a complex X' to be detected by luminescence is formed in the detection region 23, and a reference luminescent complex Y' is formed in the reference region 24.

[0073] In the embodiment of the present application, the second labeled region 20 of the chip is pre-set with a powder of the second labeled particle A. The dissolving solution can be distilled water or a diluent. Injecting the dissolving solution dissolves the second labeled particle A, so that the second labeled particle A can flow through the detection region 23 and the reference region 24 of the chip 2 in sequence along the liquid flow direction and enter the waste liquid chamber 12. The second labeled particle A binds to the complex X to be detected in the detection region 23 to form a complex X' to be detected by luminescence, and binds to the reference complex Y in the reference region 24 to form a reference luminescent complex Y' for photoexcitation photochemical detection.

[0074] As a preferred embodiment, the second labeled particle A includes a photosensitive particle GG labeled by a third labeled molecule a, and the third labeled molecule a can specifically bind to the second labeled molecule c2.

[0075] The binding of the second labeling molecule c2 and the third labeling molecule a can be one of an antibody-anti-antibody such as a mouse Ig-rabbit anti-mouse Ig antibody, a hapten-antibody such as a FITC-anti-FITC Ab, a peptide tag-anti-tag antibody, and a gene-encoded polypeptide-protein reaction pair, preferably a peptide tag-anti-tag antibody.

[0076] In the embodiment of the present application, the first labeling molecule c1 such as an antigen or an antibody and the second labeling molecule c2 such as a tag peptide are used to coat the luminescent microparticles FG together, and the second labeling molecule c2 such as a tag peptide binds to the third labeling molecule a such as an anti-tag antibody on the photosensitive microparticles GG. As a result, there are bifunctional groups on the surface of the luminescent microparticles FG, one is a tag peptide and the other is an antigen or an antibody, rather than biotin in the traditional photochemiluminescence analysis; at the same time, the surface of the photosensitive microparticles GG is coated with a tag antibody, rather than a streptavidin molecule in the traditional photochemiluminescence analysis. Therefore, the binding between the photosensitive microparticles GG and the luminescent microparticles FG in the embodiment of the present application is based on the surface short peptide tag and the anti-tag antibody, and has nothing to do with the binding strength between the virus antigen to be detected and the virus antibody to be detected, thereby greatly improving the detection sensitivity.

[0077] As a preferred embodiment, the second labeling microparticle A binds to the complex X to be detected to form a luminescent complex X' to be detected. Further, the complex is formed by the binding of the second labeling molecule c2 on the luminescent microparticle FG in the complex X to be detected and the third labeling molecule a on the photosensitive microparticle GG in the second labeling microparticle A. Since the complex X to be detected has been retained in the detection area 23, the luminescent complex X' to be detected also stays in the detection area 23.

[0078] As a preferred embodiment, the second labeling microparticle A binds to the reference complex Y to form a reference luminescent complex Y'. Further, the complex is formed by the binding of the second labeling molecule c2 on the luminescent microparticle FG in the reference complex Y and the third labeling molecule a on the photosensitive microparticle GG in the second labeling microparticle A. Since the reference complex Y has been retained in the reference area 24, the reference luminescent complex Y' also stays in the reference area 24.

[0079] As an alternative embodiment, refer to Figure 5 As shown, the detection area 23 includes a plurality of detection points 230, and different first specific binders D are fixed in the plurality of detection points 230, and the different first specific binders D can bind to different target molecules W to be detected.

[0080] In the embodiment of the present application, by setting a plurality of detection points 230, the detection of multiple different target molecules W to be detected in the same sample can be realized simultaneously. And the detection of multiple different target molecules W to be detected in the same sample can be realized in the following two ways:

[0081] The first method: Each luminescent particle FG in the first labeled particles C pre-set in the first labeling area 22 has different first labeled molecules c1 but the same second labeled molecule c2. Different first labeled molecules c1 can bind to the corresponding target molecule to be detected W through the first specific binding substance D.

[0082] The second method: Each luminescent particle FG in the first labeled particles C pre-set in the first labeling area 22 has the same first labeled molecule c1 and second labeled molecule c2, but the first labeled molecule c1 includes multiple molecules that specifically recognize different target molecules. Different first labeled molecules c1 can bind to the corresponding target molecule to be detected W through the first specific binding substance D.

