Light-activated chemiluminescence detection method, detection reagent and preparation method

Abstract

The invention relates to a light-activated chemiluminescence detection method, a detection reagent and a preparation method. The method comprises the step of combining photosensitive particles around light-emitting particles to form a plurality of photosensitive particle layers with different layer spacing. According to the scheme provided by the invention, the steric hindrance effect can be reduced, the binding rate of the light-sensitive particles and the light-emitting particles is improved, and the light-excited chemiluminescence detection sensitivity is improved.

Description

Technical Field

[0001] The present application relates to the field of chemiluminescence technology, and in particular to a photoinduced chemiluminescence detection method and a detection reagent and a preparation method thereof. Background Art

[0002] Photoluminescence technology uses homogeneous system chemiluminescence detection technology. The photosensitive particles and luminescent particles involved in the reaction can form an immunoluminescent complex in the presence of target molecules. Under the excitation light of a specific wavelength, the photosensitive particles can convert oxygen in the surrounding environment into singlet oxygen, and the luminescent particles can accept singlet oxygen within the range of 200nm and emit high-energy light.

[0003] However, due to the steric hindrance effect, the binding rate between the luminescent particles and the photosensitive particles is reduced, so singlet oxygen can only transfer energy to a few surrounding luminescent particles, resulting in low utilization of singlet oxygen and limited luminescent signal value, thus affecting the sensitivity of chemiluminescence detection. Summary of the invention

[0004] In order to solve or partially solve the problems existing in the related art, the present application provides a photochemiluminescence detection method, a detection reagent and a preparation method, which can combine around the luminescent particles to form multiple photosensitive particle layers with different layer spacings, thereby reducing the steric hindrance effect, increasing the binding rate between the photosensitive particles and the luminescent particles, and thus improving the sensitivity of photochemiluminescence detection.

[0005] In a first aspect, the present application provides a photo-induced chemiluminescence detection method, comprising the steps of combining photosensitive particles around luminescent particles to form a multilayer photosensitive particle layer with different interlayer spacings.

[0006] As an optional embodiment, the interlayer spacing of the photosensitive particle layer located at the outermost layer does not exceed the distance of energy transfer between the chemiluminescent paired particles; preferably, the photosensitive particle layer consists of two layers.

[0007] As an optional embodiment, the step includes allowing an antibody or antigen labeled with a marker to combine with the luminescent particle to form an immune complex through an immune reaction mediated by a target molecule to be detected, wherein the surface of the luminescent particle in the immune complex is connected to a plurality of the markers, and the distances between each marker and the luminescent particle are not exactly the same.

[0008] As an optional embodiment, the step includes mixing the marker-chain molecule-antibody or antigen with the luminescent particle-antibody or antigen; and / or,

[0009] Mixed markers - antibodies or antigens and luminescent particles - chain molecules - antibodies or antigens;

[0010] The chain molecules have different lengths.

[0011] The second aspect of the present application provides a photo-induced chemiluminescence detection reagent, comprising luminescent microspheres, photosensitive microspheres, and labeled antibodies or antigens, wherein the marker of the labeled antibody or antigen is connected to the antibody or antigen by chain molecules of different lengths; and / or, the surface of the luminescent microparticles is coated with antibodies or antigens, and the luminescent microparticles are connected to the antibodies or antigens by chain molecules of different lengths.

[0012] As an optional embodiment, the length of the chain molecule includes L1 and L2, L1 is greater than L2, and L1 does not exceed the distance of energy transfer between chemiluminescent paired particles; preferably, the chain molecule includes one of polyethylene glycol, polylysine and dextran.

[0013] As an alternative embodiment:

[0014]

[0015] In the formula, R is the diameter of the luminescent particles, and r is the diameter of the photosensitive particles; preferably, R=r; further preferably, R is 200-400 nm.

[0016] As an optional embodiment, the labeled antibody or antigen obtained by combining the chain molecule with a chain length of L1 with the antibody or antigen accounts for 5%-25% of the total amount of the labeled antibody or antigen in the reagent; and / or,

[0017] The antibodies or antigens connected to the surface of the luminescent particles through the chain molecules with a chain length of L1 account for 5%-25% of the total amount of antibodies or antigens connected to the surface of the luminescent particles.

[0018] The third aspect of the present application provides a method for preparing a photochemiluminescence detection reagent, including the steps of preparing a marker-chain molecule-antibody or antigen; and / or, including the steps of preparing luminescent particles-chain molecules-antibody or antigen; the chain molecules have different lengths.

[0019] As an optional embodiment, the step of preparing the marker-chain molecule-antibody or antigen includes:

[0020] S1. combining the chain molecules of different lengths with antibodies or antigens;

[0021] S2, combining the marker with the chain molecules of different lengths to obtain the marker-chain molecule-antibody or antigen; or, combining the marker combined with the chain molecules of different lengths with an antibody or antigen to obtain the marker-chain molecule-antibody or antigen; and / or,

[0022] The steps of preparing luminescent particles-chain molecules-antibodies or antigens include:

[0023] S1', combining the chain molecules of different lengths with antibodies or antigens;

[0024] S2', combining the luminescent particles with the chain molecules of different lengths to obtain the luminescent particles-chain molecules-antibodies or antigens.

