A long afterglow homogeneous phase detection method

Abstract

This invention belongs to the technical field of in vitro detection and discloses a long-persistent homogeneous detection method. Through an immune reaction or binding in a homogeneous solution, a long-persistent immune complex is formed and immobilized on the bottom plate of the test container. First, interference or influence caused by non-immune binding is effectively removed by injecting a separation solution. Based on this, long-persistent luminescent surface imaging signal acquisition technology is used to achieve high-quality detection without background, and to simultaneously and conveniently perform rapid detection on multiple samples. The long-persistent homogeneous detection method described in this paper provides a new approach for high signal-to-noise ratio and high-throughput long-persistent homogeneous detection.

Description

Technical Field

[0001] This invention belongs to the technical field of in vitro detection, and specifically relates to a long afterglow homogeneous detection method. Background Technology

[0002] Long-persistent homogeneous detection is a homogeneous assay method for immunoassay analytes. It is based on the distance effect of energy transfer between donor and acceptor microspheres, using the intensity of the long-persistent luminescence signal to indicate the analyte concentration. Related reagents and methods are disclosed in Chinese patent CN202010347677.8. In this method, the donor microspheres encapsulate sensitizer molecules, and the acceptor microspheres encapsulate buffers and luminescent agents. The acceptor microspheres approach the donor microspheres via immune binding or other mechanisms. A light source selectively excites the donor microspheres, which then activate the acceptor microspheres through energy transfer, ultimately emitting a long-persistent luminescence signal. This signal is detected by an optical detector and used for result analysis. The energy transfer efficiency is negatively correlated with distance, with an effective range of approximately 100 nm. Donor and acceptor microspheres that do not bind via immune binding or other mechanisms emit weaker luminescence because the distance exceeds this range. Functionally, the donor microspheres are sensitizer spheres, and the acceptor microspheres are luminescent spheres. The presence and concentration of the target analyte in the sample are determined by detecting the intensity of the long-persistent luminescence signal. Homogeneous detection technology effectively avoids cumbersome steps such as elution and centrifugation. By combining with high-affinity antibodies, it can complete a series of steps from incubation to detection in a short time, achieving highly sensitive testing while reducing operating costs.

[0003] Ideally, when the sensitized and luminescent spheres are not immunoassay-bound, the distance between them is greater than 100 nm, making it difficult for singlet oxygen to effectively transfer energy, thus preventing the generation of a long-persistent luminescence signal. However, in actual detection, factors such as the free diffusion of microspheres or physical adsorption binding can reduce the distance between the sensitized and luminescent spheres to less than 100 nm, or even result in zero-distance contact, still leading to efficient long-persistent luminescence and thus generating spurious signals. Furthermore, in current homogeneous detection methods, complex background signals or spurious interference are difficult to effectively remove, making it impossible to achieve background-free, high signal-to-noise ratio detection.

[0004] Furthermore, current homogeneous detection generally employs a scanning signal acquisition mode, requiring the excitation and signal collection of each sample at each test cup position according to spatial and temporal sequence. Even with fully automated equipment, light source excitation and signal collection must be performed for each test cup position individually, conceptually similar to scanning each cup position one by one. Only one sample is tested per test cycle, and the next sample test must wait for the next cycle to run. Thus, when the number of samples is large, the total testing time is long, which effectively limits throughput. Summary of the Invention

[0005] The purpose of this invention is to provide a long-persistence homogeneous phase detection method and system, which solves the technical problems of background interference and limited throughput in homogeneous phase detection, and achieves high signal-to-noise ratio and high throughput long-persistence homogeneous phase detection.

[0006] In a first aspect, this application provides a method for detecting long afterglow homogeneous phases, which includes the following steps:

[0007] S1. Provide a long afterglow homogeneous detection reagent composition and the sample to be tested;

[0008] S2. At least a portion of the long afterglow homogeneous detection reagent composition is pre-attached to the bottom plate of the test container;

[0009] S3. Add the test sample and the remaining portion of the long afterglow homogeneous detection reagent composition to the test container for incubation;

[0010] S4. Inject the separation liquid to separate the aqueous phase solution away from the bottom plate;

[0011] S5. Irradiate the separated detection solution with excitation light;

[0012] S6. Collect long-afterglow emission signals using a detector;

[0013] S7. Based on the collected long afterglow emission signal values, the target analyte information in the sample to be tested is obtained.

[0014] In this invention, the detection liquid is a liquid obtained by reacting the separation liquid with the sample to be tested and the long-afterglow homogeneous detection reagent composition. In the step of irradiating the separated detection liquid with excitation light, all liquids in the oil phase need to be irradiated with a laser.

[0015] In this invention, a long-persistent immune complex is ultimately formed and immobilized on a substrate through an immune reaction or binding in a homogeneous solution. First, interference or influence from non-immune binding is effectively eliminated through a separation solution injection technique. Based on this, long-persistent luminescent surface imaging signal acquisition technology enables high-quality detection without background, and allows for convenient and rapid simultaneous detection of multiple samples. The method and system of this invention provide a new approach for pioneering high signal-to-noise ratio, high-throughput long-persistent homogeneous detection.

[0016] This invention provides a long-persistence luminescent surface imaging signal acquisition technology, enabling convenient and rapid simultaneous detection of multiple samples. For example, when testing single or multiple samples, an excitation light source and detector are fixed corresponding to the test cup position. The long-persistence luminescence signal is collected after the excitation light is turned off. In this case, the excitation light source is a point light source or a surface light source, preferably a surface light source; the detector is a photomultiplier tube, silicon photodiode, camera, mobile phone, CCD, or EMCCD, preferably a camera, mobile phone, CCD, or EMCCD.

[0017] This invention is also compatible with scanning excitation and signal acquisition. The separation liquid injection method is still used in this detection, retaining its significant advantages in removing interference from non-immune binding and improving the signal-to-noise ratio. For example, when only a single sample is considered for testing, the excitation source and detector are fixed to the test cup position, and the long-afterglow emission signal is collected after the excitation light is turned off. In this case, the excitation source is a point source or a surface source, preferably a point source; the detector is a photomultiplier tube, silicon photovoltaic cell, camera, mobile phone, CCD, or EMCCD, preferably a photomultiplier tube or silicon photovoltaic cell.

[0018] In the long-afterglow homogeneous detection mode, the excitation light source is first turned on by the control module. The excitation light emitted by the light source excites the long-afterglow substance in one or more test cups. The excitation light source is then turned off, and the detector receives the generated long-afterglow emission or takes a picture of the test cup area. The detector transmits this signal or image to the signal processing module, which processes the relevant signal and image according to its internal pre-stored program to obtain the content of the target analyte to be detected.

[0019] The separation liquid is a gel solution or an oil phase liquid; preferably, the separation liquid is an oil phase liquid with a density greater than that of water.

[0020] Preferably, the separation liquid is transparent; the transparent liquid is colorless or light-colored, more preferably colorless.

[0021] Preferably, the oil phase of the separation liquid contains one or more of, for example, dichloromethane, trichloromethane, carbon tetrachloride, perfluorobromoalkane, perfluoroiodoalkane, perfluorinated carbon, iodized oil, and ionic liquid.

[0022] Preferably, the perfluorobromoalkane in the separation liquid is selected from one or more of, for example, 1-bromoheptafluoropropane, 2-bromoheptafluoropropane, 1-bromononafluorobutane, 1-bromoundecafluoropentane, 1-bromotridecylfluorohexane, 1-bromopentadecafluoroheptane, 1-bromoheptafluorooctane, and 1-bromononadecafluorononane.

[0023] Preferably, the perfluoroiodoalkane in the separation liquid is selected from one or more of, for example, perfluoroiodoethane, 1-iodoheptafluoropropane, 2-iodoheptafluoropropane, 2-iodononafluorobutane, 1-iododecylfluoropentane, 1-iodotridecylfluorohexane, 1-iodopentadecafluoroheptane, 1-iodoheptafluorooctane, and 1-iodononadecafluorononane.

[0024] Preferably, the perfluorinated carbon in the separation liquid is selected from one or more of, for example, perfluoropentane, perfluorohexane, hexadecylfluoroheptane, octadecylfluorooctane, perfluorononane, perfluorodecane, perfluorocyclohexane, and perfluoro(methylcyclohexane).