[0083] The embodiments of the present application also provide another lateral flow immunoassay method based on photochemiluminescence, including:

[0084] S1: Add the sample from the sample port S of the substrate 1 to the sample area 21 of the chip 2, and mix it with the powder B such as buffer pre-set in the sample area 21.

[0085] S2: The sample flows through the first labeling area 22 of the chip 2, re-dissolves the powder of the first labeled particles C pre-set in the first labeling area 22 and binds to it to form a labeled complex.

[0086] S3: The labeled complex flows through the detection area 23 and the reference area 24 in sequence and enters the waste liquid chamber 12, and a complex X to be detected is formed in the detection area 23, and a reference complex Y is formed in the reference area 24.

[0087] S4: Add the dissolving solution from the reagent port R of the substrate 1 to the second labeling area 20 of the chip 2 to dissolve the second labeled particles A pre-set in the second labeling area 20.

[0088] S5: The second labeled particles A flow through the detection area 23 and the reference area 24 of the chip 2 in sequence and enter the waste liquid chamber 12, and a complex X' of the chemiluminescence to be detected is formed in the detection area 23, and a reference complex Y' of the chemiluminescence is formed in the reference area 24.

[0089] S6: Detect the chemiluminescence signals of the complex X' of the chemiluminescence to be detected in the detection area 23 and the reference complex Y' of the chemiluminescence in the reference area 24.

[0090] In the embodiments of the present application, the labeled complex includes a complex formed by the binding of a target molecule W to be detected, such as an antigen, and a first labeled molecule c1, such as an antibody, on the luminescent particle FG; the test complex X includes a complex formed by the binding of a first specific binder D, such as an antibody, to the first labeled molecule c1, such as an antibody, on the luminescent particle FG in the labeled complex through an immune reaction mediated by the target molecule W to be detected, such as an antigen, for example, a sandwich complex; the reference complex Y includes a complex formed by the binding of the first labeled molecule c1, such as an antibody, on the luminescent particle FG in the labeled complex and a second specific binder E, such as a secondary antibody; the test luminescent complex X' includes a complex formed by the binding of a second labeled molecule c2 on the luminescent particle FG in the test complex X and a third labeled molecule a on the photosensitive particle GG; the reference luminescent complex Y' includes a complex formed by the binding of the second labeled molecule c2 on the luminescent particle FG in the reference complex Y and a third labeled molecule a on the photosensitive particle GG.

[0091] To make the present invention easier to understand, taking the detection of respiratory antigens as an example, the principle of photochemiluminescence lateral flow immunoassay in the embodiments of the present application is described as follows:

[0092] (1) Reagent preparation

[0093] See Figure 1 As shown, from left to right on the immunoassay chip are: a second labeling area 20 pre-loaded with dry powder of photosensitive particles coated with labeled antibodies, a sample area 21 pre-loaded with a sample buffer (powder B), a first labeling area 22 pre-loaded with dry powder of luminescent particles double-coated with influenza virus antibody probes and labeled short peptides (such as Histag), a detection area 23 fixed with a monoclonal antibody against influenza virus coated thereon, a reference area 24 fixed with an antibody against sheep anti-mouse IgG coated thereon, and a waste chamber 12.

[0094] (2) Sample addition

[0095] See Figure 2 As shown, during detection, first, the sample is added to the sample port S, quickly dissolving the buffer pre-loaded in the sample area 21, and under the action of the microchannel driving force, sequentially binding to the luminescent particles FG coated with influenza virus antibodies to form a labeled complex, which continues to move to the detection area 23 and forms a sandwich complex with the monoclonal antibody against influenza virus coated in the detection area 23. The unbound or free target molecules and luminescent particles FG continue to move forward to the waste chamber 12; meanwhile, the antibody against sheep anti-mouse IgG in the reference area 24 will also capture the luminescent particles FG coated with influenza virus antibodies (the influenza virus antibody is a murine monoclonal antibody and will bind to the antibody against sheep anti-mouse IgG); the unbound or free luminescent particles FG continue to flow to the waste chamber 12 and are collected.

[0096] In addition, the reagent port R located on the left side of the sample port S is in a closed state when adding samples. It will only open to communicate with the air when adding distilled water.