[0025] The technical solution provided by the present application may include the following beneficial effects: in the process of photoluminescence detection, when there is a target molecule to be detected, such as a target antibody / antigen to be detected, the target antibody / antigen to be detected specifically binds to the labeled antibody / antigen and the luminescent particles in the detection system to form an immune complex. The photosensitive particles then bind to the immune complex to form a chemiluminescent complex. During the formation of the chemiluminescent complex, the photosensitive particles will bind around the luminescent particles to form a multilayer photosensitive particle layer with different interlayer spacings, thereby adjusting the spatial distance between the luminescent particles and the photosensitive particles, reducing the spatial steric hindrance between the two, increasing the binding rate and activity utilization rate of the two, and enhancing the specific binding effect, thereby making full use of singlet oxygen, increasing the luminescent signal value, and improving the detection sensitivity.

[0026] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The above and other objects, features and advantages of the present application will become more apparent by describing in more detail exemplary embodiments of the present application in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments of the present application.

[0028] Figure 1 It is a schematic diagram of the structure of the combination of luminescent particles and photosensitive particles in the related art;

[0029] Figure 2 It is a schematic diagram of the structure of a multi-layer photosensitive particle layer formed around luminescent particles shown in an embodiment of the present application.

[0030] In the figure: 1, luminescent particles; 2, photosensitive particles; 3, photosensitive particle layer; 30, inner photosensitive particle layer; 31, outer photosensitive particle layer. DETAILED DESCRIPTION

[0031] 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.

[0032] 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 of "a", "said" and "the" used in this application and the appended claims are also intended to include plural forms unless the context clearly indicates other meanings. It should also be understood that the term "and / or" used herein refers to and includes any or all possible combinations of one or more associated listed items.

[0033] It should be understood that although the terms "first", "second", "third", etc. may be used in this application to describe various information, this 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 as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of this application, the meaning of "multiple" is two or more, unless otherwise clearly and specifically defined.

[0034] Under the excitation light, the photosensitive particles can convert the oxygen in the surrounding environment into singlet oxygen. The luminescent particles can accept singlet oxygen within the range of 200nm and emit high-energy light. Figure 1 As shown, due to the steric hindrance effect, the affinity and binding rate between the luminescent particles 1 and the photosensitive particles 2 are reduced, so the singlet oxygen can only transfer energy to a few surrounding luminescent particles 1, resulting in low utilization of singlet oxygen and limited luminescent signal value, thereby affecting the sensitivity of chemiluminescence detection.

[0035] In response to the above problems, an embodiment of the present application provides a photochemiluminescence detection method, which can combine around luminescent particles to form multiple photosensitive particle layers with different interlayer spacings, thereby improving the affinity and binding rate between photosensitive particles and luminescent particles, reducing steric hindrance effects, and improving the sensitivity of photochemiluminescence detection.

[0036] The term "target molecule to be detected" used in the embodiments of the present application can be DNA, RNA, protein, polypeptide, carbohydrate, etc., preferably protein, and more preferably immune molecule, such as antigen or antibody.

[0037] The term "test substance" used in the embodiments of the present application refers to a mixture that may contain the target molecule to be tested. 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.

[0038] The term "binding" used in the embodiments of the present application refers to the direct association between two molecules due to interactions such as covalent, electrostatic, hydrophobic, ionic and / or hydrogen bonds, including but not limited to interactions such as salt bridges and water bridges.

[0039] The term "specific binding" used in the embodiments of the present application refers to the mutual recognition and selective binding reaction between two substances, which refers to the conformational correspondence between the corresponding reactants from the perspective of three-dimensional structure.

[0040] The term "biotin" used in the embodiments of the present application is widely present in animal and plant tissues. There are two ring structures on its molecule, namely the imidazolone ring and the thiophene ring, of which the imidazolone ring is the main site for binding to streptavidin. Activated biotin can be coupled with almost all known biological macromolecules, including proteins, nucleic acids, polysaccharides and lipids, under the mediation of protein cross-linking agents. The "avidin" molecule consists of 4 identical peptide chains, each of which can bind to one biotin. Therefore, each antigen or antibody can be coupled to multiple biotin molecules at the same time, thereby producing a "tentacle effect" to improve the sensitivity of the analysis.

[0041] The technical solution of the embodiments of the present application is described in detail below with reference to the accompanying drawings.

[0042] Figure 2 It is a schematic diagram of the structure of a multi-layer photosensitive particle layer formed around luminescent particles shown in an embodiment of the present application.

[0043] See also Figure 2 A photoinduced chemiluminescence detection method includes the steps of combining photosensitive particles 2 around luminescent particles 1 to form a multilayer photosensitive particle layer 3 with different layer spacings.

[0044] The interlayer spacing in the embodiment of the present application refers to the distance between the outer surface of the photosensitive particle 2 contained in the photosensitive particle layer 3 and the outer surface of the luminescent particle 1. And the luminescent particle 1 is located in the center, and the outer layer surrounds the multi-layer photosensitive particle layer 3, so that the luminescent particle 1 in the center can receive more active oxygen generated by the photosensitive particle 2, thereby greatly improving the light signal value and improving the detection sensitivity.