[0025] Preferably, the ionic liquid of the separation solution contains, for example, the structure of Formula 1 and / or Formula 2:

[0026]

[0027] The long-afterglow homogeneous detection reagent composition according to the present invention comprises sensitized spheres and luminescent spheres. A first coupling agent is attached to the surface of the sensitized spheres, and the sensitized spheres generate singlet oxygen upon photoexcitation; a second coupling agent is attached to the surface of the luminescent spheres, and the luminescent spheres react with the singlet oxygen to generate a long-afterglow luminescence signal.

[0028] The first and second conjugates can each form immune complexes with the target analyte. The mass percentage content of both the first and second conjugates can be 0.0006% to 0.1%, preferably 0.001% to 0.05%, based on the total weight of their respective components.

[0029] According to the present invention, the sensitized sphere comprises a carrier microsphere, a donor component, or a combination thereof, while the luminescent sphere comprises a carrier microsphere, an acceptor component, or a combination thereof. The donor component contains a light absorber, and the acceptor component contains a photochemical buffer and a luminescent agent. In the method of the present invention, luminescence, particularly long-afterglow luminescence, is achieved through photochemical energy transfer between the donor and acceptor components. The photochemical buffer can construct a bridge for energy exchange and storage between the luminescent agent and the light absorber, allowing the input excitation light energy to be continuously released in the form of luminescence over a certain period of time, thereby achieving long-afterglow luminescence.

[0030] In this application, light absorbers and luminescent agents are known in the prior art. A light absorber generally refers to a substance that can absorb and capture light energy from natural or artificial light sources. The selection range of light absorbers includes traditional photosensitizers and other energy donor materials. A luminescent agent generally refers to a substance that can ultimately emit energy in the form of light. A luminescent agent can be a luminescent substance capable of producing fluorescence or phosphorescence, etc.

[0031] To utilize the beneficial effects of afterglow materials, particularly improving afterglow intensity and duration, the composition of this invention clearly distinguishes between the luminescent agent and the absorber, allowing each to absorb and release light energy respectively. This, combined with a specifically selected photochemical buffer, achieves an energy utilization pathway involving energy input, energy buffering, and energy output. This also means that, in advantageous embodiments, compounds that structurally possess both absorbing and luminescent groups, thus performing both functions with a single molecule, are not luminescent or absorber according to this invention, and the superior technical effects of this invention are not achieved. On the one hand, such compounds are equivalent to bundling the absorber and luminescent agent along with their properties together, making it impossible to separately adjust the excitation and luminescence properties of the afterglow luminescent reagent. For example, when a compound is selected based on actual excitation and charging needs, the luminescence properties of the reagent are also fixed, and vice versa. On the other hand, such compounds are equivalent to fixing the ratio of absorber to luminescent agent to, for example, 1:1, making it impossible to simultaneously adjust the intensity of light absorption and the level of light emission. Moreover, materials possessing both high-efficiency light absorption and high-efficiency light emission are relatively rare.

[0032] In the combined design of this invention, the photochemical buffer and the luminescent agent can be two independent molecules, or they can be integrated into a single molecule. When the photochemical buffer and the luminescent agent are two molecules, their concentration ratio can be independently adjusted to optimize long-persistent luminescence. When the photochemical buffer and the luminescent agent are integrated into a single molecule, effective energy transfer can still be maintained at very low concentrations, producing detectable long-persistent luminescence. Preferably, the buffer molecule itself does not emit light or emits very weak light; its luminescence properties are activated or it efficiently generates an energy-transferring dark excited state after reacting with singlet oxygen.

[0033] In the luminescent reagents according to the present invention, the selection of absorbers and luminescent agents follows certain rules and standards. Generally, compounds with a large molar absorptivity are selected as absorbers, such as photosensitizers or energy donor dyes; while compounds with higher luminescence quantum efficiency are selected as luminescent agents, such as luminescent dyes. Furthermore, the absorption peak of the absorber should overlap with the emission peak of the luminescent agent as little as possible to avoid the adverse effect of the afterglow luminescence being weakened by absorption by the absorber. In long-afterglow homogeneous reagents containing sensitized spheres and luminescent spheres, when the absorber concentration ratio is too high, the long-afterglow luminescence will be weakened by absorption by the absorber; when the absorber concentration ratio is too low, the absorbed excitation light energy is relatively limited, which will also lead to weak long-afterglow luminescence.

[0034] The inventors of this application have discovered that, from the perspective of improving luminous brightness or luminous signal intensity, the absorber and luminescent agent should advantageously be at least one compound selected from the following different molecular formulas or different structures: porphyrin and phthalocyanine dyes, metal complexes, benzoxene compounds, fluoroboron dipyrrole compounds (BODIPY), quantum dots (QDs), graphene, and derivatives or copolymers of these compounds.

[0035] (1) Light absorber

[0036] Preferably, the light absorber may be selected from porphyrin and phthalocyanine dyes, transition metal complexes, quantum dots (QDs), and derivatives or copolymers of these compounds. These compounds are known to those skilled in the art, and some non-limiting examples of light absorbers are mentioned below.

[0037] The following compounds can be mentioned as examples of porphyrin dyes and their complexes:

[0038]

[0039]

[0040]

[0041] The following are examples of phthalocyanine dyes and their complexes:

[0042]

[0043]

[0044] In the structural formulas of the light-absorbing compounds shown above,

[0045] X represents halogens such as F, Cl, Br, I; and M = metallic elements such as Al, Pd, Pt, Zn, Ga, Ge, Cu, Fe, Co, Ru, Re, Os, etc.

[0046] Various substituents R such as R 1-24 This indicates H, hydroxyl, carboxyl, amino, mercapto, ester, aldehyde, nitro, sulfonic acid, halogen, or alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy, alkylamino, or combinations thereof having 1-50, preferably 1-24, such as 2-14 carbon atoms. Preferably, the above-mentioned group R is such as R 1-24 Each is independently selected from methoxy, ethoxy, dimethylamino, diethylamino, methyl, ethyl, propyl, butyl, tert-butyl, phenyl, or combinations thereof.

[0047] Transition metal complexes that can be used as light absorbers are known, and are preferably complexes of porphyrin and phthalocyanine dyes as shown above.

[0048] Suitable quantum dot materials include, for example, graphene quantum dots, carbon quantum dots, and heavy metal quantum dots.

[0049] Heavy metal quantum dots include, for example, Ag₂S, CdS, CdSe, PbS, CuInS, CuInSe, CuInGaS, CuInGaSe, and InP quantum dots. They can be encased in a shell, forming a core-shell structure. The shell can be one or more of Ag₂S, CdS, CdSe, PbS, CuInS, CuInSe, CuInGaS, and CuInGaSe, or it can be a ZnS layer.

[0050] Preferably, the quantum dots are modified with surface ligands, such as oleic acid, oleylamine, octadecene, octadecylamine, n-dodecyl mercaptan, and combinations thereof. In some more advantageous cases, the ligands on the surface of the quantum dots are partially replaced by molecular structures containing triplet states through ligand exchange strategies, such as carboxyanthracene, carboxy-3-tetraphenyl, carboxy-5-pentaphenyl, aminoanthracene, amino-3-tetraphenyl, amino-5-pentaphenyl, mercaptoanthracene, mercapto-3-tetraphenyl, mercapto-5-pentaphenyl, etc.

[0051] In a more preferred embodiment, the light-absorbing agent is preferably selected from porphyrin and phthalocyanine complexes, quantum dots (QDs), and derivatives of these compounds. Examples include one or more of the following exemplary compounds:

[0052]

[0053]

[0054] In addition, there are quantum dot materials such as graphene quantum dots, CdSe quantum dots, and PbS quantum dots.

[0055] (2) Luminescent agent

[0056] Preferably, the luminescent agent may be selected from iridium complexes, rare earth complexes, benzoxene compounds, fluoroboron dipyrrole compounds (BODIPY), and derivatives and copolymers of these compounds.