[0097] (3), Add the lysing solution (distilled water)

[0098] See Figure 3 As shown, after 1 - 2 minutes, add distilled water dropwise to the reagent port R to re - dissolve the dry powder of the photosensitive particles GG preset in the second marking area 20 and fill the micro - pipeline. The photosensitive particles GG encounter the luminescent particles FG in the test complex X and the reference complex Y in the test area 23 and the reference area 24. The anti - tag antibody on the surface of the photosensitive particles GG binds to the tag peptide on the surface of the luminescent particles FG, reducing the distance between the two types of particles and meeting the basic conditions for photochemiluminescence (the distance between the photosensitive particles GG and the luminescent particles FG is less than 200 nm). At this time, laser - excite the test area 23 and the reference area 24 and detect the light signal intensity, and combine the light signals of the test area 23 and the reference area 24 to output the test result.

[0099] Corresponding to the foregoing method embodiments for realizing application functions, the present application also provides a reagent for a lateral - flow immunoassay method for photochemiluminescence and corresponding embodiments.

[0100] The embodiments of the present application provide a reagent for the foregoing lateral - flow immunoassay method for photochemiluminescence, including:

[0101] The second labeled particles A, the powder B, the first labeled particles C, the first specific binding substance D, and the second specific binding substance E. The second labeled particles A, the powder B, and the first labeled particles C are respectively preset in the second marking area 20, the sample area 21, and the first marking area 22 of the chip 2. The first specific binding substance D and the second specific binding substance E are respectively fixed in the test area 23 and the reference area 24;

[0102] Among them, the first labeled particles C include luminescent particles FG labeled with a first labeled molecule c1 and a second labeled molecule c2. The second labeled particles A include photosensitive particles GG labeled with a third labeled molecule a. The first labeled molecule c1 can specifically bind to the first specific binding substance D through the target molecule W to be detected and can specifically bind to the second specific binding substance E. The second labeled molecule c2 can specifically bind to the third labeled molecule a.

[0103] The reagent in the embodiment of the present application is applied to a lateral flow immunoassay chip for photochemiluminescence and a detection technique. During use, the second labeled microparticles A, the powder B, and the first labeled microparticles C can be respectively pre-placed in the second labeling area 20, the sample area 21, and the first labeling area 22 of the chip 2, and the first specific binding substance D and the second specific binding substance E are respectively fixed in the detection area 23 and the reference area 24. The photochemiluminescence lateral flow immunoassay method described above is used to detect the virus antigen or antibody to be detected.

[0104] The detection process is as follows: The sample flows through the first labeling area 22 of the chip 2, re-dissolves the powder of the first labeled microparticles C pre-placed in the first labeling area 22 and binds to form a labeled complex; the labeled complex sequentially flows through the detection area 23, the reference area 24 and enters the waste liquid chamber 12, and a complex X to be detected is formed in the detection area 23, and a reference complex Y is formed in the reference area 24; The dissolving solution is added from the reagent port R of the substrate 1 to the second labeling area 20 of the chip 2 to dissolve the second labeled microparticles A pre-placed in the second labeling area 20; the second labeled microparticles A sequentially flow through the detection area 23, the reference area 24 of the chip 2 and enter the waste liquid chamber 12, and a chemiluminescent complex X' to be detected is formed in the detection area 23, and a reference chemiluminescent complex Y' is formed in the reference area 24; The chemiluminescence signals of the chemiluminescent complex X' to be detected in the detection area 23 and the reference chemiluminescent complex Y' in the reference area 24 are detected.

[0105] As an alternative embodiment, the particle size of the photosensitive microparticles GG is not greater than the particle size of the luminescent microparticles FG.

[0106] Generally, the particle sizes of the photosensitive microparticles GG and the luminescent microparticles FG are close, about 200 nm. However, in the embodiment of the present application, the particle size of the photosensitive microparticles GG is smaller than that of the luminescent microparticles FG. On the one hand, it is beneficial for the photosensitive microparticles GG to flow in the microfluidic channel. On the other hand, one luminescent microparticle FG can bind multiple photosensitive microparticles GG, thereby providing more reactive oxygen molecules and enhancing the luminescence intensity, which is beneficial for improving the detection sensitivity.

[0107] Furthermore, the particle size of the luminescent microparticles FG is 200 nm - 400 nm, and the particle size of the photosensitive microparticles GG is 50 nm - 200 nm.

[0108] In the embodiment of the present application, the particle size of the photosensitive microparticles GG is about 50 nm, and the particle size of the luminescent microparticles FG is about 200 nm.