[0045] In the process of photoluminescence detection in the embodiment of the present application, when there is a target molecule to be detected, such as a target antibody / antigen to be detected, the target antibody / antigen to be detected specifically binds to the labeled antibody / antigen in the detection system and the luminescent particle 1 to form an immune complex. The photosensitive particle 2 then binds to the immune complex to form a chemiluminescent complex. During the formation of the chemiluminescent complex, the photosensitive particle 2 will bind around the luminescent particle 1 to form a multilayer photosensitive particle layer 3 with different interlayer spacings, thereby adjusting the spatial distance between the luminescent particle 1 and the photosensitive particle 2, reducing the steric hindrance between the two, improving the binding rate and activity utilization rate of the two, and enhancing the specific binding effect, thereby making full use of singlet oxygen, increasing the luminescent signal value, and improving the detection sensitivity.

[0046] As an optional embodiment, the interlayer distance D1 of the outermost photosensitive particle layer 3 does not exceed the distance for energy transfer between the chemiluminescent paired particles.

[0047] Since the average distance that singlet oxygen propagates in the solution in photoluminescence detection is about 200nm, the luminescent particle 1 can only accept singlet oxygen and emit high-energy light when it is within about 200nm from the photosensitive particle 2. Therefore, the interlayer spacing D1 of the photosensitive particle layer 3 located at the outermost layer does not exceed the distance of energy transfer between the chemiluminescent paired particles (luminescent particle 1 and photosensitive particle 2), that is, D1 does not exceed 200nm.

[0048] As an optional embodiment, the photosensitive particle layer is two layers.

[0049] See also Figure 2 , the inner photosensitive particle layer 30 is located inside, and the outer photosensitive particle layer 31 is located outside. The interlayer distance between the photosensitive particles 2 and the luminescent particles 1 in the inner photosensitive particle layer 30 is D2, and the interlayer distance between the photosensitive particles 2 and the luminescent particles 1 in the outer photosensitive particle layer 31 is D1, D1 is greater than D2, and D1 does not exceed the distance of energy transfer between the chemiluminescent paired particles (luminescent particles 1 and photosensitive particles 2).

[0050] As an optional embodiment, the photo-induced chemiluminescence detection method of the embodiment of the present application includes the step of combining an antibody or antigen labeled with a marker with a luminescent particle to form an immune complex through an immune reaction mediated by a target molecule to be detected, wherein the surface of the luminescent particle in the immune complex is connected to multiple markers, and the distance between each marker and the luminescent particle is not exactly the same.

[0051] When the target molecule to be detected is present, the antibody or antigen labeled with the marker can be combined with the luminescent particle 1 to form an immune complex such as a double antibody sandwich complex through the immune reaction mediated by the target molecule to be detected. In addition, the surface of the luminescent particle 1 in the immune complex is connected with multiple markers, and the distances between each marker and the luminescent particle 1 are not exactly the same.

[0052] The marker may be biotin, and the biotin may include one of biotin-12-polyethylene glycol active ester Biotin-PEG12-NHS, biotin-tetra-polyethylene glycol amino Biotin-PEG4-NH2, and EZ-Link Sulfo-NHS-LC-LC-Biotin. The chain molecule may include one of polyethylene glycol, polylysine, and dextran. The length of the chain molecule may be changed by selecting chain molecules with different polymerization degrees.

[0053] As an optional embodiment, the steps include mixing marker-chain molecule-antibody or antigen and luminescent particle-antibody or antigen, and the chain molecules have different lengths.

[0054] In this embodiment, due to the different lengths of the chain molecules, the marker-chain molecule-antibody or antigen also has different lengths, so that in the immune complex formed by mixing markers-chain molecules-antibodies or antigens of different lengths with luminescent particles-antibodies or antigens, the distances between each marker and the luminescent particle are not exactly the same.

[0055] The marker may be biotin, and the biotin may include one of biotin-12-PEG active ester Biotin-PEG12-NHS, biotin-tetraethylene glycol amino Biotin-PEG4-NH2 and EZ-Link Sulfo-NHS-LC-LC-Biotin, and is a short-arm biotin.

[0056] As an optional embodiment, the steps include mixing the marker-antibody or antigen with the luminescent particle-chain molecule-antibody or antigen, and the chain molecules have different lengths.

[0057] In this embodiment, due to the different lengths of the chain molecules, the luminescent microparticles-chain molecules-antibodies or antigens also have different lengths, so that in the immune complex formed by mixing luminescent microparticles-chain molecules-antibodies or antigens of different lengths with markers-antibodies or antigens, the distances between each marker and the luminescent microparticle are not exactly the same.

[0058] As a preferred embodiment, the photoinduced chemiluminescence detection method of the embodiment of the present application also includes the step of indirectly combining the photosensitive particles 2 with the luminescent particles 1 through a surface-coupled counterpart to form a chemiluminescent complex, and the counterpart specifically recognizes and binds to the marker.

[0059] The pairing substance may be avidin, and the labeling substance may be biotin; or, one of the binding pair molecules may be fluorescein isothiocyanate FITC, and the other may be fluorescein isothiocyanate antibody anti-FITC. The pairing substance specifically recognizes and binds to the labeling substance so that the surface of the luminescent particle 1 in the chemiluminescent complex is connected to multiple photosensitive particles 2, and the distance between each photosensitive particle 2 and the luminescent particle 1 is not exactly the same. Thus, the spatial distance between the luminescent particle 1 and the photosensitive particle 2 is adjusted, the steric hindrance between the two is reduced, the binding rate and activity utilization rate between the two are increased, and the specific binding effect is enhanced, thereby making full use of singlet oxygen, increasing the luminescent signal value, and improving the detection sensitivity.