[0057] As fluoroboron dipyrrole compounds (BODIPY), the following compounds can be mentioned:

[0058]

[0059]

[0060] As compounds in the benzobenzene class, the following compounds can be mentioned:

[0061]

[0062] In the structural formulas of the luminescent compounds shown above,

[0063] n = an integer greater than or equal to 0, such as 0, 1, 2, and 3;

[0064] Various substituents R such as R 1-16 This indicates H, hydroxyl, carboxyl, amino, mercapto, ester, aldehyde, nitro, sulfonic acid, halogen, or alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy, alkylamino, or combinations thereof having 1-50, preferably 1-24, such as 2-14 carbon atoms. Preferably, the group R is used. 1-16 It is selected from methoxy, ethoxy, dimethylamino, diethylamino, methyl, ethyl, propyl, butyl, tert-butyl, phenyl; or combinations thereof.

[0065] In iridium complexes suitable as luminescent reagents, the ligands can be composed of one or more different combinations of ligands. Structural diagrams and examples of some CN, NN, OO, and ON ligands are shown below (where CN, NN, OO, and ON ligands are simplified structural diagrams, and are highlighted to indicate coordination with iridium atoms Ir via C and N atoms, two N atoms, two O atoms, and O and N atoms, respectively. This representation is familiar and understood by those skilled in the art):

[0066]

[0067] (DMSO is dimethyl sulfoxide)

[0068]

[0069] The CN ligand can have, for example, the following structures:

[0070]

[0071] ON ligands can have structures such as the following:

[0072]

[0073] NN ligands can have structures such as the following:

[0074]

[0075] Rare earth complexes used as luminescent agents can have structures such as those where the central atom is a lanthanide element, and the ligands are coordinated to the central atom by O or N. Common central atom types include Eu, Tb, Sm, Yb, Nd, Dy, Er, Ho, and Pr. The coordination numbers of these rare earth complexes are approximately 3 to 12, preferably 6 to 10. In practical rare earth complexes, the types of ligands, the number of each ligand, and the total coordination number can vary. For examples of rare earth complexes and their ligands, see, for instance, Jean-Claude G. Bünzli's review paper, Coord. Chem. Rev., 2015, 293-294, 19-47.

[0076] In a more preferred embodiment, the luminescent agent is selected from iridium complexes, rare earth complexes, fluoroboron dipyrrole compounds (BODIPY), perylene, and derivatives of these compounds. Examples include one or more of the following exemplary compounds:

[0077]

[0078] (3) Photochemical buffer

[0079] The primary function of photochemical buffers is the conversion of photochemical energy. Unlike luminescent agents, whose main function is luminescence, buffer molecules themselves do not emit light or emit very weak light, and their molecular structures generally do not contain directly luminescent groups or conjugated structures. In particular, the photochemical buffers according to the present invention are distinguished from luminescent or absorbent agents, especially those luminescent or absorbent substances listed in this invention. In photochemical reactions, through addition, rearrangement, or bond-breaking reaction steps, the energy extraction process of transitions between energy levels is activated.

[0080] In particular, the inventors have discovered that certain caching compound compounds are especially suitable for preparing luminescent reagents with stable and long-lasting luminescence properties. Caching compounds particularly suitable for this invention are selected from the following structural formulas (Formula I):

[0081]

[0082] in,

[0083] G and T are heteroatoms selected from O, S, Se and N;

[0084] R1' and R2', and R4' to R8', are each independently selected from H, hydroxyl, carboxyl, amino, mercapto, ester, nitro, sulfonic acid, halogen, amide, or alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, aryl, aralkyl, heteroaryl or heteroaryl having N, O, or S, diphenylamino, or combinations thereof, wherein the aryl, aralkyl, heteroaryl, or heteroaryl optionally has one or more substituents L; and

[0085] L is selected from hydroxyl, carboxyl, amino, mercapto, ester, nitro, sulfonic acid, halogen, amide, or alkyl, alkenyl, alkynyl, alkoxy, and alkylamino groups having 1-50, preferably 1-24, such as 2-14 or 6-12 carbon atoms, or combinations thereof;

[0086] And R3' is a phenyl group, an electron-withdrawing group, or an aryl group containing an electron-withdrawing group.

[0087] In the context of this application, "aryl" refers to a group or ring formed from aromatic compounds, distinct from aliphatic compounds, which are directly connected to another structural group or fused to another ring structure via one or more single bonds, thus distinguishing them from groups connected to another structural group via spacers such as "aralkyl," "aryloxy," or "aryl ester." Similarly, "heteroaryl" also applies, which can be considered as a group formed by replacing the ring carbon atom on an aryl group with a heteroatom N, S, Se, or O, or by replacing the carbon atom on an aliphatic ring such as a cycloalkene with said heteroatom. Furthermore, unless otherwise indicated, "aryl" or "heteroaryl" also includes aryl or heteroaryl groups substituted or fused with aryl or heteroaryl groups, such as biphenyl, phenylthiophene, or benzothiazolyl. Additionally, "aryl" or "heteroaryl" may also include groups formed from aromatic or heteroaromatic compounds having functional groups such as ether or carbonyl groups, such as anthrone, diphenyl ether, or thiazolyl. Advantageously, the "aryl" or "heteroaryl" according to the invention has 4 to 30, more preferably 5 to 24, for example 6 to 14 or 6 to 10 carbon atoms. The term "fused" means that the two aromatic rings share a common edge.

[0088] In the context of this application, the terms "alkyl," "alkoxy," or "alkathioyl" refer to a straight-chain, branched, or cyclic saturated aliphatic hydrocarbon group connected to other groups by single bonds, oxygen groups, or thioyl groups, preferably having 1-50, more preferably 1-24, such as 1-18 carbon atoms. The terms "alkenyl" or "alkynyl" refer to a straight-chain, branched, or cyclic unsaturated aliphatic hydrocarbon group having one or more C-C double or triple bonds, preferably having 2-50, more preferably 2-24, such as 4-18 carbon atoms.

[0089] In the context of this application, the term "alkylamino" refers to one or more alkyl-substituted amino groups, including monoalkylamino or dialkylamino groups, such as methylamino, dimethylamino, diethylamino, dibutylamino, etc.

[0090] In the context of this application, the term "halogen" includes fluorine, chlorine, bromine, and iodine.

[0091] In the context of this application, the term "electron-withdrawing group" is understood to be a group that reduces the electron cloud density of an aromatic or heteroaromatic ring when it replaces a hydrogen atom on the ring. Such groups are widely known in the field of chemistry. Preferably, in this invention, the electron-withdrawing group is selected from nitro, halogen, haloalkyl, sulfonic acid, cyano, acyl, carboxyl, and / or combinations thereof.

[0092] Furthermore, in the context of this application, the selected groups in the definitions of the various substituents listed can combine with each other to form new substituents that conform to the valence bond principle. This means that, for example, C1-C6 alkyl ester vinyl groups (C1-6 alkyl-OC(=O)-C=C-) formed by the combination of alkyl, ester and vinyl groups are also in the definition of the relevant substituents.

[0093] In a preferred embodiment, the ring portion Can be selected

[0094] In a preferred embodiment, R1' and R2' and R4' to R8' are each independently selected from alkyl, alkoxy, alkylamino, or aryl groups or combinations thereof having 1-18, preferably 1-12, more preferably 1-16 carbon atoms, wherein the aryl group may be substituted or unsubstituted by one or more groups L and is preferably a phenyl group substituted or unsubstituted by one or more groups L.

[0095] Preferably, L is selected from hydroxyl, sulfonic acid, halogen, nitro, straight-chain or branched alkyl, alkoxy, alkylamino, amino, or combinations thereof having 1-12, more preferably 1-6 carbon atoms.

[0096] More preferably, the groups R1' and R2' and R4' to R8' are selected from methoxy, ethoxy, dimethylamino, diethylamino, dibutylamino, methyl, ethyl, propyl, butyl, tert-butyl, or combinations thereof.

[0097] More preferably, group R3' is selected from electron-withdrawing groups or aryl groups containing electron-withdrawing groups, wherein the electron-withdrawing groups are preferably nitro, cyano, halogen, haloalkyl, and / or combinations thereof. Accordingly, the aryl group containing the electron-withdrawing group preferably comprises an aryl group having one or more substituents selected from nitro, cyano, halogen, and / or haloalkyl on the ring, preferably phenyl, such as fluorophenyl or perfluorophenyl.