[0109] Corresponding to the foregoing embodiment of the application function implementation method, the present application also provides a lateral flow immunoassay chip for photochemiluminescence and a corresponding embodiment.

[0110] See Figures 4 to 6, an embodiment of the present application provides a lateral flow immunoassay chip for chemiluminescence, including a substrate 1 and a chip 2. The substrate 1 is provided with a liquid injection port 10, a diversion groove 11, a receiving groove 13, and a waste liquid chamber 12; the chip 2 is embedded in the receiving groove 13 and seals the diversion groove 11 and the waste liquid chamber 12; and a functional area is provided on the surface of the chip 2 located in the diversion groove 11. The functional area includes a second labeling area 20, a sample area 21, a first labeling area 22, a detection area 23, and a reference area 24 arranged in sequence along the liquid flow direction.

[0111] The lateral flow immunoassay chip for chemiluminescence in the embodiment of the present application can integrate the simple and rapid lateral flow immunoassay microfluidic technology and the highly sensitive chemiluminescence analysis technology, and innovate the chemiluminescence analysis to make it more in line with the requirements of lateral flow analysis. First, the unbound or free first labeled particles C are washed and separated by lateral flow; then, a test luminescent complex X' is formed in the detection area 23, and a reference luminescent complex Y' is formed in the reference area 24. A laser beam excites the detection area 23 and the reference area 24, inducing the test luminescent complex X' and the reference luminescent complex Y' to undergo a chemiluminescence process, generating a light signal for detection, thereby realizing the rapid detection of samples such as respiratory virus antigens and improving the detection sensitivity.

[0112] As an optional embodiment, refer to Figure 4 As shown, a waste liquid chamber 12 is recessed on the side of the substrate 1 close to the chip 2. The waste liquid chamber 12 is communicated with the diversion groove 11, and the waste liquid chamber 12 is located downstream of the reference area 24 along the liquid flow direction.

[0113] In the embodiment of the present application, by setting the waste liquid chamber 12, before adding the photosensitive particle GG solution, the process of lateral flow has been passed through, and the unreacted and free luminescent particles FG have been washed and removed and collected in the waste liquid collection area. This washing mode is mainly realized by the lateral flow movement caused by the capillary force of microfluidics. Therefore, the embodiment of the present application innovates the chemiluminescence analysis technology to make it more suitable for the lateral flow microfluidic technology platform.

[0114] Furthermore, an absorbent material is provided in the waste liquid chamber 12 for collecting waste liquid and assisting in driving the liquid to flow in the diversion groove 11.

[0115] The absorbent material in the embodiment of the present application can be filter paper, sponge, silica gel, etc., and the absorbent material can have a certain thickness and occupy the space of the diversion groove 11, thereby driving the liquid to flow from the sample area 21 to the waste liquid chamber 12 and sucking the liquid flowing into the waste liquid chamber 12 area, such as the unreacted and free luminescent particles FG, into the waste liquid chamber 12.

[0116] As an alternative embodiment, the width of the flow guiding groove 11 is 3 - 5 mm, and the height is 15 - 20 μm.

[0117] The microfluidic chip of the embodiment of the present application is formed by embedding a biochip and a plastic substrate. After the two are embedded, a micron-level flat flow guiding groove 11 is formed. The flow guiding groove 11 has a width of 3 - 5 mm and a height of 15 - 20 μm. Tiny liquid flows slowly in the micron channels along the pre-set paths, and the substances to be detected distributed in the liquid phase bind to the capture molecules in the detection area 23 in a lateral flow mode. The entire microfluidic chip has the advantages of small volume, short reaction time, less consumption of reagents and samples, and the ability to achieve point-of-care testing.

[0118] As an alternative embodiment, referring to Figure 4 and Figure 6 as shown, there are two liquid injection ports 10, and the two liquid injection ports 10 correspond to the second labeling area 20 and the sample area 21 respectively.

[0119] The embodiment of the present application belongs to the POCT operation mode and does not require special additional reagent components. There are two liquid injection ports 10 on the substrate 1, namely the sample port S and the reagent port R. The reagent port R is located upstream of the sample port S ( Figure 1 the left liquid injection port 10 in ). Detection can be carried out by first dropping the sample and then adding distilled water (to dissolve the second labeled microparticles A preset in the second labeling area 20).