[0060] As an optional embodiment, the photoluminescence detection method of the embodiment of the present application may include:

[0061] S30: Quantitatively aspirate the analyte, the luminescent microparticles-antibody or antigen, and markers-chain molecules-antibody or antigen of different lengths into a reaction container.

[0062] S31: Markers - chain molecules - antibodies or antigens of different lengths combine with luminescent particles - antibodies or antigens through an immune reaction mediated by the target molecule in the test object to form an immune complex. The surface of the luminescent particle in the immune complex is connected to multiple markers, and the distance between each marker and the luminescent particle is not exactly the same.

[0063] S32: Quantitatively aspirate the photosensitive microparticles into the reaction container. The photosensitive microparticles are indirectly combined with the luminescent microparticles via the surface-coupled counterparts to form chemiluminescent complexes, wherein the counterparts specifically recognize and bind to the markers.

[0064] S33: The photosensitive particles are excited to generate active oxygen, and the luminescent particles react with the active oxygen they come into contact with to generate chemiluminescent signals.

[0065] S34: Detect the intensity of the chemiluminescent signal and analyze the concentration of the analyte.

[0066] As an optional embodiment, the photoinduced chemiluminescence detection method of the embodiment of the present application may also include:

[0067] S30': Quantitatively aspirate the analyte, luminescent particles-chain molecules-antibodies or antigens of different lengths, and markers-antibodies or antigens into a reaction container.

[0068] S31': The marker-antibody or antigen combines with the luminescent microparticle-chain molecule-antibody or antigen of different lengths through the immune reaction mediated by the target molecule in the test object to form an immune complex. The surface of the luminescent microparticle in the immune complex is connected with multiple markers, and the distance between each marker and the luminescent microparticle is not exactly the same.

[0069] S32': Quantitatively aspirate the photosensitive microparticles into the reaction container. The photosensitive microparticles are indirectly combined with the luminescent microparticles via the surface-coupled counterparts to form chemiluminescent complexes, wherein the counterparts specifically recognize and bind to the markers.

[0070] S33': The photosensitive particles are excited to generate active oxygen, and the luminescent particles react with the active oxygen they come into contact with to generate chemiluminescent signals.

[0071] S34': Detect the intensity of the chemiluminescent signal and analyze the concentration of the analyte.

[0072] In the process of photoluminescence detection, when there is a target molecule to be detected in the test object, such as a target antibody / antigen to be detected, the target antibody / antigen to be detected specifically binds to the marker-chain molecule-antibody or antigen of different lengths in the detection system, and the luminescent microparticle-antibody or antigen to form immune complexes of different lengths, and the photosensitive microparticles then bind to the luminescent microparticles in the immune complex to form a chemiluminescent complex. Alternatively, in the process of photoluminescence detection, when there is a target molecule to be detected in the test object, such as a target antibody / antigen to be detected, the target antibody / antigen to be detected specifically binds to the marker-antibody or antigen in the detection system, and the luminescent microparticle-chain molecule-antibody or antigen of different lengths to form immune complexes of different lengths, and the photosensitive microparticles then bind to the luminescent microparticles in the immune complex to form a chemiluminescent complex. During the formation of the chemiluminescent complex, after the luminescent particle-antibody or antigen forms an inner photosensitive particle layer by binding the photosensitive particle 2 through the short marker-chain molecule-antibody or antigen, the long marker-chain molecule-antibody or antigen can also extend beyond the inner photosensitive particle layer and further bind the luminescent particle-antibody or antigen to form an outer photosensitive particle layer; or, after the short luminescent particle-chain molecule-antibody or antigen forms an inner photosensitive particle layer by binding the photosensitive particle 2 through the marker-antibody or antigen, the long luminescent particle-chain molecule-antibody or antigen can also form an outer photosensitive particle layer by binding the photosensitive particle 2 through the marker-antibody or antigen. Even if the photosensitive particle 2 in the outer photosensitive particle layer is far away from the luminescent particle 1, the singlet oxygen generated by it can be transferred to the luminescent particle 1, thereby adjusting the spatial distance between the photosensitive particle 2 and the luminescent particle 1 and improving the signal value and detection sensitivity.

[0073] Corresponding to the aforementioned application function realization method embodiment, the present application also provides a photoinduced chemiluminescence detection reagent and corresponding embodiments.

[0074] The photo-induced chemiluminescence detection reagent of the embodiment of the present application includes luminescent microspheres 1, photosensitive microspheres 2, and labeled antibodies or antigens, wherein the marker of the labeled antibody or antigen is connected to the antibody or antigen by chain molecules of different lengths; and / or the antibody or antigen is coated on the surface of the luminescent microparticles, and the luminescent microparticles and the antibody or antigen are connected by chain molecules of different lengths.