[0098] In a particularly preferred embodiment, the photochemical buffer is selected from the following compounds:

[0099]

[0100]

[0101]

[0102] In the long-afterglow homogeneous detection reagent of the present invention, one type of sensitized spheres and luminescent spheres are dispersed in a liquid phase reagent, while the dye component of the other type of spheres or the luminescent spheres is pre-attached to a substrate. For example, when sensitized spheres are dispersed in a liquid phase reagent, the acceptor component of the luminescent spheres or the luminescent spheres is pre-attached to the substrate; similarly, when luminescent spheres are dispersed in a liquid phase reagent, the donor component of the sensitized spheres or the sensitized spheres is pre-attached to the substrate. The substrate is the substrate of a homogeneous testing container, such as the substrate of a sample testing cuvette or a sample testing cup.

[0103] The method of pre-attaching the luminescent or sensitized spheres to the substrate includes connecting the luminescent or sensitized spheres to the substrate through interactions such as chemical bonds, biological binding, and immune binding; doping the luminescent or sensitized spheres in the substrate; or doping the luminescent or sensitized spheres in a transparent film and fixing them to the surface of the substrate.

[0104] The method of pre-attaching the acceptor component in the luminescent sphere or the donor component in the sensitized sphere to the substrate includes doping the acceptor component in the luminescent sphere or the donor component in the sensitized sphere into the substrate, or doping the acceptor component in the luminescent sphere or the donor component in the sensitized sphere into a transparent film and fixing it to the surface of the substrate.

[0105] The method of doping the sensitized spheres, luminescent spheres, donor components, or acceptor components into a substrate or transparent film includes blending integral molding and post-doping. The post-doping method includes pre-preparing a substrate or transparent film, reacting the substrate or transparent film with a swelling agent to induce swelling, adding sensitized spheres, luminescent spheres, donor components, or acceptor components, removing the swelling agent, and then curing.

[0106] The substrate and transparent film doped with the sensitized spheres or donor components are connected to a first coupling agent on their surfaces; the substrate and transparent film doped with the luminescent spheres or acceptor components are connected to a second coupling agent on their surfaces.

[0107] The substrate and the transparent film surface are connected to the first or second coupling agent via chemical bonds. For example, the substrate and the transparent film surface contain carboxyl, aldehyde, or epoxy groups, and the first or second coupling agent contains amino groups. The carboxyl, aldehyde, or epoxy groups react with the amino groups to form chemical bonds, thereby achieving the connection.

[0108] In addition to the necessary and preferred ingredients and components as described above, the long afterglow homogeneous detection reagent composition according to the present invention may also contain other additive components commonly used in immunoassays, such as reagents for diluting samples, reagents for maintaining system stability, etc.

[0109] Other components include, for example, salts, stabilizers, signal amplification components, surfactants, water, preservatives, nucleic acids, peptides, and pH adjusters. Optionally, such components may also include diluents, such as at least one of buffer PB, PBS, PBST, BBS, MES, Tris, TES, and HEPES, which can hemolyze and dilute whole blood samples and control the pH, salt concentration, etc., of the reaction system.

[0110] In a preferred embodiment, the sensitized sphere or the donor component in the sensitized sphere is pre-configured and fixed on the bottom plate of the test container, and the luminescent sphere is disposed in an aqueous reagent.

[0111] In a preferred embodiment, the luminescent sphere or the receptor component in the luminescent sphere is pre-configured and fixed on the bottom plate of the test container, and the sensitization sphere is disposed in an aqueous reagent.

[0112] When the reagent composition is formulated into a kit, these other components may optionally be combined separately with the reagents as described above to formulate and dispense into different reagent packages in the kit.

[0113] In some preferred embodiments, the sample to be tested includes, for example, whole blood, urine, peripheral blood, serum, plasma, body fluids, and / or cerebrospinal fluid. The information includes the type, concentration, pH value, and composition of the target analyte.

[0114] In some preferred embodiments, thorough mixing is typically performed to obtain a homogeneous solution system, during which the target analyte undergoes an immunoreaction with the first and second conjugates. The mixing methods include magnetic stirring, oscillation, and shaking. The incubation time is generally selected to be between 1 and 10 minutes.

[0115] In some preferred embodiments, advantageously, the wavelength of the excitation light used for irradiation in step S5 is selected from the range of 365-1532 nm. More preferably, the wavelength of the excitation light is 1064 nm, 980 nm, 915 nm, 808 nm, 785 nm, 830 nm, 680 nm, 630 nm, 532 nm, 488 nm, 450 nm, 405 nm, or 365 nm.

[0116] Preferably, the excitation light can be turned off after an irradiation time of 0.1 ms to 5 s. Since the detection reagent composition of the present invention contains a long-persistent luminescent material, it can continuously emit a long-persistent signal for a period of time after the light source is turned off.

[0117] Advantageously, in the above steps, long afterglow emission is generated after irradiation with excitation light, the center wavelength of the long afterglow emission being X, wherein X is selected from 400-800 nm, more preferably X is selected from 645 nm, 615 nm, 545 nm or 450 nm.

[0118] In step S6, the collection time period is set to 0.001s to 300s, preferably 0.01s to 60s. Based on the mechanism of long-persistent luminescence and the separation effect of step S4, the background signal of the detection system is effectively eliminated, and the detection signal can continue for a period of time. Therefore, increasing the collection time can enhance and amplify the detection signal and improve the signal-to-noise ratio of the detection.

[0119] In the specific detection process, the excitation source can be a laser with a corresponding wavelength (such as the preferred wavelength range mentioned above). The same or different detectors can be used to simultaneously or separately detect the long-persistence signal values, as long as they have the ability to collect the long-persistence signal described above. For example, when using different detectors, the first detector can be a silicon photodiode detector, and the second detector can be a photomultiplier tube detector. In particular, long-persistence emission signals of different wavelengths generated by different long-persistence compositions can be collected by adding a filter in front of the detector, thereby enabling the joint detection of more items in the same detection process.

[0120] In an advantageous embodiment, the detection step is based on long-persistence emission wide-field imaging signal acquisition technology, which allows for convenient and rapid simultaneous detection of multiple samples. The excitation source is a point source or a surface source, preferably a surface source; the detector is a photomultiplier tube array, a silicon photodiode array, a camera, a mobile phone, a CCD, or an EMCCD, preferably a camera, a mobile phone, a CCD, or an EMCCD. By utilizing the photogrammetric wide-field imaging detection mode, signal images containing multiple test containers can be obtained in a single imaging session, thereby efficiently increasing the detection throughput.

[0121] Beneficial Effects: In this invention, the final long-afterglow immune complex is fixed on the bottom plate of the test container, facilitating subsequent separation of interfering substances. During the separation process, a high-density oil-phase separation solution is injected, using gravity to isolate the aqueous solution in contact with the bottom plate to the upper part of the container, achieving natural separation. This eliminates interference from non-immune binding without complex steps. The high-density oil-phase separation solution settles to the bottom of the container, providing an oil-phase environment for the long-afterglow immune complex fixed on the bottom plate, which is beneficial for enhancing the long-afterglow luminescence signal. Based on this, long-afterglow luminescence surface imaging signal acquisition technology enables high-quality detection without background and allows for convenient and rapid simultaneous detection of multiple samples. The method and system of this invention provide a new approach for high signal-to-noise ratio and high-throughput homogeneous long-afterglow detection. Attached Figure Description

[0122] The preferred embodiments will be described below in a clear and easy-to-understand manner, with reference to the accompanying drawings, to further explain the above-mentioned characteristics, technical features, advantages, and implementation methods of a long afterglow homogeneous detection method and system.

[0123] Figure 1 This is a schematic diagram of the long afterglow homogeneous detection mechanism under conventional ideal conditions;

[0124] Figure 2 This is a schematic diagram illustrating the background or interference signal sources in actual practice for long-persistent homogeneous phase detection.