[0120] There are two liquid injection ports 10 on the substrate 1, namely the sample port S and the reagent port R. During detection, the sample to be detected is added to the sample port S. After 1 - 2 minutes, distilled water is added to the reagent port R, and it is left standing for 4 minutes. The optical signal is detected by a photochemiluminescence analyzer, and the test result is reported. This can ensure that the antigen-antibody immune reaction is preferably carried out first, and then the binding of the photosensitive microparticles GG and the luminescent microparticles FG is carried out. The sample is first added to the sample port S. At this time, the sealing film at the reagent port R is separated from the outside, ensuring that the sample can only move downstream to the right and react in a predetermined mode sequence, so that the antigen-antibody immune reaction is carried out first; after standing for 1 - 2 minutes, the sealing film at the reagent port R is punctured or torn off, and distilled water or buffer solution is added to dissolve the dry powder of the photosensitive microparticles GG, making the reagent port R communicate with the atmosphere, so that the solution of the photosensitive microparticles GG can move to the right under the action of atmospheric pressure. After encountering the luminescent microparticles FG, they bind through the His tag and the anti-His antibody, so that the binding of the photosensitive microparticles GG and the luminescent microparticles FG is carried out later.

[0121] As an alternative embodiment, the liquid injection port 10 is provided with a sealing film. When liquid injection is required, the sealing film is damaged to make the liquid injection port 10 communicate with the atmosphere, so that the liquid moves to the right under the action of atmospheric pressure.

[0122] In the embodiment of the present application, the sample port S and the reagent port R are sealed with a sealing film before testing, and can be punctured or opened to communicate with the atmosphere when adding a sample or distilled water.

[0123] In order to make the present invention easier to understand, the present invention will be further described in detail below in conjunction with examples, which are merely illustrative and are not intended to limit the scope of application of the present invention. The raw materials or components used in the present invention can be obtained by commercial routes or conventional methods unless otherwise specified.

[0124] 1. Reagent Preparation

[0125] (1) Preparation of the Second Labeled Particle A

[0126] The second labeled particle A solution was prepared by coating the photosensitive particles GG with anti-His-tag antibody, and the coating method was the same as the classical light-excited chemiluminescence process, wherein the photosensitive particles GG with particle sizes of 200 nm and 50 nm were coated respectively.

[0127] Take an appropriate amount of photosensitive microparticles GG, wash them several times with ultrapure water, resuspend them with 0.05M CBS, mix them with anti-His-tag antibody at a mass ratio of 20:1, rotate them at 37°C overnight, and after reduction and blocking, wash them with ultrapure water to remove free anti-His-tag antibody. Finally, add luminescent ball preservation solution (for example, 20mM HEPES / 0.2% BSA / 0.1% ProClin300), mix them thoroughly, and prepare a second labeled microparticle A solution with a concentration of 400mg / mL, which is stored at 4°C for later use.

[0128] (2) Preparation of sample buffer solution

[0129] PBS with a pH of 7.4, 0.1 M, and containing 10% BSA was used as the sample buffer solution.

[0130] (3) Preparation of the First Labeled Microparticle C

[0131] The first labeled particle C solution is prepared using luminescent particles FG (particle size 200 nm) coated with a mixture of respiratory virus such as influenza virus monoclonal antibodies and His tag short peptides. The coating method is the same as the preparation of the second labeled particle A solution.

[0132] The monoclonal antibody used was purchased from Shenzhen Feipeng Biotechnology Co., Ltd., and the short peptide tag was synthesized by Shanghai Shenggong, with a sequence of 6 histidines and 4 lysines added to the N-terminus. The two were mixed in equal proportions and then coated according to the conventional ratio (particle: protein = 20:1). Finally, a 400 mg / mL concentration of the first labeled particle C solution was prepared and stored at 4°C for later use.

[0133] (4) Preparation of the first specific binding substance D

[0134] Preparation of biotin-labeled influenza virus antibody solution: Take an appropriate amount of long-arm biotin, dissolve it with dimethylformamide (DMF), and mix it with Bos d 5 dissolved in 0.01M PBS at a molecular ratio of 20:1. Stir overnight at 4°C to allow sufficient reaction. Dialyze the reactant in 0.01M PBS for 24h to remove free biotin, aliquot and store at 4°C for later use (working solution concentration: 3 μg / mL).