[0075] In the process of photoluminescence detection in the embodiments of the present application, when the target molecule to be detected, such as the target antibody / antigen to be detected, is present in the analyte, the target antibody / antigen to be detected specifically binds to the marker-chain molecule-antibody or antigen of different lengths in the detection system, as well as the luminescent microparticle-antibody or antigen, to form immune complexes of different lengths, and the photosensitive microparticles then bind to the luminescent microparticles in the immune complex to form a chemiluminescent complex. Alternatively, in the process of photoluminescence detection, when the target molecule to be detected, such as the target antibody / antigen to be detected, is present in the analyte, the target antibody / antigen to be detected specifically binds to the marker-antibody or antigen, as well as the luminescent microparticle-chain molecule-antibody or antigen of different lengths in the detection system, to form immune complexes of different lengths, and the photosensitive microparticles then bind to the luminescent microparticles in the immune complex to form a chemiluminescent complex. During the formation of the chemiluminescent complex, after the luminescent particle-antibody or antigen forms an inner photosensitive particle layer by binding the photosensitive particle 2 through the short marker-chain molecule-antibody or antigen, the long marker-chain molecule-antibody or antigen can also extend beyond the inner photosensitive particle layer and further bind the luminescent particle-antibody or antigen to form an outer photosensitive particle layer; or, after the short luminescent particle-chain molecule-antibody or antigen forms an inner photosensitive particle layer by binding the photosensitive particle 2 through the marker-antibody or antigen, the long luminescent particle-chain molecule-antibody or antigen can also form an outer photosensitive particle layer by binding the photosensitive particle 2 through the marker-antibody or antigen. Even if the photosensitive particle 2 in the outer photosensitive particle layer is far away from the luminescent particle 1, the singlet oxygen generated by it can be transferred to the luminescent particle 1, thereby adjusting the spatial distance between the photosensitive particle 2 and the luminescent particle 1 and improving the signal value and detection sensitivity.

[0076] As an optional embodiment, the lengths of the chain molecules are L1 and L2, L1 is greater than L2, and L1 does not exceed the distance for energy transfer between the chemiluminescent paired particles.

[0077] As an alternative embodiment:

[0078]

[0079] In the formula, R is the diameter of the luminescent particles, and r is the diameter of the photosensitive particles; preferably, R=r; more preferably, R is 200-400 nm.

[0080] When R = r,

[0081] As a preferred embodiment, the labeled antibody or antigen obtained by combining the chain molecule with a chain length of L1 with the antibody or antigen accounts for 5%-25% of the total amount of the labeled antibody or antigen in the reagent.

[0082] The concentration of the labeled antibody or antigen is related to the particle concentration. When the concentration of luminescent particles is reduced, the number of photosensitive particles around the luminescent particles will increase, and the steric hindrance effect will be greater. Relatively speaking, longer labeled antibodies or antigens are needed as supplements. The entire reaction process involves the combined effects of the factors of luminescent particle concentration, analyte concentration, labeled antibody or antigen concentration, and photosensitive particle concentration. In general, when there are labeled antibodies or antigens of different lengths in the reagent, the concentration of the labeled antibody or antigen obtained by combining the chain molecule with a chain length of L1 with the antibody or antigen cannot be too high (preferably between 5% and 25%). Too many labeled antibodies or antigens obtained by combining the chain molecule with a chain length of L1 with the antibody or antigen will make the distance between the luminescent particles and the photosensitive particles farther, thereby reducing the sensitivity.

[0083] As a preferred embodiment, the antibodies or antigens connected to the surface of the luminescent particles through chain molecules with a chain length of L1 account for 5%-25% of the total amount of antibodies or antigens connected to the surface of the luminescent particles.

[0084] As described above, when luminescent particles-chain molecules-antibodies or antigens of different lengths exist in the reagent, the concentration of antibodies or antigens connected to the surface of the luminescent particles through chain molecules with a chain length of L1 cannot be too high (preferably between 5% and 25%). Too many antibodies or antigens connected to the surface of the luminescent particles through chain molecules with a chain length of L1 will make the distance between the luminescent particles and the photosensitive particles farther, thereby reducing the sensitivity.

[0085] As an optional embodiment, the chain molecule includes one of polyethylene glycol, polylysine and dextran. The length of the chain molecule can be changed by selecting chain molecules with different polymerization degrees.

[0086] Corresponding to the aforementioned application function realization method embodiment, the present application also provides a method for preparing a photoinduced chemiluminescence detection reagent and corresponding embodiments.

[0087] The method for preparing the photochemiluminescence detection reagent provided in the embodiment of the present application includes the steps of preparing a marker-chain molecule-antibody or antigen; and / or, includes the steps of preparing luminescent particles-chain molecules-antibody or antigen; the chain molecules have different lengths.

[0088] In order to achieve a multi-layer photosensitive particle layer with different interlayer spacings formed around the luminescent particles, the embodiment of the present application is achieved by preparing markers-chain molecules-antibodies or antigens of different lengths and / or by preparing luminescent particles-chain molecules-antibodies or antigens of different lengths, so that after the luminescent particles-antibodies or antigens are combined with photosensitive particles 2 through short markers-chain molecules-antibodies or antigens to form an inner layer of photosensitive particle layer, the long markers-chain molecules-antibodies or antigens can also extend beyond the inner layer of photosensitive particle layer, and further combine with luminescent particles-antibodies or antigens to form an outer layer of photosensitive particle layer; or, after the short luminescent particles-chain molecules-antibodies or antigens are combined with photosensitive particles 2 through markers-antibodies or antigens to form an inner layer of photosensitive particle layer, the long luminescent particles-chain molecules-antibodies or antigens can also combine with photosensitive particles 2 through markers-antibodies or antigens to form an outer layer of photosensitive particle layer. Even if the photosensitive particles 2 in the outer photosensitive particle layer are far away from the luminescent particles 1, the singlet oxygen generated by them can be transferred to the luminescent particles 1, thereby adjusting the spatial distance between the photosensitive particles 2 and the luminescent particles 1 and improving the signal value and detection sensitivity.