[0125] Figure 3 This is a schematic diagram of the long afterglow immune complex and the distribution of each component formed in the embodiments of the present invention. Figures a / c and b / d represent the cases of square and circular base plates, respectively. Figures a / b and c / d represent the cases of sensitized spheres distributed in aqueous solution and luminescent spheres distributed in aqueous solution, respectively.

[0126] Figure 4 This is a schematic diagram of the long afterglow immune complex formed in the test container according to an embodiment of the present invention, wherein Figure a represents a sample test cuvette and Figure b represents a sample test cup.

[0127] Figure 5 This is a schematic diagram of an embodiment of the present invention in which a separation solution is added after the formation of a long afterglow immune complex to separate the aqueous phase solution away from the bottom plate. The separation solution, which is immiscible with the aqueous phase, discharges the aqueous phase to the top of the test container due to gravity.

[0128] Figure 6 This is a schematic diagram of the long-persistence emission wide-field imaging signal acquisition technology in an embodiment of the present invention;

[0129] Figure 7This is a schematic diagram of an image obtained from long afterglow surface imaging signal acquisition in an embodiment of the present invention, and the signal portion corresponding to a single test container therein;

[0130] Figure 8 This is a schematic diagram of the device for automatically adding separation liquid to multiple test containers in an embodiment of the present invention;

[0131] Figure 9 This is an illustration of a functionalized 96-well plate in an embodiment of the present invention, wherein the bottom plate of each well integrates a light-emitting film component of long afterglow related components;

[0132] Figure 10 This is a diagram of a detachable functionalized 12-well test container in an embodiment of the present invention, which can be assembled on a 96-well plate support.

[0133] Explanation of reference numerals: 1-luminescent membrane, 2-sensitized membrane, 3-sensitized sphere, 31-first coupling agent, 4-luminescent sphere, 41-second coupling agent, 5-target analyte, 6-test container, 7-solution, 71-original solution, 72-separation solution, 8-light source, 9-detector, 10-test container tray, 11-area imaging signal acquisition image, 111-signal distribution image of a single test cup, 12-separation solution sample tray, 121-separation solution sample well. Detailed Implementation

[0134] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the specific implementation methods of the present invention will be described below with reference to the accompanying drawings. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings and other implementation methods can be obtained based on these drawings without any creative effort.

[0135] Example 1

[0136] like Figure 1 As shown, in a conventional, ideal homogeneous detection system, background or interference signals are generally ignored. However, in a real-world homogeneous detection system, sources of background or interference signals exist (see schematic diagram). Figure 2 (As shown), and it is usually difficult to eliminate. To illustrate and analyze this problem, which is common in homogeneous detection, an experimental explanation is provided in this embodiment.

[0137] Taking Eu complex (Eu-Complex-1 molecule) as the luminescent agent and SOF molecule as the photochemical buffer as an example, a transparent film containing acceptor components (such as...) was prepared. Figure 3(The luminescent film shown in b) was prepared by dissolving the Eu complex and SOF in tetrahydrofuran, where the concentration of the Eu complex was 50 mM and the concentration of SOF was 3 mM. 0.1 g of polyurethane particles were added to 1 mL of the solution and stirred thoroughly to dissolve and mix the particles. 0.1 mL of the resulting solution was added to a cuvette, dried in the dark, and cured to form a film on a substrate. The film was then surface-treated using plasma, and a second coupling agent was coupled to the surface of the film. The film was then sealed using BSA to obtain the desired result. Figure 4 The illustration shows a test container (e.g., a circular cuvette) containing a luminescent membrane. Correspondingly, the sensitized spheres are carboxylated polystyrene microspheres containing the absorbent PdPc, with a first conjugate coupled to their surface. The first and second conjugates are two specific antibodies against serum amyloid A (SAA), respectively.

[0138] The structural formulas of Eu-Complex-1, SOF, and PdPc mentioned above are shown below:

[0139]

[0140] The sensitized beads were dispersed in the aqueous phase solution of the detection reagent, and then the target analyte, serum amyloid A (SAA, 20 mg / L), was added and mixed thoroughly. 0.2 mL of the resulting aqueous mixture was added to the cuvette containing the luminescent membrane, and then incubated for 10 min. A schematic diagram of the resulting cuvette and solution system is shown below. Figure 4 As shown (e.g., a round cuvette), for ease of explanation, it is labeled as Test Example 1.

[0141] Repeat the experiment of Test Example 1 above, except that the target analyte SAA is replaced with a sample diluent. The resulting cuvette and solution system are labeled Test Example 2.

[0142] Repeat the experiment in Test Example 1 above, except that after completion, 0.2 mL of the separation solution 1-bromononefluorononane is added. The separation solution is immiscible with the aqueous phase and sinks to the bottom of the cuvette due to gravity, causing the aqueous phase solution to separate away from the bottom plate (see schematic diagram). Figure 5 (As shown). Therefore, the obtained cuvette and solution system are labeled as Test Example 3.

[0143] Repeat the experiment of Test Example 3 above, except that the target analyte SAA is replaced with a sample diluent. The resulting cuvette and solution system are labeled Test Example 4.

[0144] The samples were excited by irradiating them with 730 nm excitation light, and then the long-persistent luminescence signal was collected. The test conditions were kept consistent for all four test cases. The signal values ​​for Test Case 1, Test Case 2, Test Case 3, and Test Case 4 were 92000, 15200, 78000, and 500, respectively. The signal ratio of Test Case 1 (positive) to Test Case 2 (negative, equivalent to background) was 6, and the signal ratio of Test Case 3 (positive) to Test Case 4 (negative, equivalent to background) was 156. For Test Case 2, due to factors such as the free diffusion of microspheres or physical adsorption, the distance between the sensitized spheres and the luminescent film may be less than 100 nm, or even reach zero-distance contact, resulting in efficient long-persistent luminescence and generating pseudo-signals. These factors are the main sources of the background signal.

[0145] Based on the above experimental analysis, it can be seen that the injection of the separation liquid greatly reduced the background interference (the signal dropped from 15200 to 500), and the separation liquid achieved a good separation effect, significantly improving the detection signal-to-noise ratio (from 6 to 156).

[0146] Example 2

[0147] Taking BODIPY (BD-1 molecule) as the luminescent agent and SO molecule as the photochemical buffer as an example, a transparent film containing acceptor components (such as...) was prepared. Figure 3 (The luminescent film shown in Figure a). BODIPY and SO were dissolved in toluene, with BODIPY concentration at 10 mM and SO concentration at 2 mM. 0.1 g of polypropylene granules were added to 1 mL of the solution and stirred thoroughly to dissolve and mix the granules. 0.1 mL of the resulting solution was added to a cuvette, dried in the dark, and cured to form a film on a substrate. Surface treatment was then performed using plasma. A second coupling agent was then coupled to the surface of the film, and BSA was used for sealing to obtain the desired result. Figure 4 The illustration shows a test container (e.g., a square cuvette) containing a luminescent membrane. Correspondingly, the sensitized spheres are carboxylated polystyrene microspheres containing the light-absorbing agent PdPc, with a first conjugate coupled to their surface. The first and second conjugates are two specific antibodies against C-reactive protein (CRP), respectively.

[0148] The chemical structures of BD-1, SO, and PdPc mentioned above are shown below:

[0149]

[0150]

[0151] The sensitized beads were dispersed in the aqueous phase solution of the detection reagent, and then the target analyte, C-reactive protein (CRP, 20 mg / L), was added and mixed thoroughly. 0.2 mL of the resulting aqueous mixture was added to the cuvette containing the luminescent membrane, and then incubated for 10 min. A schematic diagram of the resulting cuvette and solution system is shown below. Figure 4 As shown (e.g., a square cuvette), for ease of explanation, it is labeled as Test Example 1.

[0152] Repeat the experiment of Test Example 1 above, except that the target analyte CRP is replaced with a sample diluent. The resulting cuvette and solution system are labeled Test Example 2.

[0153] Repeat the experiment of Test Example 1 above, except that 0.3 mL of the separation solution iodine oil is added after the experiment is completed. The separation solution is immiscible with the aqueous phase and sinks to the bottom of the cuvette due to gravity, causing the aqueous phase solution to separate away from the bottom plate. The cuvette and solution system obtained are thus labeled as Test Example 3.