[0135] (5) Preparation of the second specific binding conjugate E

[0136] Preparation of biotin-labeled rabbit anti-mouse Ig polyclonal antibody solution: The preparation method is the same as that of the first specific binding conjugate D.

[0137] (6) Preparation of streptavidin solution

[0138] Purchase streptavidin commercially and dilute it with PBS solution to a working solution concentration of 3 μg / mL for later use.

[0139] 2. Preparation of biochip

[0140] Prepare the biochip, which has been hydrophilized before leaving the factory; prepare the above solutions for later use.

[0141] (1) Coating with streptavidin

[0142] Spot 2 μL of streptavidin at the detection area 23 and reference area 24 of the biochip, and then place it in a closed humidity-controlled incubator and incubate statically for 1h. The humidity-controlled environment can prevent the spotting solution from drying and ensure the sufficient binding of avidin to the biochip. Then carry out washing and drying.

[0143] (2) Binding biotin antibody

[0144] Spot 2 μL of biotin-labeled influenza virus antibody solution and biotin-labeled rabbit anti-mouse Ig polyclonal antibody solution at the detection area 23 and reference area 24 of the biochip respectively, and then place it in a closed humidity-controlled incubator and incubate statically for 1h to complete the efficient binding of biotin and streptavidin. Then carry out washing and drying.

[0145] (3) Presetting the second labeled microparticles A, the first labeled microparticles C and the sample buffer

[0146] Spot 2 μL of the luminescent microparticle FG solution coated with anti-human influenza virus antibody at the first labeled area 22 of the biochip, spot 10 μL of the sample buffer at the sample area 21, and spot 2 μL of the luminescent microparticle FG solution coated with a mixture of influenza virus monoclonal antibody and His tag short peptide at the second labeled area 20. After spotting, place it in a 37°C drying oven for 40 min to allow it to dry thoroughly.

[0147] 3. Microfluidic Immunodetection Chip Chimerization

[0148] Use a tablet press to tightly press the prepared biochip and the plastic substrate with channel structures together to complete the preparation of the microfluidic immunodetection chip.

[0149] 4. Sample Detection

[0150] (1) Tear off the sealing film of the sample port S, add 35 μL of the sample to be tested to the sample port S, and let it stand for 1 - 2 minutes;

[0151] (2) Tear off the sealing film of the reagent port R, add 100 μL of distilled water to the reagent port R, and let it stand for 4 minutes;

[0152] (3) Use a microfluidic photoinduced chemiluminescence detector to read the signal value T of the detection area 23 and the signal value R of the reference area 24, and use the T / R value as the calculation result.

[0153] (4) Use S / Co as the reported result. S / Co greater than 1 is positive, and S / Co less than 1 is negative.

[0154] Among them, the Co value, that is, the Cutoff value, refers to the T / R value of the normal population. The T / R values of 30 normal people can be detected, the mean value (X) and the standard deviation (Sd) are calculated, and the Co value is the mean value plus two standard deviations.

[0155] 5. Test Results

[0156] (1) The Cutoff value of the normal population is 1.0

[0157] (2) The influence of photosensitive particles GG with different particle sizes on the detection sensitivity of influenza virus antigen (recombinant antigen) is as follows in the table:

[0158]

[0159] (3) The test results of the pharyngeal swab specimens of 8 influenza virus infected patients are as follows (taking the photosensitive particles GG with a particle size of 50 nm as an example):

[0160] Serial number 1 2 3 4 5 6 7 8 S / co 210 480 650 298 310 741 278 690

[0161] As can be seen from the test results, on the one hand, by using the microfluidic immunoassay chip of the embodiments of the present application, trace target molecules can be detected with high sensitivity. This is because the binding of the photosensitive particles GG and the luminescent particles FG in the embodiments of the present application is based on the short peptide tags and anti-tag antibodies on the surface, and is independent of the binding strength between the virus antigen to be detected and the virus antibody to be detected. Therefore, it is not affected by the concentration of the virus antigen in the sample to be detected, ensuring the luminescence efficiency and greatly improving the detection sensitivity. On the other hand, the detection results with the photosensitive particles GG having a particle size of 50 nm are more accurate and sensitive than those with the photosensitive particles GG having a particle size of 200 nm. This is because the particle size of the photosensitive particles GG is smaller than that of the luminescent particles FG. On the one hand, it is beneficial for the flow of the photosensitive particles GG in the microfluidic channel. On the other hand, one luminescent particle FG can bind multiple photosensitive particles GG, thus providing more reactive oxygen molecules and enhancing the luminescence intensity, which is beneficial for improving the detection sensitivity.