[0089] As an optional embodiment, the step of preparing the marker-chain molecule-antibody or antigen includes two schemes, one scheme A is as follows:

[0090] The markers combined with chain molecules of different lengths are combined with antibodies or antigens to obtain marker-chain molecules-antibody or antigen.

[0091] The marker in scheme A can be biotin (generally short-arm biotin). The substances obtained by combining biotin with chain molecules of different lengths include long-arm biotin and short-arm biotin. The long-arm biotin and short-arm biotin prepared in this step can be commercially available.

[0092] Another option B is as follows:

[0093] S1. Bind chain molecules of different lengths to antibodies or antigens.

[0094] S2. Combine the marker with chain molecules of different lengths to obtain marker-chain molecule-antibody or antigen.

[0095] The label in step S2 of scheme B can be biotin (generally short-arm biotin), and can be commercially available short-arm biotin. Short-arm biotin combines with long chain molecules to obtain long-arm biotin, and short-arm biotin combines with short chain molecules to still obtain short-arm biotin.

[0096] As an optional embodiment, the steps of preparing luminescent particles-chain molecules-antibodies or antigens include:

[0097] S1', combining chain molecules of different lengths with antibodies or antigens.

[0098] S2', combining the luminescent particles with chain molecules of different lengths to obtain luminescent particles-chain molecules-antibodies or antigens.

[0099] The chain molecules in the embodiments of the present application include one of polyethylene glycol, polylysine and dextran. The length of the chain molecules can be changed by selecting chain molecules with different polymerization degrees.

[0100] In addition, the steps of preparing the luminescent particles-chain molecules-antibodies or antigens in the embodiment of the present application may also include:

[0101] The luminescent particles combined with chain molecules of different lengths are combined with antibodies or antigens to obtain luminescent particles-chain molecules-antibodies or antigens.

[0102] In order to further understand the present invention, the photochemiluminescence detection method and detection reagent provided by the present application are described below in conjunction with the examples. The protection scope of the present application is not limited by the following examples.

[0103] Embodiment 1:

[0104] 1. Main experimental materials

[0105] Biotin-PEG n -NHS, molecular weight 20K, commercially available.

[0106] Biotin-PEG 12 -NHS, molecular weight 941.1, commercially available.

[0107] Biotin-PEG4-NH2, molecular weight 462.6, commercially available.

[0108] PIIINP (type III procollagen amino-terminal peptide) labeled antibody: purchased from Shanghai Suoxin Biotechnology Co., Ltd.

[0109] PIIINP coating antibody: purchased from Shanghai Suoxin Biotechnology Co., Ltd.

[0110] PIIINP antigen: purchased from Holmes (Beijing) Biotechnology Co., Ltd.

[0111] Luminescent particles and photosensitive particles (diameter 200 nm) were purchased from Shanghai Suoxin Biotechnology Co., Ltd.

[0112] 2. Reagent Preparation

[0113] 1) Prepare Bio-long-chain-PIIINP solution by two methods:

[0114] Method A:

[0115] The PIIINP-labeled antibody was dialyzed with 0.1M NaHCO3 and diluted to 1 mg / mL; 10 mM Biotin-PEG was prepared with DMSO (dimethylformamide) n -NHS (molecular weight 20K); take 13.4uL Biotin-PEG n -NHS (molecular weight 20K) was added to 1 mg PIIINP labeled antibody solution, mixed and placed at 4°C overnight; the protein was dialyzed and purified using 100 mM phosphate buffer (PH7.0).

[0116] Method B:

[0117] The PIIINP-labeled antibody was dialyzed with 0.1M NaHCO3 and diluted to 1mg / mL; 200mg / mL NHS-PEG was prepared with 0.1M NaHCO3 n -NHS; take 20uL NHS-PEG n -NHS to 1mg PIIINP labeled antibody solution, mix well and place at 4℃ overnight; ultrafiltration is used to purify the protein to remove unreacted NHS-PEG n -NHS molecules; prepare 100mM Biotin-PEG4-NH2 with DMSO (dimethylformamide); add 1.34uL Biotin-PEG4-NH2 to every 1mg of the above antibody reaction, mix well and place at 4°C overnight; dialysis purify the protein with 100mM phosphate buffer (PH 7.0) to remove the chain molecules not coupled to the antibody.

[0118] 2) Prepare Bio-short-chain-PIIINP solution: dialyze the PIIINP labeled antibody with 0.1M NaHCO3 and dilute it to 1 mg / mL; prepare 100mM Biotin-PEG with DMSO (dimethylformamide) 12 -NHS; take 1.34uL Biotin-PEG 12 -NHS was added to 1 mg PIIINP labeled antibody solution, mixed and placed at 4°C overnight; the protein was dialyzed and purified using 100 mM phosphate buffer (PH7.0).