[0154] Repeat the experiment of Test Example 3 above, except that the target analyte CRP is replaced with a sample diluent. The resulting cuvette and solution system are labeled Test Example 4.

[0155] The samples were excited by irradiating them with 730 nm excitation light, and then the long-persistent luminescence signal was collected. The test conditions were kept consistent for all four test cases. The signal values ​​for Test Case 1, Test Case 2, Test Case 3, and Test Case 4 were 41000, 5700, 35000, and 300, respectively. The signal ratio of Test Case 1 (positive) to Test Case 2 (negative, equivalent to background) was 7, and the signal ratio of Test Case 3 (positive) to Test Case 4 (negative, equivalent to background) was 117. For Test Case 2, due to factors such as the free diffusion of microspheres or physical adsorption, the distance between the sensitized spheres and the luminescent film may be less than 100 nm, or even reach zero-distance contact, resulting in efficient long-persistent luminescence and generating pseudo-signals. These factors are the main sources of the background signal.

[0156] Based on the above experimental analysis, it can be seen that the injection of the separation liquid greatly reduced the background interference (the signal dropped from 5700 to 300), and the separation liquid achieved a good separation effect, significantly improving the detection signal-to-noise ratio (from 7 to 117).

[0157] Example 3

[0158] The experimental procedures of Example 1 were repeated, with the difference being in the method of preparing the luminescent film: the Plasma surface treatment process in Example 1 was replaced with a surface coating process for a functionalized coating. In this example, the surface coating process for the functionalized coating involves coating and functionalizing the transparent film surface. The coating also prevents dye leakage into the solution outside the polymer. The transparent film surface may contain a polystyrene coating encapsulated with dextran. The coating can be functionalized with -NH2, -SH, -COH, -COOH, and / or -CO-OR groups, through which it is coupled to a second coupling agent.

[0159] The sample was excited by irradiating it with 730 nm excitation light, and then the long-persistent luminescence signal was collected. The test conditions were kept consistent for the four test cases obtained in this embodiment. The signal values ​​of Test Case 1, Test Case 2, Test Case 3, and Test Case 4 were 61000, 5500, 56000, and 400, respectively. The signal ratio of Test Case 1 (positive) to Test Case 2 (negative, equivalent to background) was 11, and the signal ratio of Test Case 3 (positive) to Test Case 4 (negative, equivalent to background) was 140. For Test Case 2, due to factors such as the free diffusion of microspheres or physical adsorption, the distance between the sensitized sphere and the luminescent film may be less than 100 nm, or even reach zero-distance contact, resulting in efficient long-persistent luminescence and generating pseudo-signals. These factors are the main sources of the background signal.

[0160] Based on the above experimental analysis, it can be seen that the injection of the separation liquid greatly reduced the background interference (the signal dropped from 5500 to 400), and the separation liquid achieved a good separation effect, significantly improving the detection signal-to-noise ratio (from 11 to 140).

[0161] Example 4

[0162] The experimental procedures of Example 2 were repeated, with the difference being in the method of preparing the luminescent film: the Plasma surface treatment process in Example 2 was replaced with a surface coating process for a functionalized coating. In this example, the surface coating process for the functionalized coating involves coating and functionalizing the transparent film surface, which also prevents dye leakage into the solution outside the polymer. The transparent film surface may contain a polystyrene coating encapsulated with dextran. The coating can be functionalized with -NH2, -SH, -COH, -COOH, and / or -CO-OR groups, through which it is coupled to a second coupling agent.

[0163] The sample was excited by irradiating it with 730 nm excitation light, and then the long-persistent luminescence signal was collected. The test conditions were kept consistent for the four test cases obtained in this embodiment. The signal values ​​of Test Case 1, Test Case 2, Test Case 3, and Test Case 4 were 29000, 4100, 26000, and 200, respectively. The signal ratio of Test Case 1 (positive) to Test Case 2 (negative, equivalent to background) was 7, and the signal ratio of Test Case 3 (positive) to Test Case 4 (negative, equivalent to background) was 130. For Test Case 2, due to factors such as the free diffusion of microspheres or physical adsorption, the distance between the sensitized sphere and the luminescent film may be less than 100 nm, or even reach zero-distance contact, resulting in efficient long-persistent luminescence and generating pseudo-signals. These factors are the main sources of the background signal.

[0164] Based on the above experimental analysis, it can be seen that the injection of the separation liquid greatly reduced the background interference (the signal dropped from 4100 to 200), and the separation liquid achieved a good separation effect, significantly improving the detection signal-to-noise ratio (from 7 to 130).

[0165] Example 5

[0166] Taking PdPc molecules as the light absorber as an example, a transparent film containing a donor component (such as...) is prepared. Figure 3 (The sensitized membrane shown in d). PdPc was dissolved in toluene at a concentration of 10 μM. 0.1 g of polystyrene particles were added to 1 mL of the solution and stirred thoroughly to dissolve and mix the particles. The surface of the polystyrene particles may include carboxyl, amino, carboxylic acid ester, halogen, ester, hydroxyl, carbamide, aldehyde, chloromethyl, sulfur oxide, nitrogen oxide, epoxy, and / or toluenesulfonyl functional groups, which are used to couple with the first conjugate. 0.1 mL of the resulting solution was added to a cuvette, dried in the dark, and cured to form a film on a substrate. The first conjugate was then coupled to the surface of the film and blocked with complexin to obtain the desired result. Figure 4 The diagram shows a test container containing a luminescent membrane. Correspondingly, the luminescent spheres are aldehyde-modified polystyrene microspheres containing an Eu complex (Eu-Complex-1 molecule) as the luminescent agent and an SOF molecule as a photochemical buffer, with a second conjugate coupled to their surface. The first and second conjugates are two specific antibodies against serum amyloid A (SAA), respectively.

[0167] The sensitized beads were dispersed in the aqueous phase solution of the detection reagent, and then the target analyte, serum amyloid A (SAA, 20 mg / L), was added and mixed thoroughly. 0.2 mL of the resulting aqueous mixture was added to the cuvette containing the luminescent membrane, and then incubated for 10 min. A schematic diagram of the resulting cuvette and solution system is shown below. Figure 4As shown, for ease of explanation, it is labeled as Test Example 1.

[0168] Repeat the experiment of Test Example 1 above, except that the target analyte SAA is replaced with a sample diluent. The resulting cuvette and solution system are labeled Test Example 2.

[0169] Repeat the experiment of Test Example 1 above, except that after completion, 0.4 mL of the separation solution perfluorooctane is added. The separation solution is immiscible with the aqueous phase and sinks to the bottom of the cuvette due to gravity, causing the aqueous phase solution to separate away from the bottom plate. The cuvette and solution system obtained are thus labeled as Test Example 3.

[0170] Repeat the experiment of Test Example 3 above, except that the target analyte SAA is replaced with a sample diluent. The resulting cuvette and solution system are labeled Test Example 4.

[0171] The samples were excited using 730 nm excitation light, and then long-persistent luminescence signals were collected. The test conditions were kept consistent for all four test cases. The signal values ​​for Test Case 1, Test Case 2, Test Case 3, and Test Case 4 were 22000, 6100, 16000, and 400, respectively. The signal ratio of Test Case 1 (positive) to Test Case 2 (negative, equivalent to background) was 4, and the signal ratio of Test Case 3 (positive) to Test Case 4 (negative, equivalent to background) was 40. For Test Case 2, due to factors such as free diffusion of the microspheres or physical adsorption, the distance between the luminescent spheres and the sensitized film may be less than 100 nm, or even reach zero-distance contact, resulting in efficient long-persistent luminescence and generating pseudo-signals. These factors are the main sources of the background signal.

[0172] Based on the above experimental analysis, it can be seen that the injection of the separation liquid greatly reduces background interference (signal value drops from 6100 to 400), and the separation liquid plays a good role in achieving separation, significantly improving the detection signal-to-noise ratio (from 4 to 40).