[0162] The embodiments of the present application have been described above. The above description is exemplary and not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art in the technical field without departing from the scope and spirit of the described embodiments. The selection of the terms used herein is intended to best explain the principles of the embodiments, the practical application or the improvement of the technology in the market, or to enable other ordinary skill in the art in the technical field to understand the disclosed embodiments.

Claims

1. A lateral flow immunoassay method based on chemiluminescence induced by light, characterized in that, it includes: S1: Adding a sample from the sample port of the substrate to the sample area of the chip; S2: The sample sequentially flows through the first labeling area, the detection area, the reference area of the chip and enters the waste liquid chamber, and a test luminescent complex is formed in the detection area, and a reference luminescent complex is formed in the reference area; S3: Detecting the chemiluminescence signals of the test luminescent complex in the detection area and the reference luminescent complex in the reference area.

2. The method according to claim 1, characterized in that, first labeling particles are pre - placed in the first labeling area, and when the sample flows, the target molecule to be detected in the sample binds to the first labeling particles to form a labeled complex; the labeled complex forms a test complex in the detection area and a reference complex in the reference area.

3. The method according to claim 2, characterized in that, a first specific binding substance and a second specific binding substance are respectively fixed in the detection area and the reference area; the first labeling particles can specifically bind to the first specific binding substance through the target molecule to be detected and can also specifically bind to the second specific binding substance.

4. The method according to claim 3, characterized in that, the first labeling particles comprise luminescent particles labeled with a first labeling molecule and a second labeling molecule; the test complex includes a complex formed by the first specific binding substance binding to the first labeling molecule on the luminescent particles in the labeled complex through an immune reaction mediated by the target molecule to be detected; and / or, the reference complex includes a complex formed by the first labeling molecule on the luminescent particles in the labeled complex binding to the second specific binding substance.

5. The method according to any one of claims 1 - 4, characterized in that, step S2 further includes: S21: Adding a dissolving solution from the reagent port of the substrate to the second labeling area of the chip to dissolve the second labeling particles pre - placed in the second labeling area; S22: The second labeling particles sequentially flow through the detection area, the reference area of the chip and enter the waste liquid chamber, and the test luminescent complex is formed in the detection area, and the reference luminescent complex is formed in the reference area.

6. The method according to claim 5, characterized in that, the second labeling particles comprise photosensitive particles labeled with a third labeling molecule, and the third labeling molecule can specifically bind to the second labeling molecule.

7. The method according to claim 6, characterized in that, the test luminescent complex includes a complex formed by the second labeling molecule on the luminescent particles in the test complex binding to the third labeling molecule on the photosensitive particles; and / or, the reference luminescent complex includes a complex formed by the second labeling molecule on the luminescent particles in the reference complex binding to the third labeling molecule on the photosensitive particles.

8. The method according to any one of claims 1 - 4, characterized in that, The detection area includes a plurality of detection points, and different kinds of first specific binding substances are fixed in the plurality of detection points. The different kinds of first specific binding substances can bind to different kinds of target molecules to be detected.

9. A reagent for the lateral flow immunoassay method of photochemiluminescence according to any one of claims 1-8, characterized in that it includes: second labeled microparticles, powder, first labeled microparticles, first specific binding substances and second specific binding substances. The second labeled microparticles, powder and first labeled microparticles are respectively pre-placed in the second labeling area, sample area and first labeling area of the chip, and the first specific binding substances and second specific binding substances are respectively fixed in the detection area and reference area; wherein, the first labeled microparticles include luminescent microparticles labeled with a first labeling molecule and a second labeling molecule, the second labeled microparticles include photosensitive microparticles labeled with a third labeling molecule, the first labeling molecule can specifically bind to the first specific binding substance through the target molecule to be detected and can specifically bind to the second specific binding substance, and the second labeling molecule can specifically bind to the third labeling molecule.

10. The method or reagent according to claim 6 or 9, characterized in that the particle size of the photosensitive microparticles is not greater than that of the luminescent microparticles; preferably, the particle size of the luminescent microparticles is 200 nm - 400 nm, and the particle size of the photosensitive microparticles is 50 nm - 200 nm.

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