[0119] 3) Preparation of FG-PIIINP solution: Wash the luminescent particles with 0.1M NaHCO3; dialyze the PIIINP-coated antibody with 0.1M NaHCO3, mix the luminescent particles and the PIIINP-coated antibody at a certain concentration and ratio to form a linker, and prepare a concentrated solution of luminescent particles-coated PIIINP antibodies after blocking and washing.

[0120] 3. Reagent composition for different experimental conditions

[0121]

[0122] 4. Experimental steps

[0123] Sample addition mode: 25uL sample + 25uL reagent R1 + 25uL reagent R2 + 175uL photosensitive particle solution.

[0124] The sample arm draws 25uL of sample, and the reagent arm draws 25uL of reagent R1 and 25uL of reagent R2 into the reaction cup in turn. After reacting for a certain period of time at a specific temperature, 175uL of photosensitive particle solution is added to continue the reaction. The optical system collects light signals and analyzes and determines the experimental results.

[0125] 5. Experimental data

[0126]

[0127] 6. Explanation and analysis of experimental data

[0128] When the concentration of biotin-short chain-antibody increased from 1ug / mL to 1.2ug / mL, the light signal value RLU did not increase significantly, and the P / N (ratio of positive serum to negative serum) of the reagent for different concentrations of recombinant antigens did not change significantly. However, when 0.2ug / mL of biotin-long chain-antibody was added to biotin-short chain-antibody, the light signal value RLU increased significantly, and the P / N value of the reagent for different concentrations of recombinant antigens increased significantly. And the effects of biotin-long chain-antibodies prepared by the A / B methods were consistent.

[0129] 7. Experimental conclusion

[0130] Adding a certain amount of biotin-long-chain-antibody to the biotin-short-chain-antibody can significantly improve the sensitivity of the reagent.

[0131] Embodiment 2:

[0132] 1. Main experimental materials

[0133] FITC-PEG n -NH2, molecular weight 20K, commercially available.

[0134] FITC-PEG n -NH2, molecular weight 1K, commercially available.

[0135] Biotin-PEG 12 -NHS, molecular weight 941.1, commercially available.

[0136] PIIINP-labeled antibody: purchased from Shanghai Suoxin Biotechnology Co., Ltd.

[0137] PIIINP coating antibody: purchased from Shanghai Suoxin Biotechnology Co., Ltd.

[0138] PIIINP antigen: purchased from Holmes (Beijing) Biotechnology Co., Ltd.

[0139] Luminescent particles and photosensitive particles (diameter 200 nm) were purchased from Shanghai Suoxin Biotechnology Co., Ltd.

[0140] 2. Reagent preparation:

[0141] 1) Reagent R1-1 (short-arm coated antibody FG-PEG n -PIIINP): Wash the luminescent particles with 0.1M NaHCO3, dialyze the PIIINP-coated antibody with 0.1M sodium carbonate buffer (PH9), and prepare FITC-PEG with DMSO (dimethylamine). n -NH2 (molecular weight 1K) solution, PIIINP coated with antibodies and FITC-PEG n -NH2 (molecular weight 1K) solution was mixed in a certain ratio (molar ratio 1:50) to form a linker, and unreacted FITC-PEG was removed by dialysis with 0.1M NaHCO3 n -NH2 (molecular weight 1K); the above reactants are added to the luminescent particles in a certain concentration and ratio, and a concentrated solution is prepared after sealing and washing.

[0142] 2) Reagent R1-2 (short arm coated antibody FG-PEG n -PIIINP): Wash the luminescent particles with 0.1M NaHCO3, dialyze the PIIINP-coated antibody with 0.1M sodium carbonate buffer (PH9), and prepare FITC-PEG with DMSO (dimethylamine). n -NH2 (molecular weight 1K) solution, PIIINP coated with antibodies and FITC-PEG n -NH2 (molecular weight 1K) solution was mixed in a certain ratio (molar ratio 1:60) to form a linker, and the unreacted FITC-PEG was removed by dialysis with 0.1M NaHCO3 n -NH2 (molecular weight 1K); the above reactants are added to the luminescent particles in a certain concentration and ratio, and a concentrated solution is prepared after sealing and washing.

[0143] 3) Reagent R1-3 (Long- and short-arm mixed coated antibody FG-PEG n -PIIINP): Wash the luminescent particles with 0.1M NaHCO3, dialyze the PIIINP-coated antibody with 0.1M sodium carbonate buffer (PH9), and prepare FITC-PEG with DMSO (dimethylamine). n-NH2 (molecular weight 20K) and FITC-PEG n -NH2 (molecular weight 1K) solution, PIIINP coated with antibodies, FITC-PEG n -NH2 (molecular weight 20K) and FITC-PEG n -NH2 (molecular weight 1K) was mixed in a certain ratio (molar ratio 1:10:50) to form a linker, and the unreacted FITC-PEG was removed by dialysis with 0.1M NaHCO3 n -NH2; the above reactants are added into the luminescent particles in a certain concentration and ratio, and a concentrated solution is prepared after sealing and washing.

[0144] 4) Reagent R2 (short-arm biotin-labeled antibody Bio-PIIINP): dialyze the PIIINP-labeled antibody with 0.1M NaHCO3 and dilute it to 1 mg / mL; prepare 100mM Biotin-PEG with DMSO (dimethylformamide) 12 -NHS; take 1.34uBiotin-PEG 12 -NHS was added to 1 mg PIIINP labeled antibody solution, mixed and placed at 4°C overnight; the protein was dialyzed and purified using 100 mM phosphate buffer (PH7.0).