[0173] Example 6

[0174] Using Eu-Complex-2 molecules as the luminescent agent and SOCF molecules as the photochemical buffer, a luminescent membrane containing receptor components was prepared. The matrix of the luminescent membrane was a polystyrene film, in which the contents of Eu-Complex-2 molecules and SOCF molecules were 8.5% and 0.1% respectively, based on the total mass of the luminescent membrane. The polystyrene film contained carboxyl groups, and a second conjugate was coupled to the surface of the luminescent membrane using these functional groups and then sealed. The luminescent membrane was then attached to the bottom plate of a test container to obtain a test container containing the luminescent membrane. Correspondingly, the sensitizing spheres were carboxylated polystyrene microspheres containing the light-absorbing agent SiPc, in which the content of SiPc molecules was 1.5% based on the total mass of the sensitizing spheres. A first conjugate was coupled to the surface of the sensitizing spheres. The first and second conjugates were two specific antibodies against procalcitonin (PCT), respectively.

[0175] The structural formulas of Eu-Complex-2, SOCF, and SiPc mentioned above are shown below:

[0176]

[0177] The sensitized beads were dispersed in the aqueous phase solution of the detection reagent, and then the target analyte, procalcitonin (PCT, 2 ng / L), was added and mixed thoroughly. 0.2 mL of the resulting aqueous mixture was added to the cuvette containing the luminescent membrane, and then incubated for 10 min. The resulting cuvette and solution system are designated as Test Example 1 for ease of explanation.

[0178] Repeat the experiment of Test Example 1 above, except that the target analyte PCT is replaced with a sample diluent. The resulting cuvette and solution system are labeled Test Example 2.

[0179] Repeat the experiment of Test Example 1 above, except that after completion, 0.2 mL of the separation solution 1-bromopentafluoroheptane is added. The separation solution is immiscible with the aqueous phase and sinks to the bottom of the cuvette due to gravity, causing the aqueous phase solution to separate away from the bottom plate. The cuvette and solution system obtained are thus labeled as Test Example 3.

[0180] Repeat the experiment of Test Example 3 above, except that the target analyte PCT is replaced with a sample diluent. The resulting cuvette and solution system are labeled Test Example 4.

[0181] The samples were excited by irradiating them with 680 nm excitation light, and then the long-persistent luminescence signal was collected. The test conditions were kept consistent for all four test cases. The signal values ​​for Test Case 1, Test Case 2, Test Case 3, and Test Case 4 were 105000, 23600, 81900, and 700, respectively. The signal ratio of Test Case 1 (positive) to Test Case 2 (negative, equivalent to background) was 4, and the signal ratio of Test Case 3 (positive) to Test Case 4 (negative, equivalent to background) was 117. For Test Case 2, due to factors such as the free diffusion of microspheres or physical adsorption, the distance between the sensitized spheres and the luminescent film may be less than 100 nm, or even reach zero-distance contact, resulting in efficient long-persistent luminescence and generating pseudo-signals. These factors are the main sources of the background signal.

[0182] Based on the above experimental analysis, it can be seen that the injection of the separation liquid greatly reduced the background interference (the signal dropped from 23600 to 700), and the separation liquid achieved a good separation effect, significantly improving the detection signal-to-noise ratio (from 4 to 117).

[0183] Example 7

[0184] Example 6 was repeated, except that the separating solution was replaced with 1-iodoheptafluorooctane. The results showed that the separating solution achieved a good separation effect, and the injection of the separating solution greatly reduced background interference and significantly improved the detection signal-to-noise ratio (from 4 to 115).

[0185] Example 8

[0186] Example 6 was repeated, except that the separating liquid was replaced with 1-bromotridecylfluorohexane. The results showed that the separating liquid achieved a good separation effect, and the injection of the separating liquid greatly reduced background interference and significantly improved the detection signal-to-noise ratio (from 4 to 121).

[0187] Example 9

[0188] Example 6 was repeated, except that the separating fluid was replaced with 1-iodoheptafluorooctane. The results showed that the separating fluid achieved a good separation effect, and the injection of the separating fluid greatly reduced background interference and significantly improved the detection signal-to-noise ratio (from 4 to 105).

[0189] Example 10

[0190] Example 6 was repeated, except that the separating liquid was replaced with the ionic liquid of Formula 1. The results showed that the separating liquid achieved a good separation effect, and the injection of the separating liquid greatly reduced background interference and significantly improved the detection signal-to-noise ratio (from 4 to 85).

[0191] Example 11

[0192] Example 6 was repeated, except that the separating liquid was replaced with the ionic liquid of Formula 2. The results showed that the separating liquid achieved a good separation effect, and the injection of the separating liquid greatly reduced background interference and significantly improved the detection signal-to-noise ratio (from 4 to 98).

[0193] Example 12

[0194] First, the experiment of Example 6 was repeated to prepare multiple cuvette test containers containing luminescent films. Four cuvettes were placed close together on a sample holder (as shown in the illustration). Figure 6 (As shown in the diagram), the long-persistence compositions in all these samples are excited and energized using a light source. The excitation light source is then turned off, and a wide-field imaging acquisition method is used to simultaneously collect long-persistence signals from all cuvettes. In this embodiment, the long-persistence signal acquisition device is a camera, positioned below the bottom of the cuvettes, enabling area imaging of the wide field of view formed by all cuvettes. The imaging exposure time can be set from 10ms to 300s; in this embodiment, a 10s exposure is selected to collect the long-persistence emission signal. By processing and analyzing the acquired images, an intensity distribution map of the long-persistence emission signal can be obtained. (See diagram) Figure 7 As shown, in the image obtained by imaging this surface, the luminescence of the region to which each cuvette belongs can be recorded.

[0195] Based on wide-field imaging signal acquisition technology, multiple test cases can be detected simultaneously, and each test case is essentially an independent test without mutual interference. Therefore, the test items for the four cuvettes can be set separately. In this embodiment, the four test cases were set to the same PCT detection item, and the target analyte concentration in the samples was 2 ng / L. The results show that all four tests can be performed, and the target analyte concentration information obtained from the analysis of the four tests is basically consistent, with a deviation of less than 10%. This demonstrates that the long-persistence homogeneous detection technology based on wide-field imaging has good stability and repeatability.

[0196] Example 13

[0197] Example 12 is repeated, except that the test container is replaced: in this example, a 96-well plate with a transparent bottom is used instead of 4 test cups for long afterglow homogeneous phase testing.

[0198] In the aforementioned Example 12, testing four samples takes only about 10 seconds. In this example, a 96-well plate is used for testing. Increasing the number of cuvettes tested simultaneously to 96 does not significantly increase the testing time, because although the number of samples increases, all test information can still be obtained with a single image capture. Therefore, the method of this invention provides a novel approach and method for improving the throughput of long-afterglow homogeneous phase testing. Furthermore, to facilitate simultaneous testing of multiple samples, a fully automated auxiliary liquid addition device (e.g., illustrated in the diagram) can be used in the separation liquid injection step to facilitate simultaneous testing of multiple samples. Figure 8 (As shown). Based on this, the method and technology of the present invention can play a role in high-throughput long-persistence homogeneous testing.

[0199] Example 14

[0200] This invention also provides solutions for further improving the versatility of detection, the ease of technology promotion, and the convenience of use. For example, for detection items with the same target analyte, the luminescent membrane or sensitized membrane can be pre-integrated onto the base plate of the test container. After the long-afterglow related dye components or membranes are integrated and fused in the well plate, the protein labeling (e.g., the first or second conjugate) and blocking on the well plate surface can be performed with reference to the enzyme-linked immunosorbent assay (ELISA) method, which is a universal and mature method.

[0201] Taking a 96-well plate as an example, the luminescent film functional components are pre-integrated into the bottom of each well, thereby obtaining a functionalized well plate that can simultaneously perform long-afterglow homogeneous testing on 96 samples (e.g., Figure 9 (As shown). This functionalized perforated plate is prepared using a uniform manufacturing process, which effectively reduces the deviation between holes or between plates, making it very easy to achieve excellent system quality control.

[0202] Based on this, functionalized well plates can serve as both a general testing container for the corresponding target analytes and a general component for long-afterglow systems. During use, only a fully automated auxiliary device is needed to uniformly add the sensitizing ball reagent component and the separation liquid component, which makes them convenient to use and promote.