[0145] 3. Composition of different experimental conditions

[0146]

[0147] 4. Experimental steps

[0148] Sample addition mode: 25uL sample + 25uL reagent R1 + 25uL reagent R2 + 175uL photosensitive particle solution.

[0149] The sample arm draws 25uL of sample, and the reagent arm draws 25uL of reagent R1 and 25uL of reagent R2 into the reaction cup in turn. After reacting for a certain period of time at a specific temperature, 175uL of photosensitive particle solution is added to continue the reaction. The optical system collects light signals and analyzes and determines the experimental results.

[0150] 5. Experimental data

[0151]

[0152]

[0153] 6. Explanation and analysis of experimental data

[0154] Short arm coated antibody FG-PEG n -PIIINP coating process, PIIINP coated with antibodies and FITC-PEGn When the molar ratio of -NH2 (molecular weight 1K) was adjusted from 1:50 to 1:60, the optical signal value RLU did not increase significantly, and the P / N (ratio of positive serum to negative serum) of the reagent for different concentrations of recombinant antigens did not change significantly. n -During the PIIINP coating process, PIIINP was coated with antibodies and FITC-PEG n -NH2 (molecular weight 1K) and FITC-PEG n When the molar ratio of -NH2 (molecular weight 20K) was adjusted to 1:10:50, the optical signal value RLU was significantly improved, and the P / N value of the reagent for recombinant antigens of different concentrations was significantly improved.

[0155] 7. Experimental conclusion

[0156] Coated Antibody FG-PEG n -PIIINP, a certain amount of long-arm molecules are coupled between the luminescent particles and the PIIINP-coated antibody, which can significantly improve the sensitivity of the reagent.

[0157] The embodiments of the present application have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A photoinduced chemiluminescence detection method, characterized in that: The method comprises the steps of combining photosensitive particles around luminescent particles to form a plurality of photosensitive particle layers with different layer distances.

2. The method according to claim 1, characterized in that The interlayer spacing of the photosensitive particle layer located at the outermost layer does not exceed the distance of energy transfer between the chemiluminescent paired particles; preferably, the photosensitive particle layer consists of two layers.

3. The method according to claim 1, characterized in that The method comprises the steps of combining the antibody or antigen labeled with a marker with the luminescent particle to form an immune complex through an immune reaction mediated by the target molecule to be detected, wherein the surface of the luminescent particle in the immune complex is connected with a plurality of the markers, and the distances between each marker and the luminescent particle are not exactly the same.

4. The method according to claim 3, characterized in that The steps include mixing the marker-chain molecule-antibody or antigen with the luminescent particle-antibody or antigen; and / or, Mixed markers - antibodies or antigens and luminescent particles - chain molecules - antibodies or antigens; The chain molecules have different lengths.

5. A photochemiluminescence detection reagent, comprising luminescent microspheres, photosensitive microspheres, and labeled antibodies or antigens, characterized in that: The marker for labeling the antibody or antigen is connected to the antibody or antigen by chain molecules of different lengths; and / or the surface of the luminescent particle is coated with the antibody or antigen, and the luminescent particle is connected to the antibody or antigen by chain molecules of different lengths.

6. The method or reagent according to claim 4 or 5, characterized in that The length of the chain molecule includes L1 and L2, L1 is greater than L2, and L1 does not exceed the distance of energy transfer between the chemiluminescent paired particles; preferably, the chain molecule includes one of polyethylene glycol, polylysine and dextran.

7. The method or reagent according to claim 6, characterized in that: In the formula, R is the diameter of the luminescent particles, and r is the diameter of the photosensitive particles; preferably, R=r; further preferably, R is 200-400 nm.

8. The method or reagent according to claim 6, characterized in that The labeled antibody or antigen obtained by combining the chain molecule with a chain length of L1 with the antibody or antigen accounts for 5%-25% of the total amount of the labeled antibody or antigen in the reagent; and / or, The antibodies or antigens connected to the surface of the luminescent particles through the chain molecules with a chain length of L1 account for 5%-25% of the total amount of antibodies or antigens connected to the surface of the luminescent particles.

9. A method for preparing a photochemiluminescence detection reagent, characterized in that: The method comprises the steps of preparing a marker-chain molecule-antibody or antigen; and / or the steps of preparing a luminescent particle-chain molecule-antibody or antigen; the chain molecules have different lengths.

10. The preparation method according to claim 9, characterized in that: The step of preparing the marker-chain molecule-antibody or antigen comprises: S1. combining the chain molecules of different lengths with antibodies or antigens; S2, combining the marker with the chain molecules of different lengths to obtain the marker-chain molecule-antibody or antigen; or, combining the marker combined with the chain molecules of different lengths with an antibody or antigen to obtain the marker-chain molecule-antibody or antigen; and / or, The steps of preparing luminescent particles-chain molecules-antibodies or antigens include: S1', combining the chain molecules of different lengths with antibodies or antigens; S2', combining the luminescent particles with the chain molecules of different lengths to obtain the luminescent particles-chain molecules-antibodies or antigens.

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