[0203] Example 15

[0204] To further improve the versatility of the detection, the ease of technology promotion, and the convenience of use, in this embodiment, the prepared colorimetric cup test container containing the luminescent film is configured to be detachably assembled into a 12-well configuration of a 96-well plate (e.g., Figure 10(As shown). Based on this, eight 12-well functional plates corresponding to different detection items can be placed on the plate holder of a 96-well plate, and then multi-item rapid long-persistence imaging detection can be performed. The long-persistence compositions in all these 96-well samples are excited and charged using a light source. The excitation light source is then turned off, and a wide-field imaging acquisition method is used to simultaneously collect long-persistence signals from all cuvettes. In this embodiment, the long-persistence signal acquisition device is a cooled CCD camera, which is positioned below the bottom of the cuvettes, enabling surface imaging of the wide field of view formed by all cuvettes. The imaging exposure time can be set from 10ms to 300s; in this embodiment, a 30s exposure is selected to collect long-persistence emission signals. By processing and analyzing the acquired images, an intensity distribution map of the long-persistence emission signal can be obtained. In the image obtained from this surface imaging, the emission status of the area belonging to each cuvette can be recorded.

[0205] Based on wide-field imaging signal acquisition technology, multiple test cases can be detected simultaneously, and each test case is equivalent to an independent test without mutual interference. Therefore, the items tested in the four cuvettes can be set separately. In this embodiment, a single strip of 12 wells is set to the same detection item, and the target analyte concentration in the sample covers 12 gradients; while all eight well strips (each containing 12 wells) are set to eight different items, corresponding to eight different target analytes: human chorionic gonadotropin, anti-streptolysin O, rheumatoid factor, alpha-fetoprotein, carcinoembryonic antigen, prostate-specific antigen, troponin-I, and procalcitonin. The results show that all 96 tests can be detected, and the target analyte concentration information obtained by analyzing 12 tests per strip exhibits good linearity, with R-squared values ​​greater than 0.99. This demonstrates that the long-persistence homogeneous detection technology based on wide-field imaging has good scalability and shows broad application prospects.

[0206] It should be noted that the above embodiments can be freely combined as needed. The above description is only a preferred embodiment of the present invention. It should be pointed out that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting long afterglow homogeneous phase, characterized in that, The method includes the following steps: S1. Provide a long afterglow homogeneous detection reagent composition and the sample to be tested; S2. A portion of the long-afterglow homogeneous detection reagent composition is pre-attached to the bottom plate of the test container; the long-afterglow homogeneous detection reagent composition includes sensitized spheres and luminescent spheres, wherein a first coupling agent is attached to the surface of the sensitized spheres, and the sensitized spheres generate singlet oxygen under photoexcitation; a second coupling agent is attached to the surface of the luminescent spheres, and the luminescent spheres react with the singlet oxygen to generate a long-afterglow luminescence signal; wherein the sensitized spheres or the luminescent spheres are pre-attached to the bottom plate; S3. Add the test sample and the remaining portion of the long-afterglow homogeneous detection reagent composition to the test container for incubation, so that the long-afterglow immune complex formed by the immune reaction is fixed on the bottom plate of the test container. S4. Inject the separation liquid into the test container. The density of the separation liquid is greater than that of the aqueous solution, so that the separation liquid settles to the bottom plate and the aqueous solution is separated away from the bottom plate. S5. Irradiate the separated detection solution with excitation light; S6. Acquire long-afterglow emission signals through a detector, wherein the acquisition mode of long-afterglow emission signals includes surface imaging signal acquisition or scanning signal acquisition. S7. Based on the collected long afterglow emission signal values, the target analyte information in the sample to be tested is obtained.

2. The long afterglow homogeneous detection method as described in claim 1, characterized in that, The method by which the luminescent sphere or the sensitized sphere is pre-attached to the substrate includes one of the following methods: The luminescent sphere or the sensitized sphere is connected to the substrate through chemical bonds and immune binding interactions; Alternatively, the light-emitting sphere or the sensitized sphere may be incorporated into the substrate; Alternatively, the luminescent sphere or the sensitized sphere is doped in a transparent film and fixed on the surface of the base plate.

3. The long afterglow homogeneous detection method as described in claim 1, characterized in that: The separation liquid is a gel solution or an oil phase liquid, and the density of the separation liquid is greater than that of water.

4. The long afterglow homogeneous detection method as described in claim 1, characterized in that: The separation liquid is transparent, and the transparent color is colorless or light-colored.

5. The long afterglow homogeneous detection method as described in claim 3, characterized in that, The oil phase liquid includes one or more of dichloromethane, trichloromethane, carbon tetrachloride, perfluorinated carbon, iodized oil, and ionic liquids; wherein the ionic liquid is selected from the structure of Formula 1 and / or Formula 2: Formula 1, Formula 2.

6. The long afterglow homogeneous detection method as described in claim 5, characterized in that, The perfluorinated carbon is selected from one or more of the following: 1H-perfluoropentane, perfluorohexane, hexadecylfluoroheptane, octadecylfluorooctane, perfluorononane, perfluorodecane, perfluorocyclohexane, perfluoro(methylcyclohexane), 1-bromoheptafluoropropane, 2-bromoheptafluoropropane, 1-bromononafluorobutane, 1-bromo-1H-undecaprofluoropentane, 1-bromotridecylfluorohexane, 1-bromopentadecaprofluoroheptane, 1-bromoheptafluorooctane, 1-bromononafluorononane, perfluoroiodoethane, heptafluoro-1-iodopropane, 2-iodoheptafluoropropane, 2-iodononafluorobutane, 1-iodoperfluoropentane, 1-iodotridecylfluorohexane, 1-iodopentadecaprofluoro-n-heptane, heptafluoro-1-iodooctane, and perfluoroisononyliodine.

7. The long afterglow homogeneous detection method as described in claim 1, characterized in that, The first conjugate and the second conjugate can respectively form immune complexes with the target analyte.

8. The long afterglow homogeneous detection method as described in claim 1, characterized in that, The sensitized spheres include carrier microspheres, donor components, or a combination thereof, and the luminescent spheres include carrier microspheres, acceptor components, or a combination thereof. The donor component includes a light absorber, and the acceptor component includes a photochemical buffer and a luminescent agent.

9. The method for detecting long afterglow homogeneous phase as described in any one of claims 1-8, characterized in that, The long afterglow homogeneous detection reagent composition also includes reagents for diluting the sample and reagents for maintaining system stability.

10. The method for detecting long afterglow homogeneous phase as described in any one of claims 1-8, characterized in that, The excitation light source is a point light source or a surface light source; And / or, the detector includes one of a photomultiplier tube, a silicon photocell, a camera, a mobile phone, a CCD, and an EMCCD.

11. The method for detecting long afterglow homogeneous phase as described in any one of claims 1-8, characterized in that, The wavelength range of the excitation light is 365nm~1532nm.

12. The long afterglow homogeneous detection method as described in claim 11, characterized in that, The wavelength of the excitation light is 1064nm, 980nm, 915nm, 830nm, 808nm, 785nm, 730nm, 680nm, 630nm, 532nm, 488nm, 450nm, 405nm, or 365nm.

13. The long afterglow homogeneous detection method as described in claim 11, characterized in that, The time range of the detection liquid after separation by excitation light is 0.1ms to 5s.

14. The long afterglow homogeneous detection method as described in claim 11, characterized in that, The detector acquires long-afterglow emission signals over a time range of 0.001s to 300s.

15. The long afterglow homogeneous detection method as described in claim 14, characterized in that, The detector acquires long-afterglow emission signals over a time range of 0.01 s to 60 s.

16. The long afterglow homogeneous detection method as described in claim 11, characterized in that, The incubation process takes place over a period of 1 to 10 minutes.

17. The long afterglow homogeneous detection method as described in claim 11, characterized in that, The sample to be tested includes one or more of whole blood, urine, serum, plasma, body fluid, and cerebrospinal fluid.

18. The long afterglow homogeneous detection method as described in claim 11, characterized in that, The target analyte information in the sample to be tested includes one or more of the following: analyte type, concentration, and pH value.